Epigenetics And Targeting Mechanisms In Drosophila

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Epigenetics and targeting mechanisms in Drosophila melanogaster Margarida Figueiredo Department of Molecular Biology Umeå University 2015, Sweden This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-267-3 Cover: immunostaining in polytene chromosomes (picture by Margarida Figueiredo) with the creative design by Chris Voss. Electronic version is available at http://umu.diva-portal.org/ Printed by: Arkitetkopia Umeå, Sweden 2015 Nada é, tudo se Outra Fernando Pessoa To my parents, Julieta e Carlos, for everything CONTENTS LIST OF PUBLICATIONS ............................................................................ iii ABSTRACT ....................................................................................................... iv 1. INTRODUCTION ........................................................................................ 1 Epigenetics....................................................................................................... 1 Chromatin: the nucleosome ........................................................................... 2 Nucleosome evolution ................................................................................. 3 Histone modifications ..................................................................................... 4 DNA methylation ............................................................................................ 5 Chromatin states ............................................................................................. 6 Long non-coding RNAs ................................................................................... 7 Drosophila melanogaster............................................................................... 8 Mechanisms of targeting............................................................................... 10 2. SEGMENTAL ANEUPLOIDIES ............................................................. 11 Aneuploidy ...................................................................................................... 11 Buffering ........................................................................................................ 12 Summary and discussion of Paper I ............................................................. 14 3. HP1a TARGETING ................................................................................... 16 Chromosome-wide regulation of the 4th ...................................................... 16 The 4th chromosome .................................................................................. 16 POF ..............................................................................................................17 i HP1a in chromosome 4 gene regulation ................................................... 19 HP1a and heterochromatin formation ........................................................ 20 Su(var)3-9, Setdb1 and G9a ......................................................................... 23 Summary of Paper II .....................................................................................25 4. MSL TARGETING .................................................................................... 29 Sex chromosomes and sex determination ................................................... 29 Sex Chromosome evolution ......................................................................... 30 Dosage compensation .................................................................................... 31 MSL complex ................................................................................................ 32 roX RNAs .................................................................................................. 34 MSL-complex and heterochromatin.............................................................35 Upregulation of the male X .......................................................................... 36 Mechanisms of MSL targeting ...................................................................... 37 Links between X and 4th chromosomes ....................................................... 38 Summary of Paper III ................................................................................... 38 Summary of Paper IV ................................................................................... 43 CONCLUSIONS ..............................................................................................45 ACKNOWLEDGMENTS ............................................................................. 46 REFERENCES ............................................................................................... 48 ii LIST OF PUBLICATIONS This thesis is based on the following publications (reproduced with permission from the publishers): I Lina E Lundberg, Margarida L A Figueiredo, Per Stenberg, Jan Larsson (2012). Buffering and proteolysis are induced by segmental monosomy in Drosophila melanogaster. Nucleic Acids Res 40: 59265937. II Margarida L A Figueiredo, Philge Philip, Per Stenberg, Jan Larsson (2012). HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster. PLoS Genet 8: e1003061. III Margarida L A Figueiredo, Maria Kim, Philge Philip, Anders Allgardsson, Per Stenberg, Jan Larsson (2014). Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions. PLoS Genet 10:e1004865. IV Margarida L A Figueiredo, Philge Philip, Jan Larsson (2015). MSL interaction with non-roX non-coding RNAs. (Manuscript). iii ABSTRACT Regulation of gene expression can occur at different levels, ranging from single genes to genomic regions and even to entire chromosomes. Understanding which epigenetic mechanisms are involved in this regulation, especially how protein regulators are targeted to chromatin, has been the focus of my thesis. I show that genes in monosomic regions are buffered, i.e., expressed at a higher level than the expected 50% of the wild type level. The buffering is general, it is primarily affected by gene length and is not affected by the presence of other monosomic regions. Additionally, the expression of proteolysis genes is induced in aneuploidies. Gene expression regulation at a chromosome-wide level has so far only been described for the X chromosome and for the 4th chromosome of Drosophila melanogaster. The 4th chromosome gene expression is regulated by both POF and HP1a, which target exons of active genes. Additionally, HP1a targets promoters. I found that Setdb1 and Su(var)3-9 recruit HP1a to the 4th chromosome and to pericentromeric regions, respectively, by di- and trimethylation of H3K9. Importantly, HP1a is recruited to promoters of active genes independently of methylated H3K9. The promoters bound by HP1a are enriched in HP2, in A/T content and are DNase sensitive. We propose that HP1a is bound to the H3 histone core at promoters and that the promoter targeting functions as the nucleation site from which HP1a spreads via H3K9 methylation. MSL complex targets the male X chromosome and is partially responsible for dosage compensation, by upregulation of X chromosome gene expression almost two times. The importance of roX long non-coding RNAs in MSL recruitment has been one important focus of this thesis. I found that in absence of roX, MSL targets high affinity sites on the X chromosome. Additionally, a complete and active MSL complex is bound to six genes of the 4th chromosome. Interestingly, when roX RNAs are not present, MSL targets genomic regions enriched in Hoppel transposable elements and repeats. We propose that the heterochromatic targeting represents an ancient function of the MSL complex and that the roX RNAs evolved to restrict MSL binding to the X chromosome. I further found that MSL associates with many different RNAs when roX are absent, including a set of snoRNAs. iv INTRODUCTION CHAPTER 1 1. INTRODUCTION It´s truly fascinating to imagine that once in our life time we were nothing more than a single cell, with an incredibly unique genome. Rounds of cell divisions, organized cell movements and cell differentiation shaped us. We are the result of several different populations of cells sharing the same genome. How the different cell types express their specific sets of genes, while keeping others silenced, and how this information is passed on to the daughter cells and remembered through successive rounds of cell division is called epigenetics. Epigenetics Epigenetics is a field of research focused on understanding how changes in patterns of gene expression, not due to changes in the DNA sequence, are established and maintained through mitosis and/or meiosis. DNA methylation and histone modifications within and around specific genes are known as the main epigenetic marks that are involved in gene expression regulation and cell memory. Not only is the epigenetic information heritable through cell divisions but in some cases also from generation to generation. Transgenerational epigenetics can occur when stochastic or environmental-induced (for instance nutrition) epigenetic changes in the germline of the parents are transmitted to the offspring (CHONG AND WHITELAW 2004; HEARD AND MARTIENSSEN 2014). Additionally, epigenetic differences between maternally and paternally inherited alleles can dictate which allele will be expressed, a phenomenon called genomic imprinting (DELAVAL AND FEIL 2004). Imprinting causes some genes in the adult individual to have monoallelic expression. As an example, during gametogenesis or early embryogenesis, DNA hypermethylation at the maternal allele of an imprinted gene will ensure that only the paternally inherited allele is active in the adult (DELAVAL AND FEIL 2004). Development can be compromised in imprinting disorders, the Angelman and Prader-Willi syndromes are well known examples (BUITING et 1 INTRODUCTION al. 1995). The fact that several human diseases, like cancer and disorders of the immune, endocrine and nervous system, are caused by dysfunctional epigenetic mechanisms highlights the importance of this field of research (FALLS et al. 1999). Chromatin: the nucleosome The genome of eukaryotic cells is organized in chromosomes, which consist of linear molecules of DNA associated with histones and other proteins, in a complex called chromatin. The basic repeated unit of chromatin is the nucleosome: an octamer of histones around which 147 base pairs of DNA is wrapped in 1.65 turns (figure 1) (LUGER et al. 1997). Each nucleosome in the array is separated from each other by a short “naked” DNA segment of 2090 base pairs – linker DNA (SZERLONG AND HANSEN 2011). The eight histones in each nucleosome are organized in two dimers of H2A-H2B and one tetramer of H3-H4. Additionally, the linker histone H1 binds between the nucleosome and the wrapped-DNA, stabilizing the nucleosome, and also interacts with the linker DNA (LUGER et al. 1997). Figure 1. Nucleosome assembly. Each histone molecule (excluding linker histone H1) is formed by the histone core, consisting of three α-helices separated by two loops, and by Nterminal protruding tails. The histone loops are essential for the interaction nucleosome-DNA, and the α-helices are essential for histone-histone interaction. The tetramer H3-H4 forms a more stable core, in contrast to the two H2A-H2B dimers (SMITH AND STILLMAN 1991; LUGER et al. 1997). (adapted from (VENKATESH AND WORKMAN 2015)). 2 INTRODUCTION Different structural states of the nucleosome can be affected by the incorporation of histone variants that replace the canonical histones H3, H2A and H2B, and the linker histone H1. Histone variants can be incorporated in nucleosomes throughout the cell cycle, at specific genomic regions and may have tissue-specific expression patterns. For instance, CenH3 is a histone variant that replaces H3 specifically at centromeres and plays a role in the assembly of the kinetochore (BLACK AND CLEVELAND 2011; HENIKOFF AND SMITH 2015). In fact, CenH3 is considered a better marker for centromeres than the DNA-sequence itself, since CenH3 localizes to neo-centromeres that lack the α-satellite DNA repeats that are typical for centromeric DNA (HENIKOFF AND SMITH 2015). Another example is the histone variant H2A.Z, which has been proposed to have distinct nuclear functions including transcriptional regulation, cell-cycle control, DNA replication, DNA damage repair, chromosome segregation and genome integrity (HENIKOFF AND SMITH 2015). In embryonic stem cells, H2A.Z is preferentially associated with promoters of developmentally regulated genes that have both active and repressive marks of transcription (KU et al. 2012). Nucleosome evolution Nucleosomes play a very important role in the organization of the genome by compacting the DNA and allowing the long chromosome molecules to be packed inside the small eukaryotic cell nucleus (in humans, approximately 2 meters of total DNA are fitted in a nucleus with a diameter of 6 µm). Several lines of evidence suggest that the nucleosomes evolved in parallel with the increase in genome size. Histones are highly conserved proteins, with H2A, H2B, H3 and H4 being present in all eukaryotes. From the three domains of life, Bacteria is the only one that doesn´t have histones. However, even in Bacteria the single circular chromosome is tightly associated with proteins, forming the nucleoid. The nucleoid–associated proteins (NAPs) are involved in chromosome organization, by allowing the formation of domains with different supercoils, and modulating gene expression (SANDMAN et al. 1998; WANG et al. 2011). The evolutionary history of histones can be traced back to the Archaea domain of life, prokaryotes with a single circular chromosome. In Methanothermus fervidus for instance, the histone-related 3 INTRODUCTION HMfA and HMfB proteins form tetramers that constrain 60 bp of DNA. These studies on the origin of the nucleosome are in agreement with one suggested theory on the evolution of the first eukaryotic cells: they were the result of a fusion between Archaea and eubacterium, the first becoming the nucleus and the latter the cytoplasm (YUTIN et al. 2008). In the transition from prokaryotes to eukaryotes, as the genome size increased, the nucleosomes became essential to maintain and regulate the conformational flexibility of DNA and its reversible structural changes (MINSKY et al. 1997). As for the eukaryotes, the evolution from single-cell species to multicellular ones originated a disproportional increase in genome size relative to the nuclear volume. Larger genomes had to compact their chromatin more, and a recent study has shown that eukaryotic genome expansion was accompanied by an acquisition of arginines in H2A N-terminus and that this directly affects chromatin compaction (MACADANGDANG et al. 2014). Histone modifications There are a large number of identified reversible histone posttranslational modifications (PTMs), made by highly specific enzymes on different amino acids of the histone tails and also on the histone core. While the modifications on histone tails have been the most studied, it has been suggested that modifications on residues of the histone core lateral surface can affect more directly the interaction histone – nucleosomal DNA, altering nucleosome stability (TESSARZ AND KOUZARIDES 2014). Examples of histone modifications include: acetylation of lysines, methylation of lysines and arginines, phosphorylation of serines and threonines, ubiquitination of lysines, sumoylation of lysines, ADP-ribosylation of glutamic acids, glycosylation and citrullination of arginines (KHORASANIZADEH 2004; TESSARZ AND KOUZARIDES 2014). In the work presented in this thesis the focus has been on acetylation and methylation of histone N-terminal tails. Acetylation of lysines is generally associated with activation of transcription and it has been proposed that it neutralizes the positive charges of lysines, making the interaction between the histone and the DNA weaker and thereby increasing the accessibility of the transcriptional machinery to the DNA (LUGER et al. 1997; FENLEY et al. 2010; TROPBERGER et al. 2013). Acetylated 4 INTRODUCTION lysines are recognized by proteins with bromodomains, like histone acetyltransferases (HAT) – which themselves produce this modification, and by chromatin remodelling proteins (ZENTNER AND HENIKOFF 2013). In Drosophila melanogaster, acetylation of lysine 16 of histone H4 (H4K16ac) is highly enriched on the male X chromosome and is believed to contribute to dosage compensation by increasing the transcriptional output - this is an important topic of my thesis, discussed in more detail in chapter 4. Mono-, di- or tri- methylation can occur on lysines, by the actions of histone lysine methyltranferases (HKMT), and can be associated with activation of transcription (H3K4me and H3K36me) or with repression (H3K9me and H3K27me). Methylated lysines can be recognized by proteins with specific domains, including chromo domains (ZENTNER AND HENIKOFF 2013). As an example, H3K9me is recognized by heterochromatin protein 1 (HP1), a chromo domain containing protein, which is associated with the formation of a highly compacted chromatin structure – this is an important topic of my thesis that will be discussed in more detail in chapter 3. The existence of enzymes like histone deacetylases (HDACs) and histone lysine demethylases (KDMs) makes these posttranslational modifications reversible and contributes to dynamic changes in chromatin structure. The effect of histone modifications on chromatin structure can be direct or indirect by becoming targets for binding of non-histone proteins, “the epigenetic readers” which can act on chromatin organization. The readers can bind specifically to the histone modifications (for instance, by their bromo or chromo domains) and recruit ATP-dependent chromatin remodellers or histone chaperones (LUGER et al. 2012). DNA methylation DNA can be methylated, for example at cytosines (5-mC) of CytosineGuanine dinucleotides (usually denominated CpG) by DNA methyltransferases (DNMT) and this is an epigenetic mark that has been extensively studied in mammals. DNA methylation is conserved in fungi and plants and can occur at other nucleotides and dinucleotides than CpG (JONES 2012). Although CpG is not globally abundant in mammalian genomes, there are regions enriched in CpG, called CpG islands (SMITH AND MEISSNER 2013). 