Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in Drosophila.

Insulators mediate inter- and intrachromosomal contacts to regulate enhancer-promoter interactions and establish chromosome domains. The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive. Here, we demonstrate that Drosophila insulator proteins are poly(ADP-ribosyl)ated and that mutation of the poly(ADP-ribose) polymerase (Parp) gene impairs their function. This modification is not essential for DNA occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). However, poly(ADP-ribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo. Consistent with these findings, the nuclear clustering of CP190 complexes is disrupted in Parp mutant cells. Importantly, poly(ADP-ribosyl)ation facilitates intrachromosomal interactions between insulator sites measured by 4C. These data suggest that the role of insulators in organizing the three-dimensional architecture of the genome may be modulated by poly(ADP-ribosyl)ation.

Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila.

The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], we captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, we confirmed direct binding of Scm and PRC2 using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, we found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. We propose that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, our results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing.

Different Protein Groups Involved in Transcription Regulation in Development and Housekeeping Genes in Drosophila.

Antibodies to histone modifications and an insulator protein involved in the processes of transcription initiation and elongation are mapped in Drosophila polytene chromosomes. The CHRIZ protein (chromatin insulator) and H3K36me3 histone modification (RNA elongation) are detected only in the localization of housekeeping genes (interbands and gray bands of polytene chromosomes) and never in the regions of developmental genes (black bands and large puffs arising from them). Antibodies to H3S10P histone modification, which is associated with the initial elongation of the RNA strand during transcription, are found exclusively in small puffs, but not in housekeeping gene localization sites or large ecdysone-induced puffs, where housekeeping genes are localized. Antibodies to H4R3me2 histone modification (a co-repressor of the ecdysone receptor) are detected only in large ecdysone-induced puffs.

Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state.

In the metazoan germline, piwi proteins and associated piwi-interacting RNAs (piRNAs) provide a defense system against the expression of transposable elements. In the cytoplasm, piRNA sequences guide piwi complexes to destroy complementary transposon transcripts by endonucleolytic cleavage. However, some piwi family members are nuclear, raising the possibility of alternative pathways for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromosomes. Knockdown of Piwi in the germline increases expression of transposable elements that are targeted by piRNAs, whereas protein-coding genes remain largely unaffected. Derepression of transposons upon Piwi depletion correlates with increased occupancy of Pol II on their promoters. Expression of piRNAs that target a reporter construct results in a decrease in Pol II occupancy and an increase in repressive H3K9me3 marks and heterochromatin protein 1 (HP1) on the reporter locus. Our results indicate that Piwi identifies targets complementary to the associated piRNA and induces transcriptional repression by establishing a repressive chromatin state when correct targets are found.

In Vivo Silencing of Genes Coding for dTip60 Chromatin Remodeling Complex Subunits Affects Polytene Chromosome Organization and Proper Development in Drosophila melanogaster.

Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.

H2Av facilitates H3S10 phosphorylation but is not required for heat shock-induced chromatin decondensation or transcriptional elongation.

A model has been proposed in which JIL-1 kinase-mediated H3S10 and H2Av phosphorylation is required for transcriptional elongation and heat shock-induced chromatin decondensation. However, here we show that although H3S10 phosphorylation is indeed compromised in the H2Av null mutant, chromatin decondensation at heat shock loci is unaffected in the absence of JIL-1 as well as of H2Av and that there is no discernable decrease in the elongating form of RNA polymerase II in either mutant. Furthermore, mRNA for the major heat shock protein Hsp70 is transcribed at robust levels in both H2Av and JIL-1 null mutants. Using a different chromatin remodeling paradigm that is JIL-1 dependent, we provide evidence that ectopic tethering of JIL-1 and subsequent H3S10 phosphorylation recruits PARP-1 to the remodeling site independently of H2Av phosphorylation. These data strongly suggest that H2Av or H3S10 phosphorylation by JIL-1 is not required for chromatin decondensation or transcriptional elongation in Drosophila.

Effect of Methotrexate is Changed under Conditions of High Temperature of Ontogenesis as Exemplified by Drosophila melanogaster.

Changes in the effect of methotrexate (400 µg/kg nutrient medium) combined with high temperature (28°C) of ontogenesis were studied on two hybrid lines of Drosophila melanogaster heterozygous by the vestigial gene that underwent long-term target selection for cutting injury of the wing. Changes in the wing shape, mortality rate, and morphology of polytene chromosomes were studied.

