Diversity and functional specialization of H3K9-specific histone methyltransferases.

Histone modifications play a critical role in the control over activities of the eukaryotic genome; among these chemical alterations, the methylation of lysine K9 in histone H3 (H3K9) is one of the most extensively studied. The number of enzymes capable of methylating H3K9 varies greatly across different organisms: in fission yeast, only one such methyltransferase is present, whereas in mammals, 10 are known. If there are several such enzymes, each of them must have some specific function, and they can interact with one another. Thus arises a complex system of interchangeability, "division of labor," and contacts with each other and with diverse proteins. Histone methyltransferases specialize in the number of methyl groups that they attach and have different intracellular localizations as well as different distributions on chromosomes. Each also shows distinct binding to different types of sequences and has a specific set of nonhistone substrates.

SetDB1 and Su(var)3-9 are essential for late stages of larval development of Drosophila melanogaster.

Methylation of H3K9 histone residue is a marker of gene silencing in eukaryotes. Three enzymes responsible for adding this modification - G9a, SetDB1/Egg, and Su(var)3-9 - are known in Drosophila. To understand how simultaneous mutations of SetDB1 and Su(var)3-9 may affect the fly development, appropriate combinations were obtained. Double mutants egg; Su(var)3-9 displayed pronounced embryonic lethality, slower larval growth and died before or during metamorphosis. Analysis of transcription in larval salivary glands and wing imaginal disks indicated that the effect of double mutation is tissue-specific. In salivary gland chromosomes, affected genes display low H3K9me2 enrichment and are rarely bound by SetDB1 or Su(var)3-9. We suppose that each of these enzymes directly or indirectly controls its own set of gene targets in different organs, and double mutation results in an imbalanced developmental program. This also indicates that SetDB1 and Su(var)3-9 may affect transcription via H3K9-independent mechanisms. Unexpectedly, in double and triple mutants, amount of di- and tri-methylated H3K9 is drastically reduced, but not completely absent. We hypothesize that this residual methylation implies the existence of additional H3K9-specific methyltransferase in Drosophila.

SetDB1 and Su(var)3-9 play non-overlapping roles in somatic cell chromosomes of Drosophila melanogaster.

We explored functional roles of two H3K9-specific histone methyltransferases of Drosophila melanogaster, SetDB1 (also known as Eggless) and Su(var)3-9. Using the DamID approach, we generated the binding profile for SetDB1 in Drosophila salivary gland chromosomes, and matched it to the profile of Su(var)3-9. Unlike Su(var)3-9, SetDB1 turned out to be an euchromatic protein that is absent from repeated DNA compartments, and is largely restricted to transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of ubiquitously expressed genes. Significant SetDB1 association is also observed at binding sites for the insulator protein CP190. SetDB1 and H3K9 di- and tri-methylated (me2 and me3)-enriched sites tend to display poor overlap. At the same time, SetDB1 has a clear connection with the distribution of H3K27me3 mark. SetDB1 binds outside the domains possessing this modification, and about half of the borders of H3K27me3 domains are decorated by SetDB1 together with actively transcribed genes. On the basis of poor correlation between the distribution of SetDB1 and H3K9 methylation marks, we speculate that, in somatic cells, SetDB1 may contribute to the methylation of a broader set of chromosomal proteins than just H3K9. In addition, SetDB1 can be expected to play a role in the establishment of chromatin functional domains.

Binding of SU(VAR)3-9 Partially Depends on SETDB1 in the Chromosomes of Drosophila melanogaster.

H3K9 methylation is known to play a critical role in gene silencing. This modification is established and maintained by several enzymes, but relationships between them are not fully understood. In the present study, we decipher the interplay between two Drosophila H3K9-specific histone methyltransferases, SU(VAR)3-9 and SETDB1. We asked whether SETDB1 is required for targeting of SU(VAR)3-9. Using DamID-seq, we obtained SU(VAR)3-9 binding profiles for the chromosomes from larval salivary glands and germline cells from adult females, and compared profiles between the wild type and SETDB1-mutant backgrounds. Our analyses indicate that the vast majority of single copy genes in euchromatin are targeted by SU(VAR)3-9 only in the presence of SETDB1, whereas SU(VAR)3-9 binding at repeated sequences in heterochromatin is largely SETDB1-independent. Interestingly, piRNA clusters 42AB and 38C in salivary gland chromosomes bind SU(VAR)3-9 regardless of SETDB1, whereas binding to the same regions in the germline cells is SETDB1-dependent. In addition, we compared SU(VAR)3-9 profiles in female germline cells at different developmental stages (germarium cells in juvenile ovaries and mature nurse cells). It turned out that SU(VAR)3-9 binding is influenced both by the presence of SETDB1, as well as by the differentiation stage.

Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis.

BACKGROUND: During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. RESULTS: Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. CONCLUSIONS: Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated.

Genome-wide analysis of SU(VAR)3-9 distribution in chromosomes of Drosophila melanogaster.

Histone modifications represent one of the key factors contributing to proper genome regulation. One of histone modifications involved in gene silencing is methylation of H3K9 residue. Present in the chromosomes across different eukaryotes, this epigenetic mark is controlled by SU(VAR)3-9 and its orthologs. Despite SU(VAR)3-9 was discovered over two decades ago, little is known about the details of its chromosomal distribution pattern. To fill in this gap, we used DamID-seq approach and obtained high-resolution genome-wide profiles for SU(VAR)3-9 in two somatic (salivary glands and brain ganglia) and two germline (ovarian nurse cells and testes) tissues of Drosophila melanogaster. Analysis of tissue and developmental expression of SU(VAR)3-9-bound genes indicates that in the somatic tissues tested, as well as in the ovarian nurse cells, SU(VAR)3-9 tends to associate with transcriptionally silent genes. In contrast, in the testes, SU(VAR)3-9 shows preferential association with testis-specific genes, and its binding appears dynamic during spermatogenesis. In somatic cells, the mere presence/absence of SU(VAR)3-9 binding correlates with lower/higher expression. No such correlation is found in the male germline. Interestingly, transcription units in piRNA clusters (particularly flanks thereof) are frequently targeted by SU(VAR)3-9, and Su(var)3-9 mutation affects the expression of select piRNA species. Our analyses suggest a context-dependent role of SU(VAR)3-9. In euchromatin, SU(VAR)3-9 may serve to fine-tune the expression of individual genes, whereas in heterochromatin, chromosome 4, and piRNA clusters, it may act more broadly over large chromatin domains.

Functional dissection of Drosophila melanogaster SUUR protein influence on H3K27me3 profile.

BACKGROUND: In eukaryotes, heterochromatin replicates late in S phase of the cell cycle and contains specific covalent modifications of histones. SuUR mutation found in Drosophila makes heterochromatin replicate earlier than in wild type and reduces the level of repressive histone modifications. SUUR protein was shown to be associated with moving replication forks, apparently through the interaction with PCNA. The biological process underlying the effects of SUUR on replication and composition of heterochromatin remains unknown. RESULTS: Here we performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm we revealed SuUR-sensitive chromosomal regions that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. We tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. We found that SUUR does not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. We also ruled out the possible effect of SUUR on histone genes expression and its involvement in DSB repair. CONCLUSIONS: Obtained results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication. A model that explains major SUUR-associated phenotypes is proposed.

The effects of SUUR protein suggest its role in repressive chromatin renewal during replication in Drosophila.

Replication of chromosomes is central to heredity. To become available for replication machinery, DNA invariably needs to dissociate from chromatin proteins. Yet, chromatin landscape must be promptly re-established during or soon after replication. Although this process underlies the epigenetic inheritance, little is known about its molecular mechanisms. This mini-review is focused on Drosophila melanogaster SUppressor of UnderReplication (SUUR) protein, which is involved both in replication and chromatin maintenance in polytene tissues. Existing data suggest that it is involved in the regulation of chromatin renewal during replication. According to this model, SUUR protein moves along the chromosomes together with the replication complex. When the replication fork enters the repressed, H3K27me3- or H3K9me3-enriched, chromatin, SUUR-containing complex slows down the replisome until these histone modifications are properly placed on the newly-synthesized DNA strands. Suggested model provides an insight into the mechanism of epigenetic information inheritance. This hypothesis could be tested by further analysis of the interplay between local enrichment of repressive histone modifications and the replication fork progression rate.

DNA replication in nurse cell polytene chromosomes of Drosophila melanogaster otu mutants.

