Histone H3: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Histone H3

Synonyms - H3

Cytological map position -

Function - core histone

Keywords - chromatin

Symbol - His3

FlyBase ID: FBgn0001199

Genetic map position -

Classification - histone-fold/TFIID-TAF/NF-Y domain

Cellular location - nuclear

NCBI link: Entrez Gene
His3 orthologs: Biolitmine
Recent literature
Armstrong, R. L., Penke, T. J. R., Chao, S. K., Gentile, G. M., Strahl, B. D., Matera, A. G., McKay, D. J. and Duronio, R. J. (2019). H3K9 Promotes under-replication of pericentromeric heterochromatin in Drosophila salivary gland Polytene Chromosomes. Genes (Basel) 10(2). PubMed ID: 30700014
Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. This study employed a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9), but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.
Shindo, Y. and Amodeo, A. A. (2019). Dynamics of free and chromatin-bound Histone H3 during early embryogenesis. Curr Biol. PubMed ID: 30639105
During zygotic genome activation (ZGA), the chromatin environment undergoes profound changes, including the formation of topologically associated domains, refinements in nucleosome positioning on promoters, and the emergence of heterochromatin. In many organisms, including Drosophila, ZGA is associated with the end of a period of extremely rapid, exponential cleavage divisions that are facilitated by large maternally provided pools of nuclear components. It is therefore imperative to understand how the supply of chromatin components relative to the exponentially increasing demand affects nuclear and chromatin composition during early embryogenesis. This study examined the nuclear trafficking and chromatin dynamics of histones during the cleavage divisions in Drosophila using a photo-switchable H3-Dendra2 reporter. Total H3-Dendra2 in the nucleus decreases with each cleavage cycle. This change in nuclear composition is due to depletion of large pools (>50%) of free protein that are present in the early cycles. The per nucleus import rate halves with each cycle, and a mathematical model was constructed in which increasing histone demand determines the dynamics of nuclear H3 supply. Finally, it was shown that these changes in H3 availability correspond to a large (~ 40%) reduction in global H3 occupancy on the chromatin, which is compensated by the increased incorporation of H3.3. The observed changes in free nuclear H3 and chromatin composition may contribute to the cell-cycle slowing, changes in chromatin structure, and the onset of transcription associated with this developmental stage.
Akmammedov, A., Geigges, M. and Paro, R. (2019). Bivalency in Drosophila embryos is associated with strong inducibility of Polycomb target genes. Fly (Austin). PubMed ID: 31094269
Polycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation are bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. This study provides evidence for existence of bivalency in fly embryos. Using a recently described PcG reporter fly line, a strong reporter inducibility was observed in embryo and its sharp decrease in larval and adult stages. Analysis of the chromatin landscape of the reporter revealed a strong signal for the repressive PcG mark, H3K27me3, in all three developmental stages and, surprisingly, a strong signal for a transcriptionally activating H3K4me3 mark in embryo. Using re-ChIP experiments, bivalent domains were also uncovered at endogenous PcG targets like the Hox genes.
Chen, X., Ye, Y., Gu, L., Sun, J., Du, Y., Liu, W. J., Li, W., Zhang, X. and Jiang, C. (2019). H3K27me3 signal in the cis regulatory elements reveals the differentiation potential of progenitors during Drosophila neuroglial development. Genomics Proteomics Bioinformatics. PubMed ID: 31195140
Drosophila neural development undergoes extensive chromatin remodeling and precise epigenetic regulation. However, the roles of chromatin remodeling in establishment and maintenance of cell identity during cell fate transition remain enigmatic. This study compared the changes in gene expression, as well as the dynamics of nucleosome positioning and key histone modifications between the four major neural cell types during Drosophila neural development. The neural progenitors could be separated from the terminally differentiated cells based on their gene expression profiles, whereas nucleosome distribution in the flanking regions of transcription start sites fails to identify the relationships between the progenitors and the differentiated cells. H3K27me3 signal in promoters and enhancers can not only distinguish the progenitors from the differentiated cells but also identify the differentiation path of the neural stem cells (NSCs) to the intermediate progenitor cells to the glial cells. In contrast, H3K9ac signal fails to identify the differentiation path, although it activates distinct sets of genes with neuron-specific and glia-related functions during the differentiation of the NSCs into neurons and glia, respectively. Together, this study provides novel insights into the crucial roles of chromatin remodeling in determining cell type during Drosophila neural development.
Wooten, M., Snedeker, J., Nizami, Z. F., Yang, X., Ranjan, R., Urban, E., Kim, J. M., Gall, J., Xiao, J. and Chen, X. (2019). Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement. Nat Struct Mol Biol. PubMed ID: 31358945
Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. This study shows that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining super-resolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, old H3 was found to be preferentially incorporated by the leading strand, whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, a high incidence of unidirectional replication fork movement is detected in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.
Bonnet, J., Lindeboom, R. G. H., Pokrovsky, D., Stricker, G., Celik, M. H., Rupp, R. A. W., Gagneur, J., Vermeulen, M., Imhof, A. and Muller, J. (2019). Quantification of proteins and histone marks in Drosophila embryos reveals stoichiometric relationships impacting chromatin regulation. Dev Cell. PubMed ID: 31630981
Gene transcription in eukaryotes is regulated through dynamic interactions of a variety of different proteins with DNA in the context of chromatin. This study used mass spectrometry for absolute quantification of the nuclear proteome and methyl marks on selected lysine residues in histone H3 during two stages of Drosophila embryogenesis. These analyses provide comprehensive information about the absolute copy number of several thousand proteins and reveal unexpected relationships between the abundance of histone-modifying and -binding proteins and the chromatin landscape that they generate and interact with. For some histone modifications, the levels in Drosophila embryos are substantially different from those previously reported in tissue culture cells. Genome-wide profiling of H3K27 methylation during developmental progression and in animals with reduced PRC2 levels illustrates how mass spectrometry can be used for quantitatively describing and comparing chromatin states. Together, these data provide a foundation toward a quantitative understanding of gene regulation in Drosophila.
Prieto-Sanchez, S., Moreno-Castro, C., Hernandez-Munain, C. and Sune, C. (2020). Drosophila Prp40 localizes to the histone locus body and regulates gene transcription and development. J Cell Sci. PubMed ID: 32094262
In eukaryotes, a large amount of histones must be synthesized during the S phase of the cell cycle to package newly synthesized DNA into chromatin. The transcription and 3' end processing of histone pre-mRNA are controlled by the histone locus body (HLB), which is assembled in the H3/H4 promoter. This study identified the Drosophila Prp40 pre-mRNA processing factor (dPrp40) as a novel HLB component. dPrp40 was shown to be essential for Drosophila development, with functionally conserved activity in vertebrates and invertebrates. dPrp40 was observed to be fundamental in endocycling cells, highlighting a role for this factor in mediating replication efficiency in vivo The depletion of dPrp40 from fly cells inhibited the transcription but not the 3' end processing of histone mRNA in a H3/H4 promoter-dependent manner. These results establish that dPrp40 is an essential gene for Drosophila development that can localize to the HLB and may participate in histone mRNA biosynthesis.
Sarthy, J. F., Meers, M. P., Janssens, D. H., Henikoff, J. G., Feldman, H., Paddison, P. J., Lockwood, C. M., Vitanza, N. A., Olson, J. M., Ahmad, K. and Henikoff, S. (2020). Histone deposition pathways determine the chromatin landscapes of H3.1 and H3.3 K27M oncohistones. Elife 9. PubMed ID: 32902381
Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. This study shows that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNA replication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, this study demonstrated that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. It is proposed that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis.
De, S., Gehred, N. D., Fujioka, M., Chan, F. W., Jaynes, J. B. and Kassis, J. A. (2020). Defining the Boundaries of Polycomb Domains in Drosophila. Genetics. PubMed ID: 32948625
Polycomb group (PcG) proteins are an important group of transcriptional repressors that act by modifying chromatin. PcG target genes are covered by the repressive chromatin mark H3K27me3. Polycomb repressive complex 2 (PRC2) is a multiprotein complex that is responsible for generating H3K27me3. In Drosophila, PRC2 is recruited by Polycomb Response Elements (PREs) and then tri-methylates flanking nucleosomes, spreading the H3K27me3 mark over large regions of the genome, the "Polycomb domains". What defines the boundary of a Polycomb domain? There is experimental evidence that insulators, PolII, and active transcription can all form the boundaries of Polycomb domains. This study divided the boundaries of larval Polycomb domains into six different categories. In one category, genes are transcribed toward the Polycomb domain, where active transcription is thought to stop the spreading of H3K27me3. In agreement with this, it was shown that introducing a transcriptional terminator into such a transcription unit causes an extension of the Polycomb domain. Additional data suggest that active transcription of a boundary gene may restrict the range of enhancer activity of a Polycomb-regulated gene.
Floc'hlay, S., Wong, E., Zhao, B., Viales, R. R., Thomas-Chollier, M., Thieffry, D., Garfield, D. A. and Furlong, E. E. M. (2020). Cis-acting variation is common across regulatory layers but is often buffered during embryonic development. Genome Res. PubMed ID: 33310749
Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequence, and chromatin. How DNA mutations affecting any one of these regulatory 'layers' is buffered or propagated to gene expression remains unclear. To address this, allele-specific changes were quantified in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses at three embryonic stages, yielding a comprehensive dataset of 240 samples spanning multiple regulatory layers. Genetic variation (allelic imbalance) impacts gene expression more frequently than chromatin features, with metabolic and environmental response genes being most often affected. Allelic imbalance in cis-regulatory elements (enhancers) is common and highly heritable, yet its functional impact doesn't generally propagate to gene expression. When it does, genetic variation impacts RNA levels through H3K4me3 or independently through chromatin accessibility and H3K27ac. Changes in RNA are more predictive of variation in H3K4me3 than vice versa, suggesting a role for H3K4me3 downstream of transcription. The impact of a substantial proportion of genetic variation is consistent across embryonic stages, with 50% of allelic imbalanced features at one stage being also imbalanced at subsequent developmental stages. Crucially, buffering, as well as the magnitude and evolutionary impact of genetic variants, are influenced by regulatory complexity (i.e., number of enhancers regulating a gene), with transcription factors being most robust to cis-acting, but most influenced by trans-acting variation.
Shindo, Y. and Amodeo, A. A. (2021). Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo. Curr Biol. PubMed ID: 33848457
The DNA damage checkpoint is crucial to protect genome integrity. However, the early embryos of many metazoans sacrifice this safeguard to allow for rapid cleavage divisions that are required for speedy development. At the mid-blastula transition (MBT), embryos switch from rapid cleavage divisions to slower, patterned divisions with the addition of gap phases and acquisition of DNA damage checkpoints. The timing of the MBT is dependent on the nuclear-to-cytoplasmic (N/C ratio) and the activation of the checkpoint kinase, Chk1. How Chk1 activity is coupled to the N/C ratio has remained poorly understood. This study shows that dynamic changes in histone H3 availability in response to the increasing N/C ratio control, Chk1 activity, and thus time the MBT in the Drosophila embryo. Excess H3 in the early cycles was shown to interfere with cell-cycle slowing independent of chromatin incorporation. The N-terminal tail of H3 acts as a competitive inhibitor of Chk1 in vitro and reduces Chk1 activity in vivo. Using a H3-tail mutant that has reduced Chk1 inhibitor activity, this study showed that the amount of available Chk1 sites in the H3 pool controls the dynamics of cell-cycle progression. Mathematical modeling quantitatively supports a mechanism where titration of H3 during early cleavage cycles regulates Chk1-dependent cell-cycle slowing. This study defines Chk1 regulation by H3 as a key mechanism that coordinates cell-cycle remodeling with developmental progression.
Yang, Y., Zhao, T., Li, Z., Qian, W., Peng, J., Wei, L., Yuan, D., Li, Y., Xia, Q. and Cheng, D. (2021). Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis. Proc Natl Acad Sci U S A 118(35). PubMed ID: 34429358
Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)-mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. This study demonstrates that H3K27 methylation-mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval-pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, this study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval-pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development.
Shaukat, A., Khan, M. H. F., Ahmad, H., Umer, Z. and Tariq, M. (2021). Interplay Between BALL and CREB Binding Protein Maintains H3K27 Acetylation on Active Genes in Drosophila. Front Cell Dev Biol 9: 740866. PubMed ID: 34650987
CREB binding protein (CBP) is a multifunctional transcriptional co-activator that interacts with a variety of transcription factors and acts as a histone acetyltransferase. In Drosophila, CBP mediated acetylation of histone H3 lysine 27 (H3K27ac) is a known hallmark of gene activation regulated by trithorax group proteins (trxG). Recently, it has been shown that a histone kinase Ballchen (BALL) substantially co-localizes with H3K27ac at trxG target loci and is required to maintain gene activation in Drosophila. This study reports a previously unknown interaction between BALL and CBP, which positively regulates H3K27ac. Analysis of genome-wide binding profile of BALL and CBP reveals major overlap and their co-localization at actively transcribed genes. This study shows that BALL biochemically interacts with CBP and depletion of BALL results in drastic reduction in H3K27ac. Together, these results demonstrate a previously unknown synergy between BALL and CBP and reveals a potentially new pathway required to maintain gene activation during development.
Fefelova, E. A., Pleshakova, I. M., Mikhaleva, E. A., Pirogov, S. A., Poltorachenko, V. A., Abramov, Y. A., Romashin, D. D., Shatskikh, A. S., Blokh, R. S., Gvozdev, V. A. and Klenov, M. S. (2022). Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon. Nucleic Acids Res 50(2): 867-884. PubMed ID: 35037046