The Chriz Protein Promotes the Recruitment of the Z4 Protein to the STAT-Dependent Promoters.

Proteins Z4/putzig and Chriz/Chromator are involved in the chromatin organization on the promoters of the majority of Drosophila genes. It was shown that the Chriz protein region from aa 273 to 503 is required for the interaction with the Z4 protein. Deletion of this sequence leads to derepression of a number of STAT-dependent genes and development of melanotic tumors in flies. The results of this study suggest that the Chriz protein promotes the recruitment of the Z4 protein to chromatin.

Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain.

Drosophila Polycomb (PC), a subunit of Polycomb repressive complex 1 (PRC1), is well known for its role in maintaining repression of the homeotic genes and many others and for its binding to trimethylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. Here, we identify a novel activity of PC: inhibition of the histone acetylation activity of CREB-binding protein (CBP). We show that PC and its mammalian CBX orthologs interact directly with the histone acetyltransferase (HAT) domain of CBP, binding to the previously identified autoregulatory loop, whose autoacetylation greatly enhances HAT activity. We identify a conserved PC motif adjacent to the chromodomain required for CBP binding and show that PC binding inhibits acetylation of histone H3. CBP autoacetylation impairs PC binding in vitro, and PC is preferentially associated with unacetylated CBP in vivo. PC knockdown elevates the acetylated H3K27 (H3K27ac) level globally and at promoter regions of some genes that are bound by both PC and CBP. Conversely, PC overexpression decreases the H3K27ac level in vivo and also suppresses CBP-dependent Polycomb phenotypes caused by overexpression of Trithorax, an antagonist of Polycomb silencing. We find that PC is physically associated with the initiating form of RNA polymerase II (Pol II) and that many promoters co-occupied by PC and CBP are associated with paused Pol II, suggesting that PC may play a role in Pol II pausing. These results suggest that PC/PRC1 inhibition of CBP HAT activity plays a role in regulating transcription of both repressed and active PC-regulated genes.

Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region.

Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm0, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this "ectopic lamina," and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm0 can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.

Widespread colocalization of the Drosophila histone acetyltransferase homolog MYST5 with DREF and insulator proteins at active genes.

MYST family histone acetyltransferases play important roles in gene regulation. Here, we have characterized the Drosophila MYST histone acetyltransferase (HAT) encoded by cg1894, whose closest homolog is Drosophila MOF, and which we have termed MYST5. We found it localized to a large number of interbands as well as to the telomeres of polytene chromosomes, and it showed strong colocalization with the interband protein Z4/Putzig and RNA polymerase II. Accordingly, genome-wide location analysis by ChIP-seq showed co-occurrence of MYST5 with the Z4-interacting partner Chriz/Chromator. Interestingly, MYST5 bound to the promoter of actively transcribed genes, and about half of MYST5 sites colocalized with the transcription factor DNA replication-related element-binding factor (DREF), indicating a role for MYST5 in gene expression. Moreover, we observed substantial overlap of MYST5 binding with that of the insulator proteins CP190, dCTCF, and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains, with the two roles possibly being functionally interconnected.

Banding Pattern of Polytene Chromosomes as a Representation of Universal Principles of Chromatin Organization into Topological Domains.

Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.

Genetic Organization of Open Chromatin Domains Situated in Polytene Chromosome Interbands in Drosophila.

New data on the organization of genes entirely located in the open domains for chromatin transcription and occupying only one chromosome structure (interband) were obtained. The characteristic features of these genes are the small size (on average, 1-2 kb), depletion of the replicative complex proteins in the regulatory region, and the presence of specific motifs for binding transcription factors, as compared to the genes occupying two structures (interband and gray band). The biological function of these genes is associated primarily with the processes of gene expression and RNA metabolism.

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis.

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl-dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C-terminus to kleisin's N-terminus. Our findings imply that proteolysis-independent release of cohesin from chromatin is mediated by Wapl-dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.

Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions.

Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4th chromosome. Interestingly, our sequence analysis showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

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.

Yeti, an essential Drosophila melanogaster gene, encodes a protein required for chromatin organization.