Drosophila cell lines are used extensively to study replication timing, yet data about DNA replication in larval and adult tissues are extremely limited. To address this gap, we traced DNA replication in polytene chromosomes from nurse cells of Drosophila melanogaster otu mutants using bromodeoxyuridine incorporation. Importantly, nurse cells are of female germline origin, unlike the classical larval salivary glands, that are somatic. In contrast to salivary gland polytene chromosomes, where replication begins simultaneously across all puffs and interbands, replication in nurse cells is first observed at several specific chromosomal regions. For instance, in the chromosome 2L, these include the regions 31B-E and 37E and proximal parts of 34B and 35B, with the rest of the decondensed chromosomal regions joining replication process a little later. We observed that replication timing of pericentric heterochromatin in nurse cells was shifted from late S phase to early and mid stages. Curiously, chromosome 4 may represent a special domain of the genome, as it replicates on its own schedule which is uncoupled from the rest of the chromosomes. Finally, we report that SUUR protein, an established marker of late replication in salivary gland polytene chromosomes, does not always colocalize with late-replicating regions in nurse cells.

Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes.

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the "open," euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUUR(GAL4DBD) and HP1(GAL4DBD) results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUUR(GAL4DBD) had no measurable influence on mini-white expression, HP1(GAL4DBD) targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on the protein composition of target regions. HP1(GAL4DBD) but not SUUR(GAL4DBD) could displace an open chromatin marker, CHRIZ, from the tethering sites.

The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing.

Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

Developmental variation of the SUUR protein binding correlates with gene regulation and specific chromatin types in D. melanogaster.

Eukaryotic genomes are organized in large chromatin domains that maintain proper gene activity in the cell. These domains may be permissive or repressive to the transcription of underlying genes. Based on its protein makeup, chromatin in Drosophila cell culture has been recently categorized into five color-coded states. Suppressor of Under-Replication (SUUR) protein was found to be the major component present in all three repressive chromatin states named BLACK, BLUE, and GREEN and to be depleted from the active YELLOW and RED chromatin types. Here, we addressed the question of developmental dynamics of SUUR binding as a marker of repressed chromatin types. We established genomewide SUUR binding profiles in larval salivary gland, brain, and embryos using DNA adenine methyltransferase identification (DamID) technique, performed their pairwise comparisons and comparisons with the published data from Drosophila Kc cells. SUUR binding pattern was found to vary between the samples. Increase in SUUR binding predominantly correlated with local gene repression suggesting heterochromatin formation. Reduction in SUUR binding often coincided with activation of tissue-specific genes probably reflecting the transition to permissive chromatin state and increase in accessibility to specific transcription factors. SUUR binding plasticity accompanied by the regulation of the underlying genes was mainly observed in BLACK, BLUE, and RED chromatin types. Our results provide novel insight into the developmental dynamics of repressive chromatin and reveal a link to the chromatin-guided regulation of gene expression.

Induced transcription results in local changes in chromatin structure, replication timing, and DNA polytenization in a site of intercalary heterochromatin.

In salivary gland polytene chromosomes of Drosophila melanogaster, the regions of intercalary heterochromatin are characterized by late replication, under-replication, and genetic silencing. Using Gal4/UAS system, we induced transcription of sequences adjacent to transgene insertions in the band 11A6-9. This activation resulted in a loss of "silent" and appearance of "active" epigenetic marks, recruitment of RNA polymerase II, and formation of a puff. The activated region is now early replicating and shows increased level of DNA polytenization. Notably, all these changes are restricted to the area around the inserts, whereas the rest of the band remains inactive and late replicating. Although only a short area near the insertion site is transcribed, it results in an "open" chromatin conformation in a much broader region. We conclude that regions of intercalary heterochromatin do not form stand-alone units of late replication and under-replication. Every part of such regions can be activated and polytenized independently of other parts.

The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis.

The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions.

The SUUR protein is involved in binding of SU(VAR)3-9 and methylation of H3K9 and H3K27 in chromosomes of Drosophila melanogaster.

In the present work, we found that the SUUR protein is required for the SU(VAR)3-9 enzyme to bind to the salivary gland polytene chromosomes. The SuUR mutation results in loss of SU(VAR)3-9 on the chromosomes, whereas artificial expression of the SuUR gene restores its binding. The SUUR protein is also involved in methylation of the residues H3K9 and H3K27. However, mono-, di-, and tri-methylated forms of H3K9 and H3K27 behave differently in various chromosomal domains in response to the SuUR mutation. Euchromatin and chromosome 4 are almost completely deprived of mono-, di-, and tri-methylation of H3K9. In the chromocenter, mono-methylation is reduced, di-methylation shows no noticeable changes, and tri-methylation is lost. Furthermore, mono- and di-methylation of H3K27 are not influenced by the SuUR mutation, whereas tri-methylation is lost in the chromocenter. Artificial expression of the SuUR gene on the SuUR (-) background restores the pattern of methylated residues characteristic for the wild type.