5 INTRODUCTION CpG islands at promoters of housekeeping genes and developmentally regulated genes are usually unmethylated, correlating to their active state. In contrast, DNA methylation is enriched at satellite repeats and transposable elements and functions to silence these elements (SMITH AND MEISSNER 2013). One entire X chromosome in each mammalian female cell is in fact hypermethylated, contributing to its almost complete gene inactivation, as part of the dosage compensation system in mammals, which will be briefly discussed in chapter 4. DNA methylation is recognized by proteins with methyl-CpG binding domains, which act together with histone modification readers to recruit chromatin remodellers and affect chromatin structure. DNA methylation has been reported to be absent in yeast and C. elegans. In Drosophila melanogaster the presence of DNA methylation has been controversial. Although still debated, some studies have shown that DNA methylation can occur at non-CpG locations and at early embryonic stages (LYKO et al. 2000; KUNERT et al. 2003; WEISSMANN et al. 2003) and in retrotransposon silencing (PHALKE et al. 2009). However, the existence and potential importance of DNA methylation in Drosophila remains controversial and elusive (RADDATZ et al. 2013). Chromatin states Chromatin remodelling is a dynamic process and achieving different levels of chromatin compaction is important for fitting the long DNA molecules inside the small nucleus, for the condensation of the mitotic chromosomes, and to regulate gene expression. The primary structure of chromatin is arranged as an array of nucleosomes forming the 10 nm thick “beads-on-a-string” structure, seen under the electron microscope (OUDET et al. 1975; SZERLONG AND HANSEN 2011). The presence of the linker histone H1 is important in the formation of a higher order chromatin structure, where each nucleosome in the array can interact with other nucleosomes and with DNA, resulting in a DNA fiber compaction into a secondary structure of 30 nm. Although the 30 nm chromatin fiber has been extensively studied in vitro, its structure has not been resolved yet and it has been difficult to prove its existence in vivo (ELTSOV et al. 2008; FUSSNER 6 INTRODUCTION et al. 2012). The most compact chromatin form that exists is the 700 nm thick metaphase chromosome, which is visible under the light microscope and requires the actions of condensin and topoisomerase II (KHORASANIZADEH 2004). In an interphase chromosome there are distinguishable chromatin arrangements: a highly compacted structure called heterochromatin, and a less compacted structure called euchromatin. Heterochromatin by definition fails to decondense after telophase in the cell cycle. The majority of genes reside within euchromatin, since the open structure facilitates the targeting by RNA polymerase and transcriptional machinery. Heterochromatin is usually associated with gene silencing, is typically enriched in H3K9me and HP1, replicates late in S phase and it doesn´t recombine. Constitutive heterochromatin can be found at regions that are gene poor and highly enriched in repetitive DNA sequences, satellite repeats and transposons, like centromeres and telomeres, and is present in all cell types. Some active genes reside within constitutive heterochromatin and understanding which mechanisms allow their expression in such a highly compacted repressive environment is an interesting topic of research, which will be discussed in chapters 3 and 4. Additionally, depending on the cell type, the expression of tissue specific genes needs to be switched on or off. These genomic regions can switch between euchromatic active states and heterochromatic inactive states. Facultative heterochromatin exists in regions that are heterochromatic in some cell types, but euchromatic in other cell types. The inactive X chromosome in the cells of females and imprinted genes are good examples of facultative heterochromatin (TROJER AND REINBERG 2007). Long non-coding RNAs Eukaryotic genomes contain many genes coding for non-coding RNAs (ncRNAs). ncRNAs are involved in several cellular processes of gene regulation and chromatin modification, and it has been proposed that they have evolved with organismal complexity. ncRNAs can be transcribed antisense to protein-coding genes and from introns of protein-coding genes. A large fraction of ncRNAs are small RNAs, like for instance small nuclear RNAs (snRNAs) involved in splicing; small nucleolar RNAS (snoRNAs) involved 7 INTRODUCTION in modifying nucleotides in rRNAs and other RNAs; and micro RNAs (miRNAs) which are involved in the RNA interference pathway (MORRIS AND MATTICK 2014). Long non-coding RNAs (lncRNAs) are RNA transcripts typically longer than 200 bp, polyadenylated and without open reading frames. lncRNAs can form higher order secondary structures and bind proteins or base-pair with specific RNA or DNA targets, for instance forming RNA-DNA:DNA triplexes. lncRNAs are involved in regulation of gene expression (repressing it or activating it), typically by forming ribonucleoprotein complexes that are recruited in cis (to genes near the lncRNA transcription site) or in trans (to genes in other genomic loci). The involvement of lncRNAs in dosage compensation is evident both in mammals and in Drosophila, and it will be a topic discussed in the chapter 4 of this thesis. Xist lncRNA in mammals acts in cis, coating the entire female X chromosome from which it is transcribed and, by recruiting specific proteins, induces formation of heterochromatin and contributes to X inactivation. The two lncRNAs roX1 and roX2 in Drosophila can act both in cis and in trans, coating the entire X chromosome in males, and by recruiting MSL proteins, induce up-regulation of gene expression from X chromosomal genes. In either case it is still not known how these lncRNAs can specifically recognize one entire chromosome, especially the very distant genes (FATICA AND BOZZONI 2014). Drosophila melanogaster Also known as the fruit fly, Drosophila melanogaster is one of the best studied model organisms in genetics. Although phenotypically very different from us, approximately 75% of known disease-related genes in humans have homologs in Drosophila (BIER 2005). 143.7 megabases (Mb) of the Drosophila melanogaster genome has been fully sequenced and annotated, including some of the heterochromatic sequences in pericentromeric regions (DOS SANTOS et al. 2015). The epigenetic landscape of Drosophila melanogaster has also been extensively studied (for which the modENCODE consortium contributed significantly) and the genome-wide distribution of a large number of chromatin proteins, as well as histone modification marks, have been mapped in different tissues and/or cell types (by ChIP-chip and ChIPseq data) (CELNIKER et al. 2009; ST PIERRE et al. 2014). Gene expression profiles 8 INTRODUCTION at different developmental stages are also available (RNA-seq data). One advantage of Drosophila in genetic studies has been the possibility to easily create mutants, for which the engineered balancer chromosomes and the Pelement have proven to be very important tools. The genome of Drosophila melanogaster is constituted by three autosome pairs (2, 3 and 4) and a pair of sex chromosomes (XX or XY). The 4th and the Y chromosomes are highly heterochromatic, being enriched in repeated DNA sequences and transposons. In the study of chromosomes and epigenetics, Drosophila has a great advantage in having giant chromosomes in some tissues, called polytene chromosomes. Polytene chromosomes are formed when DNA replicates without the cell going through division (endoreplication) and in salivary glands this results in approximately 2000 copies of DNA molecules aligned together, including both homolog chromosomes (figure 2). Figure 2. Drosophila melanogaster chromosomes. Polytene chromosomes from a salivary gland nuclei in comparison with mitotic chromosomes from a brain nuclei (in the rectangle) from third instar larvae stained with DAPI and visualized with 40x and 100x lenses, respectively. Left arms (2L, 3L) and right arms (2R, 3R) of chromosomes 2 and 3 are indicated as well as the X and the 4th. Note that the Y chromosome, along with other very heterochromatic regions of the genome (pericentromeric regions and part of the 4 th) are not seen in the salivary gland nuclei since they don´t endoreplicate. The pericentromeric regions of all polytene chromosomes are joined together in the chromocenter. In the mitotic nucleus, notice the two dots corresponding to the two replicated 4th homologous chromosomes. 9 INTRODUCTION Mechanisms of targeting The main focus of my thesis is to understand how proteins are recruited to chromatin and how it affects regulation of gene expression at a larger scale rather than on single genes. In chapter 2, I will discuss how gene expression is regulated at segmental monosomies. In chapters 3 and 4, the main work of my thesis, I will discuss the two chromosome-wide targeting mechanisms in Drosophila melanogaster: the mechanisms on the 4th chromosome and on the X chromosome, respectively. In chapter 3, I will focus on the role of each HKMT in HP1a recruitment. In chapter 4, I will discuss the role of roX lncRNAs in the recruitment of MSL-complex and also the search for new RNAs that interact with MSL. 10 SEGMENTAL ANEUPLOIDIES CHAPTER 2 2. SEGMENTAL ANEUPLOIDIES Aneuploidy Aneuploidy has both been defined as an alteration of the normal number of whole chromosomes in a cell or organism (chromosomal aneuploidy), and as an alteration of the normal number of a chromosomal region in a cell or organism (segmental aneuploidy). A diploid organism, for instance, can have one or three copies of one of the chromosomes (chromosomal monosomy or trisomy) and one or three copies of a region of a chromosome (segmental monosomy or trisomy). Chromosomal aneuploidies can be caused by merotellic attachments, where a single kinetochore attaches to microtubules that arise from both poles of the spindle, by spindle assembly checkpoints defects or by chromosome cohesion defects (GORDON et al. 2012). Segmental aneuploidies can be caused by replication slippage, non-allelic homologous recombination and non-homologous end-joining (SCHRIDER AND HAHN 2010). The presence of an extra copy or the loss of a copy of a chromosome or segment is usually detrimental for single cells and organisms, because it can result in an increase or decrease in production of transcripts from those genes, or it can alter the doses of regulatory sequences, resulting in gene network deregulation and possibly leading to genomic instability (GORDON et al. 2012). Studies in yeast and in mouse embryonic fibroblasts have shown that in single cells, one extra copy of a chromosome causes slow proliferation (TORRES et al. 2007; WILLIAMS et al. 2008; PAVELKA et al. 2010). In humans, aneuploidies are the leading cause of spontaneous abortions and birth defects (COLNAGHI et al. 2011). Chromosomal monosomies are generally more deleterious than chromosomal trisomies, and only trisomy of chromosome 21 (Down´s syndrome) is viable in humans. Trisomies 13 and 18 have severe developmental impairments and usually die during the first months of life (TORRES et al. 2008). In cancers, aneuploidies do not seem to be deleterious to the cells and may actually contribute to proliferation of the cancer cells, which is demonstrated by the fact that aneuploidies are present 11 SEGMENTAL ANEUPLOIDIES in most types of cancers (80% of solid tumours and 60% of hematopoietic tumours) (STANKIEWICZ AND LUPSKI 2010). Extra copies of chromosomes can be found in healthy organisms in nature, in fact many plants and animals have cells with one or more extra copies of all their chromosomes: a condition called polyploidy (genome doubling). For instance human hepatocytes contain up to eight copies of all chromosomes (octoploidy) (STENBERG AND LARSSON 2011; GENTRIC et al. 2012). Segmental aneuploidies can result in copy number variations (CNV): differences in large genomic regions (from 1 kb to several Mb) for different individuals of the same species, due to duplication or deletion events. In fact, two individuals from the same species are likely to have dozens to hundreds of CNVs (SCHRIDER AND HAHN 2010; STANKIEWICZ AND LUPSKI 2010). CNVs along with single nucleotide polymorphisms (SNPs) and indels (insertion and deletion of short segments of nucleotides up to 50 bp) account for the genomic variation between individuals of the same species (SCHRIDER AND HAHN 2010). CNVs can also be different among human populations, for instance the gene that encodes a haptoglobin-related protein, used in defense against trypanosomes, is present in 2 copies in European populations, in 1-2 copies in Asian populations, but in 4-8 copies in 25% of African populations (HANDSAKER et al. 2015). Buffering An important question regarding chromosomal and segmental aneuploidies is how the variation in gene dose contribute to the different phenotypes: how the transcript levels and the protein dose changes. Some chromosomes can exist in monosomic condition in Drosophila melanogaster healthy individuals, for instance the X chromosome in males and the 4th chromosome. It is known that in Drosophila the viability of these two monosomic conditions depend on the male-specific lethal (MSL) ribonucleoprotein complex and the Painting of fourth (POF) protein which are responsible for transcriptional dosage compensation of the X- and 4th chromosomes, respectively (discussed in chapters 3 and 4 of this thesis). If we consider the prevalence of CNVs in healthy individuals, we can hypothesize that there might be systems that compensate for the gene dosage differences in autosomes – buffering systems. Since duplications and 12 SEGMENTAL ANEUPLOIDIES deletions provide genetic variability and are therefore driving forces of evolution, incomplete buffering may exist as a way to allow some degree of genetic variation, while buffering collapse could lead to lethality (STENBERG et al. 2009). Several studies in yeast and mammalian cell lines with extra copies of chromosomes have shown that the transcriptional response increases with the increase in gene copy number, suggesting that transcriptional dosage compensatory mechanisms do not exist (TORRES et al. 2007; WILLIAMS et al. 2008; STINGELE et al. 2012). Some of these studies claimed that the respective protein levels were not increased at the same degree as the mRNA, which suggests that there might exist a buffering effect at a posttranscriptional level (TORRES et al. 2007; STINGELE et al. 2012). One of these studies proposed that in trisomies the increased amount of proteins produced disrupt cellular physiology interfering with metabolic pathways, and the cell copes with it by increasing protein degradation (TORRES et al. 2007). In fact, mutations in the deubiquitylation enzyme UBP6 makes yeast with extra chromosomal copies grow faster (TORRES et al. 2010). Another study has found that in human trisomic and tetrasomic cell lines, protein folding is significantly impaired (DONNELLY et al. 2014). In contrast, studies using microarray in trisomic fetal cell lines, and in segmental aneuploidies (deficiencies and duplications) in both Drosophila and maize have shown that the transcriptional response was not proportional to the gene dose and thus support the existence of buffering mechanisms (FITZPATRICK et al. 2002; GUPTA et al. 2006; MAKAREVITCH et al. 2008; STENBERG et al. 2009; MCANALLY AND YAMPOLSKY 2010; ZHANG et al. 2010). In one particular study in Drosophila it was shown, using microarrays, that mRNA levels at three deficiencies on chromosome 2L and monosomies for chromosome 4 were 64% of wild type levels, instead of the expected 50% (STENBERG et al. 2009). This buffering of RNA levels seen in deficiencies didn´t occur at the studied duplications, which agrees with the fact that segmental monosomies are less well tolerated than segmental trisomies. In the cited study, only genes with expression levels that could be readily measured were included, since low expressed genes or inactive genes will look like they are being fully compensated. The buffering effect that was found was suggested to act in a general mode on all genes included in the deficiency, since the 13 SEGMENTAL ANEUPLOIDIES buffering effect was normally distributed among the genes, around a mean of 64% of wild type expression. The authors suggested that the buffering effect could also be the result of feedback regulation of a few genes which could result in a local high concentration of transcriptional factors, increasing the transcription in the entire monosomic region. Additionally, tissue-specific genes were found to be more buffered than ubiquitously expressed genes (STENBERG et al. 2009). Summary and discussion of Paper I We were interested to study the factors that affect buffering of segmental aneuploidies, at regional and individual gene levels in more detail. Additionally, we wanted to investigate the general transcriptional response to segmental aneuploidies, i.e., how the transcription of genes outside the monosomic region is affected. In this study we analysed the mRNA expression levels by microarray on adult female flies from seven lines with segmental deficiencies (Df). The Df lines we analysed differ in length, total number of genes and number of expressed genes. We created pairwise combinations of deficiencies to study the combinatorial effects of buffering. We confirmed the presence of a weak but significant buffering at the RNA level and found the mean expression of the genes in each deficiency to be between 54% and 58% of wild type (WT), instead of the expected 50%, if there would be no buffering. The fact that the buffering effect obtained was lower than what previous studies have shown (FITZPATRICK et al. 2002; GUPTA et al. 2006; MAKAREVITCH et al. 2008; STENBERG et al. 2009) is probably due to the more stringent cutoff applied on expression levels in this study. Since cancers have different combinations of aneuploidies and Drosophila can tolerate monosomy up to approximately 1% of the genome, we hypothesized that combining deficiencies could decrease the general buffering (ultimately leading to lethality). When studying the pairwise combinations of deficiencies, we didn´t find any combinatorial effect on buffering, i.e., buffering was not enhanced or reduced by combining any two deficiencies and no deficiency had a directional influence on the buffering of the other deficiencies. 