The evolutionarily conserved family of Bucentaur (BCNT) proteins exhibits a widespread distribution in animal and plants, yet its biological role remains largely unknown. Using Drosophila melanogaster as a model organism, we investigated the in vivo role of the Drosophila BCNT member called YETI. We report that loss of YETI causes lethality before pupation and defects in higher-order chromatin organization, as evidenced by severe impairment in the association of histone H2A.V, nucleosomal histones and epigenetic marks with polytene chromosomes. We also find that YETI binds to polytene chromosomes through its conserved BCNT domain and interacts with the histone variant H2A.V, HP1a and Domino-A (DOM-A), the ATPase subunit of the DOM/Tip60 chromatin remodeling complex. Furthermore, we identify YETI as a downstream target of the Drosophila DOM-A. On the basis of these results, we propose that YETI interacts with H2A.V-exchanging machinery, as a chaperone or as a new subunit of the DOM/Tip60 remodeling complex, and acts to regulate the accumulation of H2A.V at chromatin sites. Overall, our findings suggest an unanticipated role of YETI protein in chromatin organization and provide, for the first time, mechanistic clues on how BCNT proteins control development in multicellular organisms.

P/CAF-mediated spermidine acetylation regulates histone acetyltransferase activity.

Histones and polyamines are important determinants of the chromatin structure. Histones form the core of nucleosome particles and their modification by acetylation of N-terminal tails is involved in chromatin structural changes and transcriptional regulation. Polyamines, including spermidine, are also targets of both cytoplasmic and nuclear acetylation, which in turn alters their affinity for DNA and nucleosomes. Previous studies report the interplay between polyamines metabolism and levels of histone acetylation, but the molecular basis of this effect is still unclear. In this work, we have analyzed the in vitro effect of spermidine on histone H3 acetylation catalyzed by P/CAF, a highly conserved histone acetyltransferase (HAT) (E.C. 2.3.1.48). We have observed that spermidine at very low concentrations activates P/CAF, while it has an inhibitory effect at concentrations higher than 4 µM. In addition, the in vitro bimodal effect of spermidine on histone H3 acetylation was also distinctly observed in vivo on polytene chromosomes of Drosophila melanogaster. We also performed kinetic studies indicating that the activating effect of low spermidine concentrations on P/CAF-HAT activity is based on its involvement as a substrate for P/CAF to produce N8-acetylspermidine that is able in turn to increase the enzyme activity up to four fold.

CHARACTERISTIC OF THE CHROMATIN TYPE CORRESPONDING TO THIN «GREY¼ BANDS IN POLYTHENE CHROMOSOMES OF DROSOPHILA MELANOGASTER.

Recently, we developed a bioinformatic algorithm dividing drosophila genome into 4 types of chromatin which differ in protein composition. This allows us to propose a model of structural and functional organization of interphase chromosomes which postulates an existence of correlation between the chromatin types and morphological structures of polytene chromosomes. So, constantly and everywhere open chromatin type named «aquamarine¼ is characteristic of interbands, while the combinations of the other three types («lazurite¼, «malachite¼ and «ruby¼) form the bands. In this study, we characterized protein composition, genetic organization and morphological features of 39 «lazurite¼-chromatin regions in polytene chromosomes. We found out that «lazurite¼-chromatin usually form thin «grey¼ bands and more rarely ­ boundary portions of large bands. This type of chromatin contains coding parts and 3R-ends of genes and is enriched with proteins and histone modifications associated with active transcription at the stage of elongation. The expression patterns of these genes differ greatly depending on the type of chromatin in their 5R-regions.

COMPREHENSIVE APPROACH TO MAPPING LATE-REPLICATING BANDS IN POLYTHENE CHROMOSOMES OF DROSOPHILA MELANOGASTER.

Heterogeneity of chromatin structure underlies the banding pattern of Drosophila polythene chromosomes. Recently, a four-color model of chromatin has been proposed, with one chromatin type, aquamarine, largely corresponding to interbands (Zhimulev et al., 2014). In this model, most of the previously mapped regions of intercalary heterochromatin are represented by the ruby chromatin type. In the present report, we used the distal part of the chromosome arm 2R to show that all dense late-replicating bands in this region invariably encompass ruby chromatin type. We propose a comprehensive approach that combines cytology mapping data of the FlyBase-annotated genes, novel tools for predicting cytogenetic features of chromosomes based on their prothein composition and data on the sequence of replication in polythene chromosome bands. This approach allows to establish accurately the correspondence between reference late-replicating bands visible on cytology maps and the molecular map.