The SuUR gene influences the distribution of heterochromatic proteins HP1 and SU(VAR)3-9 on nurse cell polytene chromosomes of Drosophila melanogaster.

We have investigated the distribution of three heterochromatic proteins [SUppressor of UnderReplication (SUUR), heterochromatin protein 1 (HP1), and SU(VAR)3-9] in chromosomes of nurse cells (NCs) and have compared the data obtained with the distribution of the same proteins in salivary gland (SG) chromosomes. In NC chromosomes, the SU(VAR)3-9 protein was found in pericentric heterochromatin and at 223 sites on euchromatic arms, while in SG chromosomes, it was mainly restricted to the chromocenter. In NC chromosomes, the HP1 and SUUR proteins bind to 331 and 256 sites, respectively, which are almost twice the number of sites in SG chromosomes. The distribution of the HP1 and SU(VAR)3-9 proteins depends on the SuUR gene. A mutation in this gene results in a dramatic decrease in the amount of SU(VAR)3-9 binding sites in autosomes. In the X chromosome, these sites are relocated in comparison to the SuUR (+), and their total number only varies slightly. HP1 binding sites are redistributed in chromosomes of SuUR mutants, and their overall number did not change as considerably as SU(VAR)3-9. These data together point to an interaction of these three proteins in Drosophila NC chromosomes.

Vital genes in the heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster.

Heterochromatin has been traditionally regarded as a genomic wasteland, but in the last three decades extensive genetic and molecular studies have shown that this ubiquitous component of eukaryotic chromosomes may perform important biological functions. In D. melanogaster, about 30 genes that are essential for viability and/or fertility have been mapped to the heterochromatin of the major autosomes. Thus far, the known essential genes exhibit a peculiar molecular organization. They consist of single-copy exons, while their introns are comprised mainly of degenerate transposons. Moreover, about one hundred predicted genes that escaped previous genetic analyses have been associated with the proximal regions of chromosome arms but it remains to be determined how many of these genes are actually located within the heterochromatin. In this overview, we present available data on the mapping, molecular organization and function of known vital genes embedded in the heterochromatin of chromosomes 2 and 3. Repetitive loci, such as Responder and the ABO elements, which are also located in the heterochromatin of chromosome 2, are not discussed here because they have been reviewed in detail elsewhere.

Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing.

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.

Abnormal tissue-dependent polytenization of a block of chromosome 3 pericentric heterochromatin in Drosophila melanogaster.

Heterochromatic DNA sequences in the polytene chromosomes of Drosophila melanogaster salivary glands are under-replicated in wild-type strains. In salivary glands of SuUR and in the nurse cells of otu mutants, under-replication is partly suppressed and a banded structure appears within the centric heterochromatin of chromosome 3. This novel banded structure in salivary gland chromosomes was called Plato Atlantis. In order to characterize the heterochromatic component of Plato Atlantis, we constructed a fine-scale cytogenetic map of deletions with break points within centric heterochromatin (Df(3L)1-16, Df(3L)2-66, Df(3R)10-65, Df(3R)4-75 and Df(3L)6B-29 + Df(3R)6B-29). Salivary gland chromosomes show that Df(3L)1-16 removes the complete Plato Atlantis, while Df(3L)2-66 deletes the most proximal 3L regions. These deletions therefore show a substantial cytological overlap. However, in the chromosomes of nurse cells, the same deficiencies remove distinct heterochromatic blocks, with the region of overlap being almost invisible. Satellite (AATAACATAG, AAGAG) and dodecasatellite DNAs mapped in a narrow interval in salivary glands but were found in three clearly distinguishable blocks in nurse cells. The 1.688 satellite was found at a single site in salivary glands but at two sites in nurse cells. We show that newly polytenized heterochromatic structures include blocks h47-h50d of mitotic heterochromatin in salivary glands, but the additional blocks h50p, h53 and h57 are also included in nurse cell chromosomes. Tissue specificity of the patterns of abnormal heterochromatic polytenization implies differential control of DNA replication in somatic and germline cells.