14 SEGMENTAL ANEUPLOIDIES We found a weak correlation between the gene expression ratio (Df/WT) and the total number of deleted genes, i.e., the fewer the deleted genes the larger the buffering. When we analysed the buffering at gene level we found that longer genes are more buffered than shorter genes, independently on both expression level and whether the genes are ubiquitously expressed or tissue-specific. These results led us to speculate that buffering mechanisms might involve transcription elongation. Alternatively, buffering could occur by passive mechanisms, like the looping out of the monosomic region, since it is unpaired, to more transcriptional permissive environments. When analysing the transcriptional changes outside the monosomic regions, we didn´t find any spreading of the buffering effect from the monosomic region to the neighbouring diploid regions. Interestingly, we found that in the individuals carrying monosomic regions, the transcription of some genes encoding for proteins with peptidase and proteolysis activities was upregulated. This result agrees with the view that protein degradation is a general response to aneuploidies, since it´s seen in both duplications and deficiencies (TORRES et al. 2010; DONNELLY et al. 2014). It is important to note that the deficiencies used in this study have been kept in monosomic condition in stocks for years in the lab. It is thus unclear if general regulatory mechanisms that compensate for gene copy number imbalances exist naturally or if the DrosDel (RYDER et al. 2007) deficiency lines analysed in this study had time to select for increases at the transcriptional level. 15 HP1a TARGETING CHAPTER 3 3. HP1a TARGETING Chromosome-wide regulation of the 4th chromosome Chromosome-wide gene regulation is well known for the X chromosome in many species, as part of dosage compensation systems. Chapter 2 of this thesis describes buffering, and it has become evident that buffering systems at the transcriptional level exist and that they compensate for gene dosage differences at least for monosomic regions. Less is known about chromosome-specific systems that regulate gene expression in an autosomespecific manner. So far, the only well described system is the one that targets and regulates the 4th chromosome of Drosophila melanogaster. The 4th chromosomes in both males and females are specifically targeted by the gene regulatory protein Painting of Fourth (POF) – the only known autosomespecific protein (LARSSON et al. 2001). The majority of Drosophila species have a dot chromosome similar to the 4th (usually referred to as the F element) (MULLER 1940; ASHBURNER 1989) and the binding of POF to the F element is conserved in evolution (LARSSON et al. 2004). There are several evolutionary links between POF and chromosome 4 and the X chromosome dosage compensation, and this will be a topic of discussion in chapter 4 of this thesis. The 4th chromosome The fourth chromosome of Drosophila melanogaster is an atypical autosome since it is the smallest chromosome, approximately 5 Mb long, and it is highly heterochromatic (LOCKE AND MCDERMID 1993). The 4th has a pericentromeric region of 3-4 Mb which is gene-poor and consists of highly condensed heterochromatin, enriched in A-T and in simple satellite repeats, and this region is underreplicated in polytene chromosomes (LOCKE AND MCDERMID 1993; RIDDLE AND ELGIN 2006; RIDDLE et al. 2009). The rest of the 4th consists of a polytenized region of 1.28 Mb where almost all genes reside and which is also enriched in repetitive DNA, transposable elements and features typical of heterochromatin, like the presence of HP1a and H3K9me (BARIGOZZI et al. 1966; MIKLOS et al. 1988; EISSENBERG et al. 1992; CZERMIN et al. 2002; SCHOTTA 16 HP1a TARGETING et al. 2002). Other heterochromatic characteristics of the 4th is that it doesn´t recombine in meiosis (HOCHMAN 1976), it replicates late in S phase (BARIGOZZI et al. 1966) and it suppresses the expression of transgenes inserted on the 4th, by the well-studied phenomenon of position-effect variegation – see below (WALLRATH AND ELGIN 1995; WALLRATH et al. 1996). Although highly heterochromatic, the 4th chromosome has a gene density similar to the autosome arms, consisting of at least 92 genes (JOHANSSON et al. 2007a). The expression of the 4th chromosome genes in this heterochromatic environment has been proposed to be fine-tuned by both POF and HP1a which form a balancing system where POF stimulates and HP1a represses gene expression (JOHANSSON et al. 2007a; JOHANSSON et al. 2007b; JOHANSSON et al. 2012). In polytene chromosomes, while HP1a targets the pericentromeric regions of all chromosomes and the whole 4th chromosome, POF only binds to the polytenized part of the 4th (figure 3) (LARSSON et al. 2001; JOHANSSON et al. 2007a). Figure 3. POF targets the 4th (left) while HP1a targets the 4th and the chromocenter (right). Polytene chromosomes from salivary gland nuclei of wild type third instar larvae, immunostained with antibodies against POF (left) and HP1a (right). DNA is stained with DAPI (blue). POF Translocation experiments between the 4th chromosome with breakpoints in the proximal part (closer to the pericentromeric region) or more distal part (closer to the telomeric region) and other chromosomes suggested that POF 17 HP1a TARGETING initiates its targeting at the proximal part of the 4th, the nucleation site, and then spreads in cis to the distal part (LARSSON et al. 2001). These experiments also showed that POF targets the 4th chromosome with some sequence specificity since it doesn´t spread in cis to the chromosomes that are translocated to the 4th. POF was also shown to spread in trans, between the endogenous 4th and the translocated 4th when these two chromosomes pair (LARSSON et al. 2001; JOHANSSON et al. 2007a). POF is a 55 kDa protein that contains an RNA recognition motif and although RNAse treatment doesn´t affect POF targeting to the 4th (LARSSON et al. 2001; JOHANSSON et al. 2012), RNA immunoprecipitation followed by tiling arrays (RIP-chip) experiments have shown that POF has a general affinity for nascent RNAs, and more specifically to RNAs from the 4th chromosome (JOHANSSON et al. 2012). The suggestion that POF is responsible for dosage compensation of the genes on the 4th chromosome came from the fact that homozygous loss-of-function Pof mutants are lethal when they only have one 4th chromosome, but are viable and fertile if both 4th chromosomes are present (JOHANSSON et al. 2007a). Whole transcriptome studies by expression microarray experiments have further shown that homozygous Pof mutants have a significant reduction in mRNAs encoded from chromosome 4 genes (JOHANSSON et al. 2007a; LUNDBERG et al. 2013). An additional study has shown that POF stimulates the expression of 4th genes independently on chromosome 4 copy numbers (STENBERG et al. 2009). In the cited study, when fold changes in mRNA expression of haplo-4 flies, compared to wild type, were calculated based on expression microarray results, it was shown that the genes on the 4th in haplo-4 flies are buffered to 64% of wild type levels, instead of the expected 50% if there would be no buffering - a similar level of buffering to what is seen in segmental monosomies (see chapter 2). Chromosome 4 genes in triplo-4th flies were also buffered to 139% of wild type, instead of the expected 150% if there would be no buffering, contrary to what was seen for segmental trisomies (see chapter 2). Importantly, the study showed that the genes more strongly buffered in haplo-4 flies are the ones that have the largest decrease of expression in Pof mutants, compared to wild type, showing that the POF protein in this case is responsible for 4th chromosome dosage compensation (STENBERG et al. 2009). 18 HP1a TARGETING It remains elusive by which mechanism POF stimulates transcription output from the 4th chromosome. It has been shown that genes located in pericentromeric regions and chromosome 4 genes (genes enriched in HP1a) show an increased ratio between whole cell transcripts and nuclear transcripts, compared to other genes. It has been suggested that one potential role of HP1a and POF might be increasing the export of transcripts from the nucleus by taking advantage of the positioning close to the nuclear envelope (JOHANSSON et al. 2012). In fact, highly heterochromatic regions of the genome are in close proximity to the nuclear envelope and transcriptionally active genes may interact with the nuclear pore complex (LANCTÔT et al. 2007; CAPELSON et al. 2010). Whether genes located in heterochromatic regions take advantage of their location close to the nuclear periphery when activated remains an important question to answer. HP1a in chromosome 4 gene regulation The fact that chromosome 4 gene regulation is not affected only by POF but also by HP1a was first showed by the general increase in expression from 4th genes caused by RNAi knockdown of HP1a (LIU et al. 2005; JOHANSSON et al. 2007a). There is in fact a negative correlation between fold change in gene expression from the 4th caused by Pof homozygous mutations and by HP1a RNAi (JOHANSSON et al. 2007a). An expression microarray study in first instar larvae has also shown that HP1a transheterozygous mutants display an increase in gene expression level from the 4th chromosome (LUNDBERG et al. 2013). These results argue for the existence of a balancing system that regulates gene expression on the 4th chromosome: POF stimulates while HP1a represses expression (JOHANSSON et al. 2007a). POF and HP1a colocalize on the 4th chromosome and their targeting on polytenes has been shown to be interdependent, since in Pof homozygous mutants there is less HP1a targeted to the 4th and in HP1a transheterozygous mutants no POF is seen on the 4th chromosome (JOHANSSON et al. 2007a). A recent study has challenged this view on POF and HP1a interdependence, showing that in HP1a transheterozygous mutants, POF ChIP-chip enrichment values on the 4th are unaffected (RIDDLE et al. 2012). The discrepancy between polytene stainings and ChIP-chip experiments when it comes to POF binding in HP1a mutants remains to be understood. In a sensitised translocation system, POF 19 HP1a TARGETING spreading on the 4th chromosome depends on heterochromatin. In this system, increasing the amount of heterochromatin, by decreased temperature or removal of the highly heterochromatic Y chromosome, also led to an increase in POF targeting (JOHANSSON et al. 2007a). Additionally, experiments using chromatin immunoprecipitation followed by tilling arrays (ChIP-chip), in both S2 cell lines and salivary gland tissue, have shown at a high resolution that POF and HP1a bind to the same regions on the fourth chromosome, with a bias towards exons of actively transcribed genes, but interestingly, HP1a binds additionally to promoters (JOHANSSON et al. 2007b). The enrichment of HP1a in promoters of active genes is intriguing and is the main focus of paper II – see below. HP1a and heterochromatin formation HP1a was first identified in Drosophila melanogaster as a non-histone chromosomal protein associated with heterochromatin that when mutated acts as a suppressor of variegation - Su(var) (SINCLAIR et al. 1983; JAMES AND ELGIN 1986; EISSENBERG et al. 1990). Position-effect variegation is a phenomenon by which an active gene becomes silent when inserted inside or in the vicinity of a heterochromatic region, probably due to the spreading of heterochromatin (MULLER AND ALTENBURG 1930; EISSENBERG AND REUTER 2009). This happens in some cells but not in others, which leads to a mosaic (or variegated) expression of the gene in the adult. The hp1a gene, also called Su(var)2-5, when mutated suppresses the silencing of a transgene inserted in a heterochromatic environment. In other words, it suppresses positioneffect variegation (SINCLAIR et al. 1983). Several other mutations have been identified as suppressors of variegation, like mutations in Su(var)3-9 and Su(var)3-7 (SINCLAIR et al. 1983; EISSENBERG AND REUTER 2009). Su(var)3-7, Su(var)3-9 and HP1a are in different ways involved in heterochromatin assembly and function and they colocalize in the chromocenter, at some telomeres and euchromatic sites. They might be loaded on the chromatin in an hierarchical manner: Su(var)3-7 recruits Su(var)3-9 and Su(var)3-9 recruits HP1a (CLEARD et al. 1997; DELATTRE et al. 2000; SCHOTTA et al. 2002; DELATTRE et al. 2004; SPIERER et al. 2005). HP1a is an essential protein and it is well known for being involved in heterochromatin formation, being enriched at pericentromeric and telomeric regions and binding both di- and 20 HP1a TARGETING trimethylated H3K9 (JAMES AND ELGIN 1986; LU et al. 2000; LACHNER et al. 2001). HP1 is a conserved protein family found in many eukaryotic organisms besides Drosophila, for instance in yeast, plants and humans. There are several hp1 genes in each species coding for HP1 proteins with different targeting specificities and probably also different functions (VERMAAK AND MALIK 2009). For instance, in several Drosophila species, four conserved hp1 genes have been identified: hp1a, hp1b, hp1c, hp1d (LEVINE et al. 2012). In Drosophila melanogaster it has been shown that: HP1a is bound to pericentromeric and H3K9me-enriched regions; HP1b targets both euchromatin and heterochromatin; HP1c localizes primarily to euchromatin; HP1d targets heterochromatic compartments distinct from HP1a and H3K9me, and is expressed specifically in the female germline; and HP1e doesn´t seem to localize to chromatin and its expression is male germline specific (SMOTHERS AND HENIKOFF 2001; VERMAAK et al. 2005; FONT-BURGADA et al. 2008; VERMAAK AND MALIK 2009). Most HP1 studies have been focused on the mammalian HP1α and on the Drosophila melanogaster HP1a proteins. These studies have shown that HP1 proteins are constituted by a conserved chromo-domain separated from a conserved chromo-shadow domain by a less conserved hinge region (LOMBERK et al. 2006). HP1a chromo-domain is involved in the interaction with H3K9me2,3 (BANNISTER et al. 2001; LACHNER et al. 2001; NAKAYAMA et al. 2001; JACOBS AND KHORASANIZADEH 2002), whereas the chromo-shadow domain is required for protein-protein interaction, specifically for the HP1a homodimerization and its interaction with several distinct proteins (SMOTHERS AND HENIKOFF 2000). In fact, HP1a (and/or HP1α) has been shown to interact with proteins involved in varied cellular processes not related to heterochromatin formation, like for instance: transcriptional regulation, replication, DNA repair, nuclear architecture and chromosomal maintenance (LOMBERK et al. 2006). HP1a recruitment might depend on RNA, since the hinge region of HP1a interacts with RNA and is involved in heterochromatin targeting (SMOTHERS AND HENIKOFF 2001; MUCHARDT et al. 2002). Additionally, it has been shown that the hinge region can be post-translationally modified by phosphorylation, which affects HP1a targeting (BADUGU et al. 2005). 21 HP1a TARGETING The current model for heterochromatin formation postulates that two chromo-domains of a HP1a homodimer bind to H3K9me2,3 in the tails of two adjacent nucleosomes, crosslinking the nucleosomes and restricting the access to transcriptional machinery due to the formation of a more compact chromatin structure (VERMAAK AND MALIK 2009). Additionally, HP1a interacts directly with the histone methyltransferase Su(var)3-9 (see below), through its chromo-shadow domain, which leads to the deposition of more H3K9me and allows the spreading of H3K9me2,3 as well as HP1a, in a positive feedback loop that facilitates the spreading of heterochromatin in cis from a nucleation site (SCHOTTA et al. 2002; VERMAAK AND MALIK 2009). HP1a targeting to chromatin is still not fully understood, and it might be a combination of interaction with H3K9me2,3 marks, interaction with chromatin-associated proteins and a DNA sequence specific recognition (DE WIT et al. 2005). It has been shown that HP1a binding and silencing occurs preferentially at regions enriched in DNA repeats, for example, HP1a targets P-element tandem repeats (FANTI et al. 1998; DE WIT et al. 2005; DE WIT et al. 2007). Intriguingly, besides targeting the highly repetitive DNA from pericentromeric regions and the 4th chromosome, HP1a also binds to a euchromatic region of the 2L chromosome (2L:31) in polytene chromosomes from third instar larvae and it was claimed to silence those genes (HWANG et al. 2001). Additionally to being mainly associated with gene silencing, HP1a has been shown to bind active genes in both euchromatin and heterochromatin and in these cases HP1a is suggested to be required for their proper expression (WAKIMOTO AND HEARN 1990; LU et al. 2000; PIACENTINI et al. 2003; DE WIT et al. 2007; RIDDLE et al. 2011). An expression microarray study in Drosophila first instar larvae has shown that besides HP1a repressing effect on chromosome 4 active genes, HP1a has a stimulating effect on pericentromeric active genes (LUNDBERG et al. 2013). The dependence of HP1a targeting on H3K9me has been challenged in some studies. A study showed that HP1a lacking its chromo-domain is still able to target heterochromatin (SMOTHERS AND HENIKOFF 2001). Additionally, it has been suggested that HP1a can be targeted to some chromatin sites by interacting with Piwi proteins (which bind to their DNA targets by interacting with Piwi- interacting RNAs – piRNAs) in a manner independent on H3K9me 22 HP1a TARGETING (HUANG et al. 2013). HP1a has been proposed to bind DNA directly, as it seems to be the case for telomeric HP1a which doesn´t require H3K9me or the action of Su(var)3-9 for its binding (ZHAO et al. 2000; PERRINI et al. 2004). Interestingly, it has been shown that HP1 can bind nucleosomes independently on the H3 tail and that HP1 in vitro can bind with high affinity to the H3 histone-fold (ZHAO et al. 2000; NIELSEN et al. 2001; DIALYNAS et al. 2006; LAVIGNE et al. 2009). A recent study in C. elegans has shown that the genome-wide targeting of protein HP1 Like 2 (HPL-2), is not affected in HKMT mutant embryos that lack H3K9me (GARRIGUES et al. 2015). Su(var)3-9, Setdb1 and G9a In Drosophila melanogaster there are three conserved histone lysine methyltransferases (HKMTs) responsible for methylating H3K9: Su(var)3-9, Setdb1 and G9a. Su(var)3-9, discovered in Drosophila, localizes mainly to the pericentromeric regions but also to some extent to the 4th chromosome, in polytene chromosome stainings (SCHOTTA et al. 2002; LUNDBERG et al. 2013). Su(var)3-9 di- and tri-methylates H3K9 in pericentromeric regions, through its SET domain (REA et al. 2000; SCHOTTA et al. 2002). Additionally, this HKMT has a chromo-domain possibly involved in interaction with H3K9me, and interacts with HP1a through its N-terminal domain (REA et al. 2000; SCHOTTA et al. 2002). Some studies have suggested that Su(var)3-9 can interact with histone deacetylase enzymes and that deacetylation of H3K9 precedes its methylation, since Su(var)3-9 only methylates H3K9 when this residue is not acetylated (CZERMIN et al. 2001; LACHNER et al. 2001). The physical interaction between Su(var)3-9 and linker histone H1 also seems to play a role in Su(var)3-9 recruitment to chromatin and H3K9 methylation (LU et al. 2013). Su(var)3-9 and HP1a binding to chromatin was shown to be interdependent. In HP1a homozygous mutants, Su(var)3-9 relocalizes to ectopic places on chromosome arms besides its normal sites, where it causes H3K9me (SCHOTTA et al. 2002). In addition, on polytene chromosomes from Su(var)3-9 homozygous mutants, HP1a and H3K9me are lost from the chromocenter but remain on the 4th chromosome (SCHOTTA et al. 2002; EBERT et al. 2004; FIGUEIREDO et al. 2012). It has also been shown that, in Kc cells, the binding of HP1a to most genes on the 4th chromosome is independent on Su(var)3-9 (GREIL et al. 2003). 23 HP1a TARGETING In fact, from immunostaining experiments on polytene chromosomes, it was shown that Setdb1 is the enzyme responsible for H3K9 methylation and HP1a recruitment on the 4th chromosome, not acting in the chromocenter (SEUM et al. 2007b; TZENG et al. 2007). In polytene chromosomes from Setdb1 homozygous mutants, POF, HP1a and H3K9me2,3, enrichments are decreased on the 4th chromosome (TZENG et al. 2007). ChIP-chip analysis of modENCODE data also showed that in Setdb1 mutants, POF recruitment is impaired (RIDDLE et al. 2012). These results suggest that Setdb1 is required for POF binding to the 4th, and in fact POF and Setdb1 were shown to interact in vivo (TZENG et al. 2007). Whereas Su(var)3-9 and G9a are not required for normal fly development, Setdb1 is required for normal oogenesis and was claimed to be essential for survival (STABELL et al. 2006a; CLOUGH et al. 2007; SEUM et al. 2007b; YOON et al. 2008). G9a has H3K9 methylation activity in Drosophila melanogaster and like Setdb1, was discovered as a homolog to the human protein. Contrary to Su(var)3-9 and to Setdb1, which localize primarily to the chromocenter and to the 4th, respectively, G9a localizes to euchromatic bands in polytene chromosome arms and it´s role in global H3K9 methylation is still elusive (MIS et al. 2006; STABELL et al. 2006b; SEUM et al. 2007a). The three HKMTs appear to exert their methylation activities in distinct regions of the genome, and accordingly: Su(var)3-9 is a strong suppressor of variegation of transgenes inserted in pericentric heterochromatin, while Setdb1 is a strong suppressor of variegation of transgenes inserted on the 4th and G9a appears to be a weak suppressor of variegation of genes inserted in pericentric heterochromatin (BROWER-TOLAND et al. 2009). Interestingly, although Su(var)3-9 doesn´t seem to affect H3K9me or HP1a recruitment to the 4th, a study showed that both Setdb1 and Su(var)3-9 mutants display decreased gene expression from the 4th (LUNDBERG et al. 2013). In this study, it was suggested that Setdb1 and Su(var)3-9 have some degree of redundancy, since Setdb1 and Su(var)3-9 affect the expression of the same sets of genes. 24 HP1a TARGETING Summary of Paper II The roles of each HKMT in methylating H3K9 and in recruiting HP1a genomewide in Drosophila melanogaster are still elusive and previous studies were based on results from immunostaining experiments on polytene chromosomes. In this project we were interested in the division of labour between HKMTs at a high resolution, and we therefore performed ChIP-chip experiments in salivary gland tissue from third instar larvae, to compare with polytene chromosome stainings. We mapped H3K9me2, H3K9me3 and HP1a genome-wide in wild type and in mutants for Setdb1 (Setdb110.1), for Su(var)3-9 (Su(var)3-9evo/06) and for G9a (G9aRG5). Additionally we also mapped HP1a in Pof mutants (PofD119). Our mapping of HP1a and H3K9me2,3 in wild type showed that these factors are mainly enriched on chromosome 4 and in pericentromeric regions of all chromosomes. This is in agreement with previous results from others who performed ChIP-chip and DamID (DNA adenine methyltransferase fused to a chromatin protein of interest) in Drosophila cell lines, embryos and fly heads, which indicates the stability of HP1a and H3K9me2,3 throughout development (DE WIT et al. 2005; DE WIT et al. 2007; JOHANSSON et al. 2007b; FILION et al. 2010; KHARCHENKO et al. 2011; RIDDLE et al. 2011; YIN et al. 2011). In one of these studies, the authors performed DamID experiments for 53 chromatin associated proteins, in embryonic cell lines, and they found that the chromatin in Drosophila can be divided in five types (black, blue, green, red and yellow), depending on their unique combination of proteins (FILION et al. 2010). The chromatin type defined as green is enriched in HP1a, Su(var)3-9 and H3K9me2, and corresponds to pericentromeric heterochromatin and to the 4th chromosome. Our ChIP-chip and immunostaining performed in salivary gland polytene chromosomes in HKMT mutants confirm previous results from other groups, and show that: Su(var)3-9 is responsible for H3K9me2,3 and HP1a recruitment in pericentric regions of all chromosomes; Setdb1 produces H3K9me2,3 and recruits HP1a to the 4th chromosome and to the 2L:31 region; and G9a doesn´t affect H3K9me or HP1a recruitment genome-wide (SEUM et al. 2007b; BROWER-TOLAND et al. 2009). Interestingly, since region 2L:31 in wild type polytene chromosomes has been shown to ocassionaly bind POF, it seems like Setdb1 methylates H3K9 at POF-bound regions 25 HP1a TARGETING (LUNDBERG et al. 2013). It is intriguing that Su(var)3-9 doesn´t methylate H3K9 on chromosome 4 nor recruits HP1a, since it was shown that Su(var)3-9 is enriched on the 4th chromosome (SCHOTTA et al. 2002; EBERT et al. 2004; LUNDBERG et al. 2013). Interestingly, our results show that HP1a recruitment to promoters of active genes, on the 4th chromosome, 2L:31 region and pericentromeric regions, doesn´t depend on methylated H3K9. In Setdb1 mutants, H3K9me2,3 is lost from promoters of active genes at all the regions mentioned above, but HP1a remains enriched (figure 4). We suggest that the initial H3K9me independent binding of HP1a at promoters of active genes constitutes a nucleation event, and that HP1a will then spread to the gene bodies by recognition of H3K9me marks. Figure 4. HP1a at promoters of active genes on the 4th chromosome binds independently on H3K9me, on Setdb1 and on POF. Left: ChIP-chip profiles, depicted for a region on the 4th chromosome, of HP1a, H3K9me2 and H3K9me3 binding on salivary glands from third instar larvae of wild type and mutants for Pof and Setdb1. Y-axes show ChIP enrichment in log2 ratios and x-axes show chromosomal position in Mb. Right: metagene profile representing the average HP1a targeting on 4th chromosome active genes, based on ChIP-chip profiles of HP1a in wild type, and mutants for Su(var)3-9, Pof and Setdb1. Y-axes show ChIP enrichment in log2 ratios and the x-ratios represent the different gene features: IG – intergenic; P – promoter; E1-5 – exon bins; IN – intron. 26 HP1a TARGETING The most prominent H3K9me-independent HP1a promoter peaks are seen on the 4th chromosome active genes in Setdb1 mutants. In these promoters, HP1a targeting is decreased, which might be due to the fact that the lack of Setdb1 causes less H3K9me on gene bodies and consequently less HP1a being recruited. Since HP1a is essential for survival, contrary to Su(var)-3-9, G9a, and Setdb1 (Setdb1 mutants could be recovered although less viable than wild type), it might be that HP1a at promoters is essential for viability. Otherwise, the essential role of HP1a might relate to its activity in several distinct cell functions. It is intriguing that Setdb1 produces H3K9me2,3 at promoters of pericentromeric regions, since it has been reported that this enzyme does not act in these regions (SEUM et al. 2007b; TZENG et al. 2007; LUNDBERG et al. 2013). Our results on Pof mutants are intriguing since they showed that in the absence of POF, HP1a is lost from bodies of active genes on the 4th, as it was previously shown (JOHANSSON et al. 2007b), but it remains at promoters (figure 4). The metagene profile from chromosome 4 active genes for HP1a targeting is very similar between Setdb1 and Pof mutants. It was shown before that Setdb1 colocalizes with POF and HP1a on the 4th chromosome, and that in Sedtb1 mutants, POF and HP1a enrichments are decreased on the 4th chromosome (TZENG et al. 2007; LUNDBERG et al. 2013). Our results suggest that the HP1a spreading from its nucleation site at promoters depends on the presence of both Setdb1 and POF at gene bodies. The dependence on POF and Setdb1 for HP1a targeting seems to be specific to active genes. In fact, we found a region on the 4th chromosome with two unexpressed genes in which the high enrichment in HP1a doesn´t depend on Setdb1 nor on Pof. Since HP1a can bind RNA, we propose that POF interaction with nascent transcripts from the 4th stabilizes the interaction between HP1a and H3K9me. We further studied the H3K9me independent HP1a-bound promoters for the presence of a sequence motif or for the specific enrichment in chromatinassociated protein factors and/or histone marks. Using CompleteMOTIFS platform for motif search, we found that the H3K9me independent HP1a promoters, in comparison to promoter regions of 2L (excluding pericentromeric and 2L:31 regions), are enriched in A/T. This is in agreement with previous results showing that HP1 is enriched at A/T rich regions (GREIL et al. 2003). 27 HP1a TARGETING After analysing the mapping of 51 chromatin factors in S2 cells, from modENCODE, we found that the H3K9me independent binding of HP1a at promoters correlates very well with the binding of HP2 (heterochromatin protein 2). HP2 protein has been shown to colocalize with HP1a on polytenes, to be a suppressor of variegation and to interact with HP1a, and was proposed to drive HP1a dimerization (SHAFFER et al. 2002; MENDEZ et al. 2011). Since mutations that affect HP1a dimerization abolish the interactions between HP1 and non-modified H3, we suggest that HP2 interaction with HP1 is required for HP1 dimer to bind to the core of H3 (LAVIGNE et al. 2009). Our results support the model that HP1a high affinity binding to H3 histone fold happens at promoters of active genes, and is a nucleation site for HP1a chromatin targeting (DIALYNAS et al. 2006). This model suggests that HP1 binds with high affinity to the H3 histone fold when H3 becomes exposed, in regions of high transcription or replication, and subsequently HP1a can spread by binding with low affinity to H3K9me sites. It has been shown previously that HP1a binds with higher affinity to the fold region of H3 than to the methylated lysine 9 on the N-tail (NIELSEN et al. 2001). By analysing DNase hypersensitivity data from embryos (THOMAS et al. 2011), we found that on average, promoters at the 4th chromosome are more DNase sensitive than the 2L promoters, even though the 4th chromosome promoters are enriched in HP1a. In fact, it was previously suggested that HP1a opens the chromatin structure at some promoters of active genes on the 4 th (CRYDERMAN et al. 2011). The role of HP1a at promoters of active genes is still elusive. A study showed that for 4th chromosome genes from Setdb1 mutants, which have HP1a at promoters but not at gene bodies, there is an increased transcriptional output from these genes, compared to wild type (LUNDBERG et al. 2013). It was suggested that the role of HP1a at promoters is activating transcription while at gene-bodies it represses transcription. In support of this, a study has proposed that HP1a at promoters of active genes on the 4th promoters can open the chromatin structure by recruiting chromatin-remodelling complexes (CRYDERMAN et al. 2011). 28 MSL TARGETING CHAPTER 4 4. MSL TARGETING Sex chromosomes and sex determination Among species with sexual differentiation, i.e. distinct males and females, there is a large variety of sex determination mechanisms. In some animals sex is determined by the environment, for instance by the temperature of incubation of eggs. In other animals there are genetic mechanisms of sex determination, for example the existence of heteromorphic sexchromosomes. Both in mammals and in Drosophila, males are the heterogametic sex and have one X and one Y chromosome, in contrast to females which have two X chromosomes and are thus the homogametic sex. The X and the Y chromosomes are morphologically and genetically different and are believed to have evolved from a homologous pair of autosomes. In mammals, sex determination is conferred by the gene SRY (sex determining region Y) on the Y chromosome, which is the master trigger of male embryonic differentiation (LAHN et al. 2001). In Drosophila melanogaster, the primary determinant for sex is the ratio X-chromosomes: Autosomes (X:A), or more specifically, the number of X-chromosomes relative to the number of precellular nuclear divisions (CLINE AND MEYER 1996; MARIN AND BAKER 1998; ERICKSON AND QUINTERO 2007). Females will develop from XX:AA embryos with an X:A ratio of 1.0, and males will develop from XY:AA embryos with an X:A ratio of 0.5. When comparing flies, with two pairs of autosomes, and humans: XXY flies are viable and fertile females whereas XXY humans are sterile males with Klinefelter syndrome; XO flies are sterile males, whereas humans XO are sterile females with Turner syndrome. In female flies, the X-linked gene sex-lethal (sxl) is expressed in its active form, and is responsible for somatic sex determination, dosage compensation and oogenesis. The components of the X:A ratio signal are proposed to be numerators, denominators and maternal genes, and will act by activating or repressing the sxl RNA (SCHUTT AND NOTHIGER 2000). According to the model, numerator genes are located on the X chromosome and are responsible for activation of sxl. If numerator genes are duplicated in XY:AA 29 MSL TARGETING embryos, it leads to activation of sxl causing male lethality; when numerator are deleted in XX embryos, it prevents activation of sxl leading to female lethality. SXL is a splicing factor and an RNA binding protein, which is activated early in XX embryos and initiates its own positive feedback regulation. SXL also regulates downstream genes essential for female differentiation: it activates transformer (tra), represses translation of malespecific lethal 2 (msl2) transcript and activates genes involved in oogenesis (SCHUTT AND NOTHIGER 2000). The TRA protein is a splicing factor involved in the regulation of double sex (dsx) transcript, which is a transcription factor that in the spliced form, in females, represses male-specific genes and activates female-specific genes, and in the unspliced form, in males, does the opposite. MSL2 is part of the dosage compensation system, which will only function later in development, and it is active only in males – see below. Sex chromosome evolution The prevalent theory for the evolution of sex chromosomes postulates that the acquisition of a male-determining gene on a chromosome from an homologous pair (for instance, the sry in humans) gives rise to the proto-Y chromosome, which, since it will only be transmitted through males, will likely accumulate male advantageous mutations. The proto-Y will diverge from the proto-X and to avoid intersex there will be a selection for suppression of recombination, for which inversions contribute. The loss of recombination will in turn lead to the accumulation of more deleterious mutations, mobile elements, and loss of genes, which promotes progressive proto-Y degeneration. Despite the fact that the formation of sex chromosomes occurred independently several times in evolution, the Y chromosomes display similarities among species, often (but not always) being small, gene-poor and rich in repetitive sequences (LAHN et al. 2001). The view that X and Y in Drosophila evolved from a pair of autosomes has been challenged since these two chromosomes share very little homology, contrary to mammals that have many homologous regions through which the X and the Y recombine (CARVALHO 2002). The Y chromosome in Drosophila is constituted by repetitive sequences and fewer than 20 protein coding genes mainly involved 30 MSL TARGETING in spermatogenesis, and apparently there is no homology between them and genes on the X chromosome (CARVALHO 2002; CARVALHO et al. 2009). The only homologous region between the sequenced portions of Drosophila X and Y chromosomes is the rDNA locus, which helps in their pairing in meiosis (CARVALHO 2002; TSAI AND MCKEE 2011). Interestingly, the Y chromosome genes in Drosophila have paralogs on autosomes, which led to the hypothesis that the Y chromosome might have evolved through the accumulation of male-related genes from autosomes on a B-chromosome (B chromosomes are additional dispensable chromosomes that are present in a fraction of individuals) (CARVALHO 2002; CARVALHO et al. 2009). Dosage compensation Y chromosome degeneration leads to a gene dose problem since genes on the male X chromosome are in only one copy, when compared to autosomal genes and to X chromosomal genes in females, which are in two copies. Different animals have evolved distinct mechanisms of dosage compensation which balance the relative expression levels of X-linked genes between sexes and in accordance to the autosomes. It has been proposed that an increased expression of the monosomic X-linked genes in males was the first event in dosage compensation (OHNO 1967; LARSSON AND MELLER 2006). In fact, studies in mammals have shown that expression from X genes is on average twice that from autosomal genes, although this is still in debate (GUPTA et al. 2006; NGUYEN AND DISTECHE 2006; YILDIRIM et al. 2012; FAUCILLION AND LARSSON 2015). A recent study has also showed that both in male and female human cell lines, the average half-lives for transcripts from the X chromosome is longer than for transcripts from autosomes and the ribosome density is higher on X-linked transcripts (FAUCILLION AND LARSSON 2015). The increase in X-linked gene expression in females would create the need to compensate for the presence of two X chromosomes, which in mammals is achieved by silencing one of the X chromosomes in each female cell (LARSSON AND MELLER 2006). In Drosophila, the dose problem is solved by a ribonucleoprotein complex – the MSL (male-specific lethal) complex, which is only formed in males, due to the male specific production of one protein of the complex: MSL2 (BASHAW AND BAKER 1995). The MSL complex specifically recognizes the male X 31 MSL TARGETING chromosome and contributes to the 2-fold upregulation of almost all its genes (HAMADA et al. 2005; DENG AND MELLER 2006; ZHANG et al. 2010). MSL complex The MSL complex is formed by the association of five proteins and two long non-coding RNAs. The five genes encoding for the proteins of the complex were discovered in screens for mutations with male-specific lethal phenotypes (BELOTE 1983), and their names are suggestive for that: male specific lethal 1 (msl1), male specific lethal 2 (msl2), male specific lethal 3 (msl3), maleless (mle) and male absent on first (mof). The two redundant long non-coding RNAs are RNA on the X 1 (roX1), and RNA on the X 2 (roX2), and their genes are located on the X chromosome, more than 7 Mb apart. Contrary to the protein components of the complex, roX1 or roX2 alone are not essential for survival of males, but if both roX RNAs are removed than the male flies die (MELLER AND RATTNER 2002). Immunostainings on polytene chromosomes for each of the proteins of the complex and RNA in situ hybridizations for each roX show that they specifically target the X chromosome in males with similar distributions (seen for MSL3 in Figure 5). Figure 5. MSL complex targets the male X chromosome. MSL3 immunostaining on polytene chromosomes from salivary glands from male third instar larvae. All the other components of the complex colocalize with MSL3. The protein components of the MSL complex are conserved and have orthologues in other species including humans, while the roX RNAs have low degree of conservation, even in Drosophila related species (PARK et al. 2003; 32 MSL TARGETING SMITH et al. 2005). The conservation of MSL-complex proteins suggests that they might have had a function outside dosage compensation before they were recruited to the monosomic male X (STENBERG AND LARSSON 2011). MSL1 and MSL2 are the core components of the complex, being required for its assembly. In contrast, in msl3, mof or mle mutants, and in roX1 roX2 double mutants, partial MSL complexes form at some locations on the male X chromosome, although the majority of MSL targeting to the X chromosome is lost (PALMER et al. 1994; LYMAN et al. 1997; KAGEYAMA et al. 2001; MELLER AND RATTNER 2002). It has been shown that in wild type females, MLE, MSL3 and MOF are produced, contrary to MSL1 and MSL2 (PALMER et al. 1994). If in females MSL2 is expressed ectopically or is induced by mutation of Sxl, the MSL complex is formed and targets both X chromosomes (KELLEY et al. 1995; LYMAN et al. 1997). MSL2 is the limiting component for formation of the MSL complex, since its translation is repressed in males by SXL and, as it has been proposed, the msl2 transcript associates with free MSL complexes in the nucleus and regulates the production of complexes by a feedback mechanism (JOHANSSON et al. 2011). It has also been shown that MSL2 positively regulates MSL1 translation or protein stability (PALMER et al. 1994). Besides the major role of MSL2 in the complex formation, MSL2 requires MSL1, since in msl1 homozygous mutant males no MSL2 staining is seen on the X chromosome and MSL2 expression is decreased (LYMAN et al. 1997). MSL2 and MSL1 have also been shown to interact and form a heteromer which binds DNA (COPPS et al. 1998; FAUTH et al. 2010). MSL3 protein contains a chromo-domain and has been shown to bind nucleosomes marked with H3K36me3 in vitro, a mark of active chromatin, and this targeting is suggested to be important for MSL recruitment to the male X chromosome (LARSCHAN et al. 2007). MOF is a histone lysine acetyltransferase responsible for acetylation of H4K16 at a high level on the male X chromosome (AKHTAR AND BECKER 2000; MORALES et al. 2004). MOF has functions outside the MSL complex, and ChIPchip and ChIP-seq experiments in Drosophila cell lines have shown that MOF is enriched in promoters of autosomal genes (KIND et al. 2008; CONRAD et al. 2012). MOF at promoters of autosomal genes is part of the Non-Specific Lethal (NSL) complex, formed by six other proteins, and it seems to promote gene expression (PRESTEL et al. 2010; RAJA et al. 2010). Interestingly, there is 33 MSL TARGETING a high degree of conservation between the Drosophila MOF and the mammalian MOF, which also produces H4K16ac and is involved in embryogenesis and oncogenesis (TAIPALE et al. 2005; GUPTA et al. 2008). Additionally, in humans, MOF has been suggested to be involved in upregulation of the X chromosome (DENG et al. 2013). MLE is a DNA/RNA helicase and has been shown to be essential for incorporating roX1 and roX2 in the MSL complex, by binding to stem-loop structures on the roXs in an ATP-dependent manner (PARK et al. 2007; PARK et al. 2008; ILIK et al. 2013; MAENNER et al. 2013). roX RNAs The role of roX redundant long non-coding RNAs in MSL complex targeting to the male X chromosome is still very intriguing. Although roX1 and roX2 have little sequence homology, they share conserved domains that are important for their interaction with MSL proteins (PARK et al. 2007; PARK et al. 2008; ILIK et al. 2013). The redundancy of roX1 and roX2 is interesting and it has been suggested that only one roX (roX1 or roX2) is present in each MSL complex (ILIK et al. 2013). It has been proposed that roX RNAs are incorporated in the MSL complex co-transcriptionally since they are unstable when not bound to the MSL complex (MELLER et al. 2000). MSL2 regulates roX transcription (RATTNER AND MELLER 2004) and it has been shown that roX1 transcription is regulated by an autoregulatory loop (LIM AND KELLEY 2012). Interestingly, roX RNAs ectopically expressed from autosomes in roX1 roX2 double mutant males, can recruit MSL, which spreads together with roX RNA in cis around the transgene, and can also recognize the X chromosome in trans and recruit MSL to the X chromosome (KELLEY et al. 1999; MELLER et al. 2000; PARK et al. 2002; ALEKSEYENKO et al. 2008; STRAUB et al. 2013). It has been suggested that the role of roX RNAs is to mediate the spreading of the MSL complex on the whole X chromosome (KELLEY et al. 1999). MSL spreading ectopically to the genes surrounding the transgene seems to increase the expression of those genes (KELLEY AND KURODA 2003; VENKEN et al. 2010). The importance of roX RNAs for correct MSL targeting to the X chromosome was further shown by the fact that in roX1 roX2 double mutants, MSL2 and H4K16ac are decreased on the X chromosome and target new genomic 34 MSL TARGETING regions like the chromocenter, the 4th chromosome and a few other autosomal sites (MELLER AND RATTNER 2002; DENG AND MELLER 2006; JOHANSSON et al. 2011). This is an important topic of study in paper III. The role of roX RNAs in MSL targeting shows parallels with the mammalian dosage compensation system. In mammal females, the Xist long non-coding RNA is expressed from the Xic locus on one of the X chromosomes, coats the entire chromosome in cis, and recruits several proteins and chromatin modifiers that, for instance by producing DNA methylation, lead to a complete compaction of the chromatin structure and silencing of the majority of genes on that chromosome (HEARD AND DISTECHE 2006). MSL-complex and heterochromatin The male X chromosome seems to require heterochromatin associated proteins to keep its normal shape. Using DamID technique it has been shown that HP1a is enriched on the male X chromosome compared to an autosome and compared to the female X chromosome (DE WIT et al. 2005). Interestingly, in HP1a and in Su(var)3-7 mutants, the X chromosome loses compaction, shows a bloated phenotype, and this phenotype depends on a functional MSL complex (SPIERER et al. 2005). Another link between the MSL complex and heterochromatin is provided by JIL-1, an essential protein kinase involved in Histone 3 Serine 10 (H3S10) phosphorylation, which was shown to colocalize and physically interact with the MSL complex (JIN et al. 2000; WANG et al. 2013; LINDEHELL et al. 2015). JIL-1 targets all chromosomes in females but is highly enriched on the male X chromosome dependent on a functional MSL complex (JIN et al. 1999; JIN et al. 2000). JIL-1 is actually increased on the female X chromosome when MSL2 is expressed ectopically (JIN et al. 2000). JIL-1 on the male X chromosome was proposed to be important for a normal transcription, since genome-wide mapping of JIL-1 showed that its binding correlates with marks of active chromatin (H3K36me3 and H4K16ac) (REGNARD et al. 2011), and RNAi mediated knockdown of JIL-1 caused decreased transcription from the male X chromosome (REGNARD et al. 2011). It has also been shown that in JIL-1 null mutants, HP1a and H3K9me2 enrichments are increased on the male and female X chromosome(s) (ZHANG et al. 2006). Additionally, Su(var)3-7 loses its chromocenter specific binding and is ectopically targeted to chromosome 35 MSL TARGETING arms in JIL-1 null mutants (DENG et al. 2010). Decreasing the dose of Su(var)37 or Su(var)3-9 rescues the lethality of JIL-1 null mutants (DENG et al. 2007; DENG et al. 2010). Some studies suggested that Su(var)3-7 acts downstream of Su(var)3-9 and H3K9me in heterochromatic formation and gene silencing, and that JIL-1 counteracts this, repressing the spreading of heterochromatin, possibly helping to keep an open chromatin structure on the male X chromosome (DENG et al. 2010; REGNARD et al. 2011). Upregulation of the male X It is still unclear how dosage compensation upregulates gene expression from the male X chromosome approximately two times. H4K16ac has been proposed to open the chromatin but it´s still elusive how this affects transcription (AKHTAR AND BECKER 2000). It has been suggested that the MSL complex acts by facilitating transcription elongation by reducing the pausing of RNA polymerase II (RNA Pol II) (LUCCHESI 1998). Supporting this view, it was shown by ChIP-chip experiments that MSL complex targeting is linked to active transcription since MSL complex and H4K16ac are enriched in active gene bodies with a 3´ end bias (ALEKSEYENKO et al. 2006; GILFILLAN et al. 2006; ALEKSEYENKO et al. 2008; KIND et al. 2008; PRESTEL et al. 2010). In contrast to the transcription elongation theory, it has been claimed that MSL acts by increased transcription initiation, through increased recruitment of RNA Pol II to promoters of X linked genes, in comparison to autosomes and female X chromosomal genes (CONRAD et al. 2012), but this is still controversial (FERRARI et al. 2013; STRAUB AND BECKER 2013; VAQUERIZAS et al. 2013). Several lines of evidence suggest that MSL is not alone responsible for the full dosage compensation of X chromosomal genes. Only 75% of the actively transcribed genes on the male X are bound by MSL (CONRAD AND AKHTAR 2012). Gene expression studies suggest that MSL only accounts for ~1.4-fold increase in transcriptional output from the X chromosome, and it has been suggested that the rest of the compensation, up to the final 2-fold is, at least partly, accomplished by general buffering effects that act on all monosomic regions, discussed in chapter 2 (STENBERG et al. 2009; ZHANG et al. 2010; LUNDBERG et al. 2012). In fact it was shown that 15% of the expressed genes on the male X chromosome are dosage compensated but unbound by MSL and have reduced levels of H4K16ac (PHILIP AND STENBERG 2013). It has been 36 MSL TARGETING hypothesized that, during the evolution of sex chromosomes, the MSL complex targeting was a secondary adaptation, while buffering mechanisms were the initial response to the monosomic state of the X chromosome in males (STENBERG AND LARSSON 2011). It´s important to keep in mind that the MSL complex and other compensatory mechanisms can cause dosage compensation not only by regulating transcription, but also by regulating for instance RNA stability, translation or protein stability (FAUCILLION AND LARSSON 2015). Mechanisms of MSL targeting It´s still elusive how the MSL complex specifically recognizes and targets the male X chromosome, but the prevailing model postulates that it is recruited in a sequence-dependent manner to approximately 250 High Affinity Sites (HAS), defined by the enrichment of a GA-rich DNA sequence motif, and then spreads to neighbouring genes that are being actively transcribed (LARSCHAN et al. 2007; ALEKSEYENKO et al. 2008; STRAUB et al. 2008; GELBART AND KURODA 2009; CONRAD AND AKHTAR 2012; STRAUB et al. 2013). HAS, initially called chromatin entry sites, were defined as the regions on the male X that still bind MSL1 or MSL2, when one of the other proteins of the complex is removed (in msl3, mle or mof mutants) (LYMAN et al. 1997; KELLEY et al. 1999). Another characteristic of HAS is that they can recruit MSL when inserted on an autosome in males (OH et al. 2004; ALEKSEYENKO et al. 2008). roX1 and roX2 genes are themselves two of the strongest HAS on the X chromosome (KELLEY et al. 1999; ALEKSEYENKO et al. 2008; STRAUB et al. 2013). Other features besides the DNA sequence will likely contribute to HAS recognition by the MSL complex, since the GA-rich motif enriched at HAS is found elsewhere in the genome, and by itself it cannot predict HAS (ALEKSEYENKO et al. 2008). In fact one feature typical for HAS is that they have reduced nucleosome density (ALEKSEYENKO et al. 2008; STRAUB et al. 2008; STRAUB et al. 2013). The spreading beyond HAS requires an MSL complex constituted by all five proteins and at least one roX RNA (LYMAN et al. 1997; MELLER AND RATTNER 2002). Besides the preference of MSL to target actively transcribed genes, and regions enriched in H3K36me3, other features might affect the binding of the complex (ALEKSEYENKO et al. 2006; STRAUB et al. 2008). 37 MSL TARGETING Links between X and 4th chromosomes Several lines of evidence suggest an evolutionary relationship between the X and the 4th chromosomes. The X and the 4th are the only chromosomes that have a chromosome-wide gene regulatory mechanism that compensate for dose differences. In several species related to Drosophila melanogaster, POF targets the F element (equivalent to the 4th chromosome) and the MSL complex targets the X chromosome, like in Drosophila simulans, Drosophila pseudoobscura and Drosophila virilis. However, this is not the case for all Drosophila species. For instance, in Drosophila ananassae and Drosophila malerkotliana, both POF and the MSL complex target the male X chromosome, while the F element is targeted only by POF and in both males and females. Interestingly, in Drosophila busckii, the F element is located at the base of the X and the Y chromosomes, and POF targets the entire male X chromosome which is also enriched in H4K16ac (including the F element part) (LARSSON et al. 2001; LARSSON et al. 2004; STENBERG AND LARSSON 2011). These facts led to the suggestion that the 4th and the X chromosomes have the same origin and that POF originated as part of an ancient dosagecompensation mechanism (STENBERG AND LARSSON 2011). Summary of Paper III To study the role of roX RNAs in the recruitment of MSL complex to chromatin, we performed immunostaining experiments and ChIP-seq experiments on polytene chromosomes from third instar larvae to map MSL1, MSL2 and MOF, in wild type or double mutants for roX1 and roX2. Our ChIP-seq results correlate very well with the immunostainings and show that in roX1 roX2 double mutants, MSL is restricted to the HAS on the X chromosome, and that MSL in addition targets ectopic places: the chromocenter and specifically three regions on the 4th chromosome (Figure 6). 38 MSL TARGETING Figure 6. MSL complex targets three bands on the 4th chromosome in the absence of roX. ChIP-seq enrichment profiles showing that MOF and MSL1 are enriched on three regions of the 4th chromosome (top) that correspond to the three bands of MSL2 immunostaining seen in the polytene 4th chromosome (down). Our results clearly show that the role of the protein components of the MSL complex is distinct from the role of roX RNAs. While both in roX1 roX2 and in msl3 mutants the complex loses most of its binding from the male X, in roX1 roX2 mutants the complex is relocated to ectopic sites. In order to test if the pericentromeric targeting of the MSL complex was restricted to polytene chromosomes, we analysed the colocalization between the 1.686 pericentromeric repeat (enriched in pericentromeric regions of chromosomes 2 and 3) and MSL by combined DNA in situ hybridization and immunostaining in brain cells from third instar larvae. We found a high degree of co-localization between the pericentromeric probe and the MSL complex in roX mutants but not in wild type or mof mutants, confirming the results from polytenes. We speculate that in interphase chromosomes of roX1 roX2 mutants, the pericentromeric regions are in close proximity to the X chromosome, in a region where local MSL concentration is high. One study has previously shown that HAS are in closer proximity to each other in males than in females (GRIMAUD AND BECKER 2009). We didn´t find colocalization between MSL and the 1.686 repeat on mitotic chromosomes. Additionally, MSL targeting on mitotic chromosomes was restricted to the euchromatic part of the X chromosome, in both wild type and roX mutants. The high 39 MSL TARGETING compaction of the heterochromatic regions of the mitotic chromosomes might repress the MSL targeting to heterochromatin, although this still remains elusive. Importantly, we show that all five proteins of the complex colocalize at the ectopic places on the chromocenter and on the 4th chromosome in roX1 roX2 mutants and that H4K16ac also increases in these regions. These results show that the MSL complex doesn´t require roX RNAs for complex assembly and activity. When we looked at the mRNA levels from the 4th genes specifically targeted by MSL in roX mutants, by quantitative PCR in third instar larvae, we found that their mean expression levels are not significantly different from wild type. Our results suggest that the presence of H4K16ac is not enough to stimulate transcription, at least from the 4th chromosome. It´s important to keep in mind that on the 4th chromosome, gene expression is regulated by both POF and HP1a which might counteract the effect caused by H4K16ac. MSL targeting on the 4th didn´t affect HP1a or POF immunostaining pattern (data not shown). We found that JIL-1 is also targeted to the chromocenter and 4th chromosome in roX1 roX2 mutants. JIL-1 seems to be part of the MSL complex or to be recruited to chromatin by the MSL complex. Intriguingly, if MSL1 and MSL2 are overexpressed in males, MSL2 targets also the chromocenter and the 4th chromosome (DEMAKOVA et al. 2003). The chromocenter and the 4th chromosome are among the most heterochromatic regions of the genome. Our results suggest that the levels of roX RNAs relatively to the levels of MSL protein components dictate where the MSL complex will bind. MSL might have an intrinsic affinity to heterochromatin, and when there is excess of MSL protein components relatively to the roX RNAs, as in roX1 roX2 mutants or when MSL1 and MSL2 are overexpressed, the complex is recruited to heterochromatin. It has been proposed that heterochromatin functions as a sink to limit the quantities of silencing factors (EISSENBERG AND REUTER 2009). It was shown that increasing the amount of Y chromosomes (or the 4th or the heterochromatic portion of the X) suppresses PEV, possibly because the amount of heterochromatin compaction elsewhere in the genome decreases. Since it was shown that in roX double mutants the flies that escape the lethality are more likely to be roX1 roX2/0 than roX1 roX2/Y, it might be the case that 40 MSL TARGETING increasing compaction of heterochromatin in the genome (i.e. in X/0 males) makes the MSL complex leave the chromocenter and the 4th and target the X. To test the MSL dependence on heterochromatin, we performed MSL immunostainings on polytene chromosomes from null mutants for the heterochromatin proteins: Su(var)3-7, Su(var)3-9, HP1a and HP2 (data not shown). It was reported that in mutants lacking Su(var)3-7, MSL targeted the 4th and the chromocenter (SPIERER et al. 2008); however, we couldn´t reproduce these results. In the heterochromatin mutants tested we couldn´t see any distinct difference compared to wild type: MSL always targeted preferentially the male X chromosome. We also found HP1a and Su(var)3-7 targeting on polytenes not to be affected by the lack of roX RNAs (data not shown). We couldn´t determine if HP1a was required for MSL targeting to the 4th chromosome, since roX1 roX2; Setdb1 triple mutants died before reaching third instar larval stage. Since the 4th and the X chromosomes, as well as POF and MSL, share evolutionary links, it might be that the 4th specific genes targeted by MSL were on the X chromosome in Drosophila melanogaster related species, but we haven´t found any evidence for this possibility. By analysing our ChIP-seq data we found that all the enrichment peaks for MSL on the X chromosome in roX mutants corresponded to the previously mapped HAS (ALEKSEYENKO et al. 2008; STRAUB et al. 2008; STRAUB et al. 2013). Out of 263 reported HAS, we found 208 to be independent on roX RNAs. Additionally, by performing motif analysis using MEME software tool, we show that the GA rich motif found in the roX independent HAS, is identical to the one previously described for wild type (ALEKSEYENKO et al. 2008), and that this motif is conserved, since it was also enriched in the MSL1 peaks from Drosophila simulans (ChIP-seq for MSL1). Since partial MSL complexes target HAS in absence of MSL3, MLE or MOF, and as we found now, in absence of roX1 and roX2 RNAs, it seems that MSL1 and MSL2 are the only components of the complex required for HAS targeting. It´s still elusive which characteristics of HAS determine this targeting. We were very intrigued by the link between MSL and heterochromatin and this led us to study the properties of the heterochromatic regions targeted by MSL in roX mutants. By doing motif search (using MEME) on the heterochromatic MSL-bound regions of chromosomes 2, 3 and 4 in roX 41 MSL TARGETING mutants (which mainly include pericentromeric sequences), we found a motif containing repeats from the Hoppel (1360) transposable element. Hoppel are non-autonomous DNA transposons highly enriched in pericentromeric heterochromatin and on chromosome 4 (COELHO et al. 1998). When we mapped the motif containing Hoppel repeats along the chromosome arms we confirmed it to be enriched towards the centromeres. We performed in situ DNA hybridization with a probe containing the motif combined with MSL immunostaining and found them to colocalize on the three bands on the 4th in roX mutants. The affinity for Hoppel was further confirmed when we aligned all the ChIP-seq reads (including highly repetitive sequences) to sequences from repeats included in the Repbase Update database. We found that in roX mutants MSL is enriched at PROTOP_B, PROTOP_A and NTS (Non-transcribed Spacer). Interestingly, PROTOP is a family of autonomous DNA transposons that have been suggested to be ancestors of Hoppel and P-element. Since both Hoppel and the other transposable elements are usually in repeated copies in the genome, we wondered if MSL has affinity to repeats in general. In roX mutants MSL also binds to some autosomal sites, and we asked if these were also enriched in repeats. Indeed, we found that the percentage of repeat masked sequences (from UCSC) is higher around autosomal genes targeted by MSL in roX mutants, than around autosomal genes not targeted by MSL. To test if MSL has affinity to any sequence when repeated in tandem, we analysed flies carrying 7 or 2 copies in tandem of the P[lacW] transgene. Very interestingly, we found that in a roX mutant background, MSL is recruited to the 7 tandem repeat transgene but not to the 2 tandem repeat transgene. In contrast, in a wild type background, no MSL targeting was seen to these repeat transgenes. These results show that sequences in tandem are enough to recruit MSL when roX RNAs are not present. It´s still unclear if the sequence plays a role in the targeting, since P[lacW] contains a mini-white gene (X-linked). We propose a model in which the heterochromatic targeting of the MSL complex represents an ancient targeting of the complex, before specializing in dosage compensation. MSL at heterochromatin could for instance play an important role in allowing transcription of genes in repressive environment. In this model, roX are evolutionary younger than the MSL and they act in 42 MSL TARGETING restricting the MSL targeting to the X chromosome, which in a monosomic state required mechanisms to upregulate its expression. Summary of Paper IV Although the absence of roX1 and roX2 causes male lethality, there are some escapers (MELLER AND RATTNER 2002; DENG et al. 2005; MENON AND MELLER 2009). roX1 roX2 mutant male escapers might survive due to the incorporation of additional roX RNAs in the MSL complex which could restore dosage compensation. In wild type individuals, such RNAs have not been identified (OH et al. 2003; ILIK AND AKHTAR 2009; JOHANSSON et al. 2011). It is known that MLE requires RNA to target chromatin, since RNase treatment causes MLE dissociation from wild type polytene chromosomes (RICHTER et al. 1996). Interestingly, when roX RNAs are absent, MLE, together with the other proteins of the MSL complex, targets heterochromatic regions of the genome (FIGUEIREDO et al. 2014). Therefore, we hypothesized that in roX mutants MSL is recruited to chromatin by recognition of non-roX RNA molecules, possibly via MLE. In order to test if there are other RNA species that associate with the MSL complex and target it to heterochromatin in roX mutants, and if there are additional roX RNAs, we performed RNA immunoprecipitation followed by deep sequencing (RIP-seq) with an antibody against MSL2, in wild type and in roX1 roX2 mutant male larvae. As expected, we found MSL2 to be enriched in roX1 and roX2 RNAs in wild type. There is no evidence for the existence of additional roX RNAs, confirming previous results. We found several RNAs, transcribed from genes on the X and on other chromosomes, to be MSL2 bound in roX mutants and the majority corresponds to snoRNAs. We next calculated the MSL2 RIP/input average ratio for each gene of D. melanogaster genome and obtained a list with the top MSL2-bound RNAs enriched in wild type and the top MSL2-bound RNAs enriched in roX mutants. From this list we selected some RNAs for further analysis, depending on concordance between the RIP/input average values and the RIP/input enrichment profiles seen in integrated genome browser (IGB). We found Sgs3 transcript to be enriched in MSL2 in wild type, and the transcripts from Lsp1ß, 7SLRNA:CR32864, snoRNA:SC35-a and snoRNA:Psi28S-2562 to be enriched in MSL2 in roX mutants. We tested the targeting of these RNAs in 43 MSL TARGETING polytene chromosomes and in brain cells nuclei, by RNA in situ hybridization (FISH) with antisense RNA probes against each RNA, combined with MSL2 immunostaining. None of the RNAs tested targeted chromatin in polytene chromosomes or colocalized with MSL2 in brain cells nuclei. snoRNA:Psi28S2562 and snoRNA:SC35-a target the nucleolus in polytene chromosomes and in brain cells nuclei, 7SLRNA:CR864 targets the cytoplasm, and both Sgs3 and Lsp1ß showed a diffused pattern in the nucleus. In order to select only the RNAs that associate with chromatin, we analysed the MSL2-bound RNAs in roX mutants that were previously shown to be chromatin associated (caRNAs) in embryos (SCHUBERT et al. 2012). From the list of 28 caRNAs, we selected the ones which showed consistency between the MSL2 RIP/input average ratio values and the MSL2 RIP/input enrichment profiles seen in IGB. We tested each selected RNA by RNA-FISH combined with MSL2 immunostaining and none them was seen targeting chromatin in polytenes or colocalizing with MSL2 in brain cells nuclei. The majority of the selected caRNAs bound by MSL2 are snoRNAs, which was seen for Rps7, Uhg4, CG10576, Uhg2, Rps8 and CG13900 that target the nucleolus in both polytene chromosomes and brain cells nuclei. Rps16 transcript showed a dispersed pattern in the nucleus. Our results indicate that MSL binding to heterochromatin probably doesn´t occur via recognition of specific RNAs. Alternatively, the MSL complex might incorporate several RNAs promiscuously and this binding can be lower than the limit of detection by in situ hybridization and immunostaining. Additionally, we can´t exclude the possibility that other non-tested RNA molecules might target MSL to chromatin. Since the majority of RNAs bound by MSL2 are snoRNAs, we can speculate that snoRNAs have similar stem-loop structures to the roX RNAs. Also, since we found before that in roX mutants MSL has affinity for non-transcribed spacer sequences (NTS) present in ribosomal genes, one possibility is that the higher local concentration of MSL in the rDNA locus, and in the nucleolus, promotes MSL binding to snoRNAs. 44 CONCLUSIONS CONCLUSIONS Paper I     Genes in monosomic condition are buffered. The buffering is general and is not affected by other monosomic regions. Gene length is the primary determinant for buffering: longer genes are more strongly buffered than shorter genes. Genes involved in proteolysis are upregulated in individuals carrying segmental monosomies. Paper II     Setdb1 is responsible for H3K9me dependent recruitment of HP1a to chromosome 4 and to region 2L:31. Su(var)3-9 is responsible for H3K9me dependent recruitment of HP1a to pericentromeric regions. HP1a targets promoters of active genes in a manner that is independent of H3K9me and of POF. HP1a promoter peaks are enriched in AT content, in HP2 and are DNase sensitive. Paper III  MSL recruitment to HAS is independent on roX RNAs.  A complete and active MSL complex forms at six genes in the 4th chromosome, independently from roX RNAs.  In the absence of roX RNAs, MSL has affinity to Hoppel transposable elements and to repeats in general. Paper IV  In the absence of roX, MSL associates with several RNAs, especially with snoRNAs.  The RNAs associated with MSL don´t seem to target chromatin. 45 ACKNOWLEDGMENTS ACKNOWLEDGMENTS I am glad I chose to do my PhD studies in the cold far-north Umeå, there has been a lot of exciting research and so much fun outside the lab. The beautiful white cold winters and the fresh green summers will be missed, along with the walks in the forests, the cycling, the skiing and the swimming in the lakes. Many people were important during these last five years. I want to thank you Jan, if you wouldn´t have accepted me as a master student in your lab, I would have missed the chance to experience all of this. Workwise I have to thank you so much for wanting to share your great scientific knowledge with me during these PhD years. I hope I have learned something from you, like how to do good and honest research, how to always do my best, how to be patient and last but not least, how to be fair and kind to people. The scientific world would be a better place with more people like you. Also, thank you for giving me freedom. I also want to thank the people in Jan Larsson´s and Per Stenberg´s groups that I have been closely working and socializing with. Philge, thank you for your great bioinformatics knowledge, for the help throughout the years and for the kindness. I was really lucky that you were sitting in the office next door and that I could always come to discuss the projects. Masha, thank you for sharing your scientific knowledge with me, for all the help in the lab and in the project we had together, for being always available to discuss science and other subjects too. Thank you Per for all your ideas and comments during group meetings and project discussions, they were really important. Without Philge, Masha, Per and Jan, this thesis wouldn´t be possible. Anna-Mia, thank you for being such a good colleague, for all the encouraging words! Your smile and positive energy are contagious! Marie-Line, thank you for sharing your excitement about science, but also for all the lunches, outdoor activities, game nights and trips. Lina, thank you so much for all the great times at work, for our long conversations, travels and so much more! It was never the same here without you. I am so happy that we are friends and that we always keep in touch! To Anders and to Henrik: thank you for the good mood! I want to thank everyone in Yuri Schwartz´s group and in Per Stenberg´s group for the interesting journal clubs, scientific discussions and gatherings! Good luck to EpiCoN! 46 ACKNOWLEDGMENTS I also want to thank everyone in the fly floor for the good environment! Thank you Erik for the good times at work and outside work: everything gets funnier when you are around! Thank you for your friendship! Thank you Fredrik for the entertaining and loud conversations: it was fun teaching with you! Thank you Hande for the good times teaching together and outside work too. I want to thank everyone in the friday fika group for the delicious cakes, specially to the fika king John! Also, to the people that I hanged out with in the beer corner, it was fun! Sveta, thank you so much for all the great times inside and outside the department and especially for all our adventurous travels! So many good memories! I am so happy that we are friends! I also want to thank the friends outside work that I spent my time with in Umeå, you made the dark cold winters warmer and brighter! A special thanks to the band members for the fun and for teaching me! I loved that one music day per week! Thank you Johanna, you are the best flatmate! Thank you Kathi for all the fun we had together! Come back to Portugal soon! Melanie, thank you so much for your friendship! We had so many good times together and Umeå was never the same without you. Your words really motivate me, in science and in life. Fariba, thank you so much for your friendship, for caring for me, for all the good times we had together and for the motivation words! I hope we will live close to each other in the future! Aos amigos de Portugal, obrigada por estarem longe mas sempre tão perto! Bárbara, obrigada por estares sempre aí para mim! A tua amizade é inigualável. Tânia, Pequita, Anahí, Filipa, Clara, Lúcia, gosto muito de voces! Ir a Portugal é tão bom por saber que nos vamos encontrar! Obrigada Sílvia, Joana, Melissa e Diana pela amizade. À minha família, aos tios, primos e irmão, que sempre se preocuparam e de quem eu gosto muito, muito obrigada! O maior agradecimento de todos vai para os meus pais, Julieta e Carlos, não há palavras para descrever todo o vosso amor, apoio e motivacão desde sempre! Esta tese não seria possivel sem voces! desculpem estar longe, mas trago-vos sempre comigo!! Chris, your love is all I ever dreamed of. Thank you for all the fun, caring and support in this last step of my PhD. I hope the days come easy and the moments pass slow, and each road leads us where we want to go. 47 REFERENCES REFERENCES Akhtar, A., and P. B. Becker, 2000 Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila. Mol Cell 5: 367-375. Alekseyenko, A. A., E. Larschan, W. R. Lai, P. J. Park and M. I. Kuroda, 2006 High-resolution ChIP-chip analysis reveals that the Drosophila MSL complex selectively identifies active genes on the male X chromosome. Genes Dev. Alekseyenko, A. A., S. Peng, E. Larschan, A. A. Gorchakov, O. K. Lee et al., 2008 A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome. Cell 134: 599-609. Ashburner, M., 1989 Drosophila: A laboratory handbook. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Badugu, R., Y. Yoo, P. B. Singh and R. Kellum, 2005 Mutations in the heterochromatin protein 1 (HP1) hinge domain affect HP1 protein interactions and chromosomal distribution. Chromosoma 113: 370-384. Bannister, A. J., P. Zegerman, J. F. Partridge, E. A. Miska, J. O. Thomas et al., 2001 Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120-124. Barigozzi, C., S. Dolfini, M. Fraccaro, G. R. Raimondi and L. Tiepolo, 1966 In vitro study of the DNA replication patterns of somatic chromosomes of Drosophila melanogaster. Exp Cell Res 43: 231-234. Bashaw, G. J., and B. S. Baker, 1995 The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development 121: 3245-3258. Belote, J. M., 1983 Male-Specific Lethal mutations of Drosophilamelanogaster .2. parameters of gene-action during male development. Genetics 105: 881-896. Bier, E., 2005 Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet 6: 9-23. Black, B. E., and D. W. Cleveland, 2011 Epigenetic centromere propagation and the nature of CENP-a nucleosomes. Cell 144: 471-479. Brower-Toland, B., N. C. Riddle, H. Jiang, K. L. Huisinga and S. C. Elgin, 2009 Multiple SET methyltransferases are required to maintain normal heterochromatin domains in the genome of Drosophila melanogaster. Genetics 181: 1303-1319. 48 REFERENCES Buiting, K., S. Saitoh, S. Gross, B. Bittrich, S. Schwartz et al., 1995 Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting center on human-chromosome-15 (Vol 9, Pg 395, 1995). Nat Genet 10: 249-249. Capelson, M., Y. Liang, R. Schulte, W. Mair, U. Wagner et al., 2010 Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell 140: 372-383. Carvalho, A., L. Koerich and A. Clark, 2009 Origin and evolution of Y chromosomes: Drosophila tales. Trends Genet: 270-277. Carvalho, A. B., 2002 Origin and evolution of the Drosophila Y chromosome. Curr Opin Genet Dev 12: 664-668. Celniker, S. E., L. A. Dillon, M. B. Gerstein, K. C. Gunsalus, S. Henikoff et al., 2009 Unlocking the secrets of the genome. Nature 459: 927-930. Chong, S. Y., and E. Whitelaw, 2004 Epigenetic germline inheritance. Curr Opin Genet Dev 14: 692-696. Cleard, F., M. Delattre and P. Spierer, 1997 SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation. EMBO J 16: 5280-5288. Cline, T. W., and B. J. Meyer, 1996 Vive la difference: male vs females in flies vs worms. Annu. Rev. Genet. 30: 637-702. Clough, E., W. Moon, S. Wang, K. Smith and T. Hazelrigg, 2007 Histone methylation is required for oogenesis in Drosophila. Development 134: 157165. Coelho, P. A., J. Queiroz-Machado, D. Hartl and C. E. Sunkel, 1998 Pattern of chromosomal localization of the Hoppel transposable element family in the Drosophila melanogaster subgroup. Chromosome Res 6: 385-395. Colnaghi, R., G. Carpenter, M. Volker and M. O'Driscoll, 2011 The consequences of structural genomic alterations in humans: Genomic Disorders, genomic instability and cancer. Seminars in Cell & Developmental Biology 22: 875-885. Conrad, T., and A. Akhtar, 2012 Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription. Nat Rev Genet 13: 123-134. Conrad, T., F. M. Cavalli, J. M. Vaquerizas, N. M. Luscombe and A. Akhtar, 2012 Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters. Science 337: 742-746. 49 REFERENCES Copps, K., R. Richman, L. M. Lyman, K. A. Chang, J. Rampersad-Ammons et al., 1998 Complex formation by the Drosophila MSL proteins: role of the MSL2 RING finger in protein complex assembly. EMBO J 17: 5409-5417. Cryderman, D. E., M. W. Vitalini and L. L. Wallrath, 2011 Heterochromatin protein 1a is required for an open chromatin structure. Transcription 2: 9599. Czermin, B., R. Melfi, D. McCabe, V. Seitz, A. Imhof et al., 2002 Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111: 185-196. Czermin, B., G. Schotta, B. B. Hülsmann, A. Brehm, P. B. Becker et al., 2001 Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep 2: 915-919. de Wit, E., F. Greil and B. van Steensel, 2005 Genome-wide HP1 binding in Drosophila: developmental plasticity and genomic targeting signals. Genome Res 15: 1265-1273. de Wit, E., F. Greil and B. van Steensel, 2007 High-resolution mapping reveals links of HP1 with active and inactive chromatin components. PLoS Genet 3:e38. Delattre, M., A. Spierer, Y. Jaquet and P. Spierer, 2004 Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation. J Cell Sci 117: 6239-6247. Delattre, M., A. Spierer, C. H. Tonka and P. Spierer, 2000 The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1. J Cell Sci 113 Pt 23: 4253-4261. Delaval, K., and R. Feil, 2004 Epigenetic regulation of mammalian genomic imprinting. Curr Opin Genet Dev 14: 188-195. Demakova, O. V., I. V. Kotlikova, P. R. Gordadze, A. A. Alekseyenko, M. I. Kuroda et al., 2003 The MSL complex levels are critical for its correct targeting to the chromosomes in Drosophila melanogaster. Chromosoma 112: 103-115. Deng, H., X. Bao, W. Zhang, J. Girton, J. Johansen et al., 2007 Reduced levels of Su(var)3-9 but not Su(var)2-5 (HP1) counteract the effects on chromatin structure and viability in loss-of-function mutants of the JIL-1 histone H3S10 kinase. Genetics 177: 79-87. Deng, H., W. Cai, C. Wang, S. Lerach, M. Delattre et al., 2010 JIL-1 and Su(var)3-7 interact genetically and counteract each other's effect on position-effect variegation in Drosophila. Genetics 185: 1183-1192. 50 REFERENCES Deng, X., J. B. Berletch, W. Ma, D. K. Nguyen, J. B. Hiatt et al., 2013 Mammalian X upregulation is associated with enhanced transcription initiation, RNA half-life, and MOF-mediated H4K16 acetylation. Dev Cell 25: 55-68. Deng, X., and V. H. Meller, 2006 roX RNAs are required for increased expression of X-linked genes in Drosophila melanogaster males. Genetics 174: 1859-1866. Deng, X., B. P. Rattner, S. Souter and V. H. Meller, 2005 The severity of roX1 mutations is predicted by MSL localization on the X chromosome. Mech Dev 122: 1094-1105. Dialynas, G. K., D. Makatsori, N. Kourmouli, P. A. Theodoropoulos, K. McLean et al., 2006 Methylation-independent binding to histone H3 and cell cycledependent incorporation of HP1beta into heterochromatin. J Biol Chem 281: 14350-14360. Donnelly, N., V. Passerini, M. Durrbaum, S. Stingele and Z. Storchova, 2014 HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells. EMBO J 33: 2374-2387. Dos Santos, G., A. J. Schroeder, J. L. Goodman, V. B. Strelets, M. A. Crosby et al., 2015 FlyBase: introduction of the Drosophila melanogaster Release 6 reference genome assembly and large-scale migration of genome annotations. Nucleic Acids Res 43: D690-697. Ebert, A., G. Schotta, S. Lein, S. Kubicek, V. Krauss et al., 2004 Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18: 2973-2983. Eissenberg, J. C., T. C. James, D. M. Foster-Hartnett, T. Hartnett, V. Ngan et al., 1990 Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc Natl Acad Sci U S A 87: 9923-9927. Eissenberg, J. C., G. D. Morris, G. Reuter and T. Hartnett, 1992 The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. Genetics 131: 345-352. Eissenberg, J. C., and G. Reuter, 2009 Cellular mechanism for targeting heterochromatin formation in Drosophila. Int Rev Cell Mol Biol 273: 1-47. Eltsov, M., K. M. MacLellan, K. Maeshima, A. S. Frangakis and J. Dubochet, 2008 Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ. Proc Natl Acad Sci USA 105: 19732-19737. 51 REFERENCES Erickson, J. W., and J. J. Quintero, 2007 Indirect effects of ploidy suggest X chromosome dose, not the X:A ratio, signals sex in Drosophila. PLoS Biol 5: e332. Falls, J. G., D. J. Pulford, A. A. Wylie and R. L. Jirtle, 1999 Genomic imprinting: implications for human disease. Am J Pathol 154: 635-647. Fanti, L., D. R. Dorer, M. Berloco, S. Henikoff and S. Pimpinelli, 1998 Heterochromatin protein 1 binds transgene arrays. Chromosoma 107: 286292. Fatica, A., and I. Bozzoni, 2014 Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet 15: 7-21. Faucillion, M. L., and J. Larsson, 2015 Increased expression of X-linked genes in mammals is associated with a higher stability of transcripts and an increased ribosome density. Genome Biol Evol. Fauth, T., F. Muller-Planitz, C. Konig, T. Straub and P. B. Becker, 2010 The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome. Nucleic Acids Res 38: 3209-3221. Fenley, A. T., D. A. Adams and A. V. Onufriev, 2010 Charge state of the globular histone core controls stability of the nucleosome. Biophys J 99: 1577-1585. Ferrari, F., Y. L. Jung, P. V. Kharchenko, A. Plachetka, A. A. Alekseyenko et al., 2013 Comment on "Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters". Science 340: 273. Figueiredo, M. L., M. Kim, P. Philip, A. Allgardsson, P. Stenberg et al., 2014 Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions. PLoS Genet 10: e1004865. Figueiredo, M. L., P. Philip, P. Stenberg and J. Larsson, 2012 HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster. PLoS Genet 8: e1003061. Filion, G. J., J. G. van Bemmel, U. Braunschweig, W. Talhout, J. Kind et al., 2010 Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143: 212-224. FitzPatrick, D. R., J. Ramsay, N. I. McGill, M. Shade, A. D. Carothers et al., 2002 Transcriptome analysis of human autosomal trisomy. Hum Mol Genet 11: 3249-3256. Font-Burgada, J., D. Rossell, H. Auer and F. Azorín, 2008 Drosophila HP1c isoform interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression. Genes Dev 22: 3007-3023. 52 REFERENCES Fussner, E., M. Strauss, U. Djuric, R. Li, K. Ahmed et al., 2012 Open and closed domains in the mouse genome are configured as 10-nm chromatin fibres. EMBO Rep 13: 992-996. Garrigues, J. M., S. Sidoli, B. A. Garcia and S. Strome, 2015 Defining heterochromatin in C. elegans through genome-wide analysis of the heterochromatin protein 1 homolog HPL-2. Genome Res 25: 76-88. Gelbart, M. E., and M. I. Kuroda, 2009 Drosophila dosage compensation: a complex voyage to the X chromosome. Development 136: 1399-1410. Gentric, G., C. Desdouets and S. Celton-Morizur, 2012 Hepatocytes polyploidization and cell cycle control in liver physiopathology. Int J Hepatol 2012: 282430. Gilfillan, G. D., T. Straub, E. de Wit, F. Greil, R. Lamm et al., 2006 Chromosome-wide gene-specific targeting of the Drosophila dosage compensation complex. Genes Dev 20: 858-870. Gordon, D. J., B. Resio and D. Pellman, 2012 Causes and consequences of aneuploidy in cancer. Nat Rev Genet 13: 189-203. Greil, F., I. van der Kraan, J. Delrow, J. F. Smothers, E. de Wit et al., 2003 Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev 17: 2825-2838. Grimaud, C., and P. B. Becker, 2009 The dosage compensation complex shapes the conformation of the X chromosome in Drosophila. Gene Dev 23: 2490-2495. Gupta, A., T. G. Guerin-Peyrou, G. G. Sharma, C. Park, M. Agarwal et al., 2008 The mammalian ortholog of Drosophila MOF that acetylates histone H4 lysine 16 is essential for embryogenesis and oncogenesis. Mol Cell Biol 28: 397-409. Gupta, V., M. Parisi, D. Sturgill, R. Nuttall, M. Doctolero et al., 2006 Global analysis of X-chromosome dosage compensation. Journal of Biology 5: 3. Hamada, F. N., P. J. Park, P. R. Gordadze and M. I. Kuroda, 2005 Global regulation of X chromosomal genes by the MSL complex in Drosophila melanogaster. Gene Dev 19: 2289-2294. Handsaker, R. E., V. Van Doren, J. R. Berman, G. Genovese, S. Kashin et al., 2015 Large multiallelic copy number variations in humans. Nat Genet 47: 296-303. Heard, E., and C. M. Disteche, 2006 Dosage compensation in mammals: finetuning the expression of the X chromosome. Gene Dev 20: 1848-1867. 53 REFERENCES Heard, E., and R. A. Martienssen, 2014 Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157: 95-109. Henikoff, S., and M. M. Smith, 2015 Histone variants and epigenetics. Cold Spring Harb Perspect Biol 7. Hochman, B., 1976 The fourth chromosome of Drosophila melanogaster, pp. 903-928 in The Genetics and biology of Drosophila, edited by M. Ashburner and E. Novitski. Academic Press. Huang, X. A., H. Yin, S. Sweeney, D. Raha, M. Snyder et al., 2013 A major epigenetic programming mechanism guided by piRNAs. Dev Cell 24: 502516. Hwang, K. K., J. C. Eissenberg and H. J. Worman, 2001 Transcriptional repression of euchromatic genes by Drosophila heterochromatin protein 1 and histone modifiers. Mol Biol Cell 12: 356a-356a. Ilik, I., and A. Akhtar, 2009 roX RNAs: non-coding regulators of the male X chromosome in flies. RNA Biol 6: 113-121. Ilik, I. A., J. J. Quinn, P. Georgiev, F. Tavares-Cadete, D. Maticzka et al., 2013 Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila. Mol Cell 51: 156-173. Jacobs, S. A., and S. Khorasanizadeh, 2002 Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science 295: 2080-2083. James, T. C., and S. C. R. Elgin, 1986 Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophilamelanogaster and its gene. Mol Cell Biol 6: 3862-3872. Jin, Y., Y. M. Wang, J. Johansen and K. M. Johansen, 2000 JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associates with the male specific lethal (MSL) dosage compensation complex. J Cell Biol 149: 1005-1010. Jin, Y., Y. M. Wang, D. L. Walker, H. Dong, C. Conley et al., 1999 JIL-1: A novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Mol Cell 4: 129-135. Johansson, A. M., A. Allgardsson, P. Stenberg and J. Larsson, 2011 msl2 mRNA is bound by free nuclear MSL complex in Drosophila melanogaster. Nucleic Acids Res 39: 6428-6439. Johansson, A. M., P. Stenberg, A. Allgardsson and J. Larsson, 2012 POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA. Mol Cell Biol 32: 2121-2134. 54 REFERENCES Johansson, A. M., P. Stenberg, C. Bernhardsson and J. Larsson, 2007a Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster. EMBO J 26: 2307-2316. Johansson, A. M., P. Stenberg, F. Pettersson and J. Larsson, 2007b POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation. PLoS Genet 3: e209. Jones, P. A., 2012 Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13: 484-492. Kageyama, Y., G. Mengus, G. Gilfillan, H. G. Kennedy, C. Stuckenholz et al., 2001 Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site. EMBO J 20: 2236-2245. Kelley, R. L., and M. I. Kuroda, 2003 The Drosophila roX1 RNA gene can overcome silent chromatin by recruiting the male-specific lethal dosage compensation complex. Genetics 164: 565-574. Kelley, R. L., V. H. Meller, P. R. Gordadze, G. Roman, R. L. Davis et al., 1999 Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98: 513-522. Kelley, R. L., I. Solovyeva, L. M. Lyman, R. Richman, V. Solovyev et al., 1995 Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 81: 867-877. Kharchenko, P. V., A. A. Alekseyenko, Y. B. Schwartz, A. Minoda, N. C. Riddle et al., 2011 Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471: 480-485. Khorasanizadeh, S., 2004 The nucleosome: from genomic organization to genomic regulation. Cell 116: 259-272. Kind, J., J. M. Vaquerizas, P. Gebhardt, M. Gentzel, N. M. Luscombe et al., 2008 Genome-wide analysis reveals MOF as a key regulator of dosage compensation and gene expression in Drosophila. Cell 133: 813-828. Ku, M., J. D. Jaffe, R. P. Koche, E. Rheinbay, M. Endoh et al., 2012 H2A.Z landscapes and dual modifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions. Genome Biol 13: R85. Kunert, N., J. Marhold, J. Stanke, D. Stach and F. Lyko, 2003 A Dnmt2-like protein mediates DNA methylation in Drosophila. Development 130: 50835090. Lachner, M., D. O'Carroll, S. Rea, K. Mechtler and T. Jenuwein, 2001 Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410: 116-120. 55 REFERENCES Lahn, B. T., N. M. Pearson and K. Jegalian, 2001 The human Y chromosome, in the light of evolution. Nat Rev Genet 2: 207-216. Lanctôt, C., T. Cheutin, M. Cremer, G. Cavalli and T. Cremer, 2007 Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet 8: 104-115. Larschan, E., A. A. Alekseyenko, A. A. Gortchakov, S. Peng, B. Li et al., 2007 MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism. Mol Cell 28: 121-133. Larsson, J., J. D. Chen, V. Rasheva, A. Rasmuson Lestander and V. Pirrotta, 2001 Painting of fourth, a chromosome-specific protein in Drosophila. Proc Natl Acad Sci U S A 98: 6273-6278. Larsson, J., and V. H. Meller, 2006 Dosage compensation, the origin and the afterlife of sex chromosomes. Chromosome Res. 14: 417-431. Larsson, J., M. J. Svensson, P. Stenberg and M. Mäkitalo, 2004 Painting of fourth in genus Drosophila suggests autosome-specific gene regulation. Proc Natl Acad Sci U S A 101: 9728-9733. Lavigne, M., R. Eskeland, S. Azebi, V. Saint-André, S. M. Jang et al., 2009 Interaction of HP1 and Brg1/Brm with the globular domain of histone H3 is required for HP1-mediated repression. PLoS Genet 5: e1000769. Levine, M. T., C. McCoy, D. Vermaak, Y. C. G. Lee, M. A. Hiatt et al., 2012 Phylogenomic Analysis Reveals Dynamic evolutionary history of the Drosophila Heterochromatin Protein 1 (HP1) gene family. Plos Genet 8. Lim, C. K., and R. L. Kelley, 2012 Autoregulation of the Drosophila Noncoding roX1 RNA Gene. PLoS Genet 8: e1002564. Lindehell, H., M. Kim and J. Larsson, 2015 Proximity ligation assays of protein and RNA interactions in the male-specific lethal complex on Drosophila melanogaster polytene chromosomes. Chromosoma. Liu, L. P., J. Q. Ni, Y. D. Shi, E. J. Oakeley and F. L. Sun, 2005 Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription. Nat Genet 37: 1361-1366. Locke, J., and H. McDermid, 1993 Analysis of Drosophila chromosome four by pulse field electrophoresis. Chromosoma 102: 718-723. Lomberk, G., D. Bensi, M. E. Fernandez-Zapico and R. Urrutia, 2006 Evidence for the existence of an HP1-mediated subcode within the histone code. Nat Cell Biol 8: 407-415. Lu, B. Y., P. C. Emtage, B. J. Duyf, A. J. Hilliker and J. C. Eissenberg, 2000 Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila. Genetics 155: 699-708. 56 REFERENCES Lu, X. W., S. N. Wontakal, H. Kavi, B. J. Kim, P. M. Guzzardo et al., 2013 Drosophila H1 regulates the genetic activity of heterochromatin by recruitment of Su(var)3-9. Science 340: 78-81. Lucchesi, J. C., 1998 Dosage compensation in flies and worms: the ups and downs of X-chromosome regulation. Curr Opin Genet Dev 8: 179-184. Luger, K., M. L. Dechassa and D. J. Tremethick, 2012 New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat Rev Mol Cell Biol 13: 436-447. Luger, K., A. W. Mäder, R. K. Richmond, D. F. Sargent and T. J. Richmond, 1997 Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389: 251-260. Lundberg, L. E., M. L. Figueiredo, P. Stenberg and J. Larsson, 2012 Buffering and proteolysis are induced by segmental monosomy in Drosophila melanogaster. Nucleic Acids Res 40: 5926-5937. Lundberg, L. E., P. Stenberg and J. Larsson, 2013 HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster. Nucleic Acids Res 41: 4481-4494. Lyko, F., B. H. Ramsahoye and R. Jaenisch, 2000 DNA methylation in Drosophila melanogaster. Nature 408: 538-540. Lyman, L. M., K. Copps, L. Rastelli, R. L. Kelley and M. I. Kuroda, 1997 Drosophila male-specific lethal-2 protein: structure/function analysis and dependence on MSL-1 for chromosome association. Genetics 147: 17431753. Macadangdang, B. R., A. Oberai, T. Spektor, O. A. Campos, F. Sheng et al., 2014 Evolution of histone 2A for chromatin compaction in eukaryotes. Elife 3. Maenner, S., M. Muller, J. Frohlich, D. Langer and P. B. Becker, 2013 ATPDependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins. Mol Cell 51: 174-184. Makarevitch, I., R. L. Phillips and N. M. Springer, 2008 Profiling expression changes caused by a segmental aneuploid in maize. BMC Genomics 9: 7. Marin, I., and B. S. Baker, 1998 The evolutionary dynamics of sex determination. Science 281: 1990-1994. McAnally, A. A., and L. Y. Yampolsky, 2010 Widespread transcriptional autosomal dosage compensation in Drosophila correlates with gene expression level. Genome Biol Evol 2: 44-52. 57 REFERENCES Meller, V. H., P. R. Gordadze, Y. Park, X. Chu, C. Stuckenholz et al., 2000 Ordered assembly of roX RNAs into MSL complexes on the dosagecompensated X chromosome in Drosophila. Curr Biol 10: 136-143. Meller, V. H., and B. P. Rattner, 2002 The roX genes encode redundant malespecific lethal transcripts required for targeting of the MSL complex. EMBO J 21: 1084-1091. Mendez, D. L., D. Kim, M. Chruszcz, G. E. Stephens, W. Minor et al., 2011 The HP1a disordered C terminus and chromo shadow domain cooperate to select target peptide partners. Chembiochem 12: 1084-1096. Menon, D. U., and V. H. Meller, 2009 Imprinting of the Y chromosome influences dosage compensation in roX1 roX2 Drosophila melanogaster. Genetics 183: 811-820. Miklos, G. L. G., M. T. Yamamoto, J. Davies and V. Pirrotta, 1988 Microcloning reveals a high frequency of repetitive sequences characteristic of chromosome foour and the b-heterochromatin of Drosophila melanogaster. Proc Natl Acad Sci USA 85: 2051-2055. Minsky, A., R. Ghirlando and Z. Reich, 1997 Nucleosomes: a solution to a crowded intracellular environment? J Theor Biol 188: 379-385. Mis, J., S. S. Ner and T. A. Grigliatti, 2006 Identification of three histone methyltransferases in Drosophila: dG9a is a suppressor of PEV and is required for gene silencing. Mol Genet Genomics 275: 513-526. Morales, V., T. Straub, M. F. Neumann, G. Mengus, A. Akhtar et al., 2004 Functional integration of the histone acetyltransferase MOF into the dosage compensation complex. EMBO J 23: 2258-2268. Morris, K. V., and J. S. Mattick, 2014 The rise of regulatory RNA. Nat Rev Genet 15: 423-437. Muchardt, C., M. Guilleme, J. S. Seeler, D. Trouche, A. Dejean et al., 2002 Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1 alpha. EMBO Rep 3: 975-981. Muller, H. J., 1940 Bearings on the Drosophila work on systematics, pp. 185268 in The new systematics, edited by J. Huxley. Clarendon Press, Oxford. Muller, H. J., and E. Altenburg, 1930 The Frequency of Translocations Produced by X-Rays in Drosophila. Genetics 15: 283-311. Nakayama , J., J. C. Rice, B. D. Strahl, C. D. Allis and S. I. Grewal, 2001 Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292: 110-113. Nguyen, D. K., and C. M. Disteche, 2006 Dosage compensation of the active X chromosome in mammals. Nat Genet 38: 47-53. 58 REFERENCES Nielsen, A. L., M. Oulad-Abdelghani, J. A. Ortiz, E. Remboutsika, P. Chambon et al., 2001 Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins. Mol Cell 7: 729-739. Oh, H., J. R. Bone and M. I. Kuroda, 2004 Multiple classes of MSL binding sites target dosage compensation to the X chromosome of Drosophila. Curr Biol 14: 481-487. Oh, H., Y. Park and M. I. Kuroda, 2003 Local spreading of MSL complexes from roX genes on the Drosophila X chromosome. Gene Dev 17: 1334-1339. Ohno, S., 1967 Sex chromosomes and sex-linked genes. Monographs on endocrinology 1. Oudet, P., M. Gross-Bellard and P. Chambon, 1975 Electron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell 4: 281-300. Palmer, M. J., R. Richman, L. Richter and M. I. Kuroda, 1994 Sex-specific regulation of the male-specific lethal-1 dosage compensation gene in Drosophila. Gene Dev 8: 698-706. Park, S. W., Y. I. Kang, J. G. Sypula, J. Choi, H. Y. Oh et al., 2007 An evolutionarily conserved domain of roX2 RNA is sufficient for induction of H4-Lys16 acetylation on the Drosophila X chromosome. Genetics 177: 14291437. Park, S. W., M. I. Kuroda and Y. Park, 2008 Regulation of histone H4 Lys16 acetylation by predicted alternative secondary structures in roX noncoding RNAs. Mol Cell Biol 28: 4952-4962. Park, Y., R. L. Kelley, H. Oh, M. I. Kuroda and V. H. Meller, 2002 Extent of chromatin spreading determined by roX RNA recruitment of MSL proteins. Science 298: 1620-1623. Park, Y., G. Mengus, X. Bai, Y. Kageyama, V. H. Meller et al., 2003 Sequencespecific targeting of Drosophila roX genes by the MSL dosage compensation complex. Mol Cell 11: 977-986. Pavelka, N., G. Rancati, J. Zhu, W. D. Bradford, A. Saraf et al., 2010 Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast. Nature 468: 321-325. Perrini, B., L. Piacentini, L. Fanti, F. Altieri, S. Chichiarelli et al., 2004 HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila. Mol Cell 15: 467-476. Phalke, S., O. Nickel, D. Walluscheck, F. Hortig, M. C. Onorati et al., 2009 Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2. Nat Genet 41: 696-702. 59 REFERENCES Philip, P., and P. Stenberg, 2013 Male X-linked genes in Drosophila melanogaster are compensated independently of the Male-Specific Lethal complex. Epigenetics & Chromatin 6. Piacentini, L., L. Fanti, M. Berloco, B. Perrini and S. Pimpinelli, 2003 Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin. J Cell Biol 161: 707-714. Prestel, M., C. Feller, T. Straub, H. Mitlöhner and P. B. Becker, 2010 The activation potential of MOF is constrained for dosage compensation. Mol Cell 38: 815-826. Raddatz, G., P. M. Guzzardo, N. Olova, M. R. Fantappie, M. Rampp et al., 2013 Dnmt2-dependent methylomes lack defined DNA methylation patterns. Proc Natl Acad Sci USA 110: 8627-8631. Raja, S. J., I. Charapitsa, T. Conrad, J. M. Vaquerizas, P. Gebhardt et al., 2010 The nonspecific lethal complex is a transcriptional regulator in Drosophila. Mol Cell 38: 827-841. Rattner, B. P., and V. H. Meller, 2004 Drosophila male-specific lethal 2 protein controls sex-specific expression of the roX genes. Genetics 166: 1825-1832. Rea, S., F. Eisenhaber, D. O'Carroll, B. D. Strahl, Z. W. Sun et al., 2000 Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: 593-599. Regnard, C., T. Straub, A. Mitterweger, I. K. Dahlsveen, V. Fabian et al., 2011 Global analysis of the relationship between JIL-1 kinase and transcription. PLoS Genet 7: e1001327. Richter, L., J. R. Bone and M. I. Kuroda, 1996 RNA-dependent association of the Drosophila maleless protein with the male X chromosome. Genes Cells 1: 325-336. Riddle, N. C., and S. C. Elgin, 2006 The dot chromosome of Drosophila: insights into chromatin states and their change over evolutionary time. Chromosome Res. 14: 405-416. Riddle, N. C., Y. L. Jung, T. Gu, A. A. Alekseyenko, D. Asker et al., 2012 Enrichment of HP1a on Drosophila Chromosome 4 genes creates an alternate chromatin structure critical for regulation in this heterochromatic domain. PLoS Genet 8: e1002954. Riddle, N. C., A. Minoda, P. V. Kharchenko, A. A. Alekseyenko, Y. B. Schwartz et al., 2011 Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin. Genome Res 21: 147-163. 60 REFERENCES Riddle, N. C., C. D. Shaffer and S. C. Elgin, 2009 A lot about a little dot - lessons learned from Drosophila melanogaster chromosome 4. Biochem Cell Biol 87: 229-241. Ryder, E., M. Ashburner, R. Bautista-Llacer, J. Drummond, J. Webster et al., 2007 The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource. Genetics 177: 615-629. Sandman, K., S. L. Pereira and J. N. Reeve, 1998 Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome. Cell Mol Life Sci 54: 1350-1364. Schotta, G., A. Ebert, V. Krauss, A. Fischer, J. Hoffmann et al., 2002 Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J 21: 1121-1131. Schrider, D. R., and M. W. Hahn, 2010 Gene copy-number polymorphism in nature. Proc Biol Sci 277: 3213-3221. Schubert, T., M. C. Pusch, S. Diermeier, V. Benes, E. Kremmer et al., 2012 Df31 protein and snoRNAs maintain accessible higher-order structures of chromatin. Mol Cell 48: 434-444. Schutt, C., and R. Nothiger, 2000 Structure, function and evolution of sexdetermining systems in Dipteran insects. Development 127: 667-677. Seum, C., S. Bontron, E. Reo, M. Delattre and P. Spierer, 2007a Drosophila G9a is a nonessential gene. Genetics 177: 1955-1957. Seum, C., E. Reo, H. Peng, F. J. Rauscher, P. Spierer et al., 2007b Drosophila SETDB1 is required for chromosome 4 silencing. PLoS Genet 3: e76. Shaffer, C. D., G. E. Stephens, B. A. Thompson, L. Funches, J. A. Bernat et al., 2002 Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin. Proc Natl Acad Sci USA 99: 14332-14337. Sinclair, D., R. Mottus and T. Grigliatti, 1983 Genes which suppress positioneffect variegation in Drosophila melanogaster are clustered. Mol Gen Genet 191: 326-333. Smith, E. R., C. Cayrou, R. Huang, W. S. Lane, J. Cote et al., 2005 A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16. Mol Cell Biol 25: 91759188. Smith, S., and B. Stillman, 1991 Stepwise assembly of chromatin during DNA replication in vitro. EMBO J 10: 971-980. Smith, Z. D., and A. Meissner, 2013 DNA methylation: roles in mammalian development. Nat Rev Genet 14: 204-220. 61 REFERENCES Smothers, J. F., and S. Henikoff, 2000 The HP1 chromo shadow domain binds a consensus peptide pentamer. Curr Biol 10: 27-30. Smothers, J. F., and S. Henikoff, 2001 The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins. Mol Cell Biol 21: 2555-2569. Spierer, A., F. Begeot, P. Spierer and M. Delattre, 2008 SU(VAR)3-7 links heterochromatin and dosage compensation in Drosophila. PLoS Genet 4: e1000066. Spierer, A., C. Seum, M. Delattre and P. Spierer, 2005 Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation. J Cell Sci 118: 5047-5057. St Pierre, S. E., L. Ponting, R. Stefancsik, P. McQuilton and C. FlyBase, 2014 FlyBase 102--advanced approaches to interrogating FlyBase. Nucleic Acids Res 42: D780-788. Stabell, M., M. Bjørkmo, R. B. Aalen and A. Lambertsson, 2006a The Drosophila SET domain encoding gene dEset is essential for proper development. Hereditas 143: 177-188. Stabell, M., R. Eskeland, M. Bjørkmo, J. Larsson, R. B. Aalen et al., 2006b The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development. Nucleic Acids Res 34: 4609-4621. Stankiewicz, P., and J. R. Lupski, 2010 Structural variation in the human genome and its role in disease. Annu Rev Med 61: 437-455. Stenberg, P., and J. Larsson, 2011 Buffering and the evolution of chromosome-wide gene regulation. Chromosoma 120: 213-225. Stenberg, P., L. E. Lundberg, A. M. Johansson, P. Rydén, M. J. Svensson et al., 2009 Buffering of segmental and chromosomal aneuploidies in Drosophila melanogaster. PLoS Genet 5: e100302. Stingele, S., G. Stoehr, K. Peplowska, J. Cox, M. Mann et al., 2012 Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells. Mol Syst Biol 8: 608. Straub, T., and P. B. Becker, 2013 Comment on "Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters". Science 340: 273. Straub, T., C. Grimaud, G. D. Gilfillan, A. Mitterweger and P. B. Becker, 2008 The chromosomal high-affinity binding sites for the Drosophila dosage compensation complex. PLoS Genet 4: e1000302. Straub, T., A. Zabel, G. D. Gilfillan, C. Feller and P. B. Becker, 2013 Different chromatin interfaces of the Drosophila dosage compensation complex revealed by high-shear ChIP-seq. Genome Res 473-85. 62 REFERENCES Szerlong, H. J., and J. C. Hansen, 2011 Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structure. Biochem Cell Biol 89: 24-34. Taipale, M., S. Rea, K. Richter, A. Vilar, P. Lichter et al., 2005 HMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells. Mol Cell Biol 25: 6798-6810. Tessarz, P., and T. Kouzarides, 2014 Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol 15: 703-708. Thomas, S., X. Y. Li, P. J. Sabo, R. Sandstrom, R. E. Thurman et al., 2011 Dynamic reprogramming of chromatin accessibility during Drosophila embryo development. Genome Biol 12: R43. Torres, E. M., N. Dephoure, A. Panneerselvam, C. M. Tucker, C. A. Whittaker et al., 2010 Identification of aneuploidy-tolerating mutations. Cell 143: 7183. Torres, E. M., T. Sokolsky, C. M. Tucker, L. Y. Chan, M. Boselli et al., 2007 Effects of aneuploidy on cellular physiology and cell division in haploid yeast. Science 317: 916-924. Torres, E. M., B. R. Williams and A. Amon, 2008 Aneuploidy: cells losing their balance. Genetics 179: 737-746. Trojer, P., and D. Reinberg, 2007 Facultative heterochromatin: Is there a distinctive molecular signature? Mol Cell 28: 1-13. Tropberger, P., S. Pott, C. Keller, K. Kamieniarz-Gdula, M. Caron et al., 2013 Regulation of transcription through acetylation of H3K122 on the lateral surface of the histone octamer. Cell 152: 859-872. Tsai, J. H., and B. D. McKee, 2011 Homologous pairing and the role of pairing centers in meiosis. Journal of Cell Science 124: 1955-1963. Tzeng, T. Y., C. H. Lee, L. W. Chan and C. K. Shen, 2007 Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1. Proc Natl Acad Sci USA 104: 12691-12696. Wakimoto, B. T., and M. G. Hearn, 1990 The effects of chromosome rearrangements on the expression of heterochromatic genes in chromosome 2L of Drosophila melanogaster. Genetics 125: 141-154. Wallrath, L. L., and S. C. Elgin, 1995 Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev 9: 1263-1277. Wallrath, L. L., V. P. Guntur, L. E. Rosman and S. C. Elgin, 1996 DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues of Drosophila melanogaster. Chromosoma 104: 519527. 63 REFERENCES Wang, C. I., A. A. Alekseyenko, G. Leroy, A. E. Elia, A. A. Gorchakov et al., 2013 Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nat Struct Mol Biol 20: 202-209. Wang, W., G. W. Li, C. Chen, X. S. Xie and X. Zhuang, 2011 Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333: 1445-1449. Vaquerizas, J. M., F. M. Cavalli, T. Conrad, A. Akhtar and N. M. Luscombe, 2013 Response to comments on "Drosophila Dosage Compensation Involves Enhanced Pol II Recruitment to Male X-Linked Promoters". Science 340: 273. Weissmann, F., I. Muyrers-Chen, T. Musch, D. Stach, M. Wiessler et al., 2003 DNA hypermethylation in Drosophila melanogaster causes irregular chromosome condensation and dysregulation of epigenetic histone modifications. Mol Cell Biol 23: 2577-2586. Venkatesh, S., and J. L. Workman, 2015 Histone exchange, chromatin structure and the regulation of transcription. Nat Rev Mol Cell Biol. Venken, K. J., E. Popodi, S. L. Holtzman, K. L. Schulze, S. Park et al., 2010 A molecularly defined duplication set for the X chromosome of Drosophila melanogaster. Genetics 186: 1111-1125. Vermaak, D., S. Henikoff and H. S. Malik, 2005 Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila. PLoS Genet 1: 96-108. Vermaak, D., and H. S. Malik, 2009 Multiple roles for heterochromatin protein 1 genes in Drosophila. Annu Rev Genet 43: 467-492. Williams, B. R., V. R. Prabhu, K. E. Hunter, C. M. Glazier, C. A. Whittaker et al., 2008 Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science 322: 703-709. Yildirim, E., R. I. Sadreyev, S. F. Pinter and J. T. Lee, 2012 X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription. Nat Struct Mol Biol 19: 56-61. Yin, H., S. Sweeney, D. Raha, M. Snyder and H. Lin, 2011 A high-resolution whole-genome map of key chromatin modifications in the adult Drosophila melanogaster. PLoS Genet 7: e1002380. Yoon, J., K. S. Lee, J. S. Park, K. Yu, S. G. Paik et al., 2008 dSETDB1 and SU(VAR)3-9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster. PLoS One 3: e2234. Yutin, N., K. S. Makarova, S. L. Mekhedov, Y. I. Wolf and E. V. Koonin, 2008 The deep archaeal roots of eukaryotes. Mol Biol Evol 25: 1619-1630. 64 REFERENCES Zentner, G. E., and S. Henikoff, 2013 Regulation of nucleosome dynamics by histone modifications. Nat Struct Mol Biol 20: 259-266. Zhang, W., H. Deng, X. Bao, S. Lerach, J. Girton et al., 2006 The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila. Development 133: 229-235. Zhang, Y., J. H. Malone, S. K. Powell, V. Periwal, E. Spana et al., 2010 Expression in aneuploid Drosophila S2 cells. PLoS Biol in press. Zhao, T., T. Heyduk, C. D. Allis and J. C. Eissenberg, 2000 Heterochromatin protein 1 binds to nucleosomes and DNA in vitro. J Biol Chem 275: 2833228338. 65