Eukaryotic genomes harbor hundreds of rRNA genes, many of which are transcriptionally silent. In Drosophila, some rDNA repeats contain insertions of the R2 retrotransposon, which is capable to be transcribed only as part of pre-rRNA molecules. rDNA units with R2 insertions are usually inactivated, although R2 expression may be beneficial in cells with decreased rDNA copy number. This study found that R2-inserted rDNA units are enriched with HP1a and H3K9me3 repressive mark, whereas disruption of the heterochromatin components slightly affects their silencing in ovarian germ cells. Surprisingly, a dramatic upregulation of R2-inserted rRNA genes was observed in ovaries lacking Udd (Under-developed) or other subunits (TAF1b and TAF1c-like) of the SL1-like complex, which is homologues to mammalian Selective factor 1 (SL1) involved in rDNA transcription initiation. Derepression of rRNA genes with R2 insertions was accompanied by a reduction of H3K9me3 and HP1a enrichment. It is suggested that the impairment of the SL1-like complex affects a mechanism of selective activation of intact rDNA units which competes with heterochromatin formation. It is also proposed that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.
Chaouch, A., Berlandi, J., Chen, C. C. L., Frey, F., Badini, S., Harutyunyan, A. S., Chen, X., Krug, B., Hebert, S., Jeibmann, A., Lu, C., Kleinman, C. L., Hasselblatt, M., Lasko, P., Shirinian, M. and Jabado, N. (2021). Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks. Mol Cell. PubMed ID: 34739871
Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. This study shows that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon derepression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. These results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks (Chaouch, 2021).
Cheng, Q., Xie, H., Zhang, X. Y., Wang, M. Y., Bi, C. L., Wang, Q., Wang, R. and Fang, M. (2022). An essential role for PTIP in mediating Hox gene regulation along PcG and trxG pathways. Febs J. PubMed ID: 35655387
During Drosophila development, Polycomb-group and Trithorax group proteins function to ensure correct maintenance of transcription patterns by epigenetically repressing or activating target gene expression. To get a deep insight into the PcG and trxG pathways, this study investigated a BRCT domain-containing protein called PTIP, which was generally identified as a transcriptional coactivator and belongs to the TRR complex. At the genome scale, given PTIP-binding peaks were sorted into two groups: PTIP/TRR-cobound and PTIP/PC-cobound peaks. In particular, it was found that PTIP mediates the molecular switch between H3K4me3/H3K27ac and H3K27me3 histone modifications at TRR or PC occupied regions. Thus, it is suggested that PTIP is a mediator rather than a dedicated co-activator along PcG and trxG pathways. This hypothesis is further supported by the genetic assay: PTIP interacts genetically with either PcG or TrxG in a dosage-dependent manner, suggesting that PTIP functions as a co-factor of PcG/TrxG proteins. In addition, in accordance with the analysis of ChIP-seq, these genetic interactions correlate with modified ectopic HOX protein levels in imaginal discs, which reveals an essential role for PTIP in PcG-mediated Hox gene repression. Hence, this study revealed a novel role for PTIP in the epigenetic regulation of gene expression along PcG and trxG pathways.
Huang, W., Zhu, J. Y., Fu, Y., van de Leemput, J. and Han, Z. (2022). Lpt, trr, and Hcf regulate histone mono- and dimethylation that are essential for Drosophila heart development. Dev Biol 490: 53-65. PubMed ID: 35853502
Mammalian KMT2C, KMT2D, and HCFC1 are expressed during heart development and have been associated with congenital heart disease, but their roles in heart development remain elusive. This study found that the Drosophila Lpt and trr genes encode the N-terminal and C-terminal homologs, respectively, of mammalian KMT2C or KMT2D. Lpt and trr mutant embryos showed reduced cardiac progenitor cells. Silencing of Lpt, trr, or both simultaneously in the heart led to similar abnormal cardiac morphology, tissue fibrosis, and cardiac functional defects. Like KMT2D, Lpt and trr were found to modulate histone H3K4 mono- and dimethylation, but not trimethylation. Investigation of downstream genes regulated by mouse KMT2D in the heart showed that their fly homologs are similarly regulated by Lpt or trr in the fly heart, suggesting that Lpt and trr regulate an evolutionarily conserved transcriptional network for heart development. Moreover, this study showed that cardiac silencing of Hcf, the fly homolog of mammalian HCFC1, leads to heart defects similar to those observed in Lpt and trr silencing, as well as reduced H3K4 monomethylation. These findings suggest that Lpt and trr function together to execute the conserved function of mammalian KMT2C and KMT2D in histone H3 lysine K4 mono- and dimethylation required for heart development. Possibly aided by Hcf, which plays a related role in H3K4 methylation during fly heart development.
Cheng, Q. and Xie, H. (2022). Genome-wide analysis of bivalent histone modifications during Drosophila embryogenesis. Genesis: e23502. PubMed ID: 36125264
In eukaryotes, histone modifications are key epigenetic regulators that are associated with distinct chromatin features. Bivalent histone modifications describe a situation where a subset of promoters have with both activating (H3K4me3) and repressive (H3K27me3) markers in pluripotent cells (e.g., ESCs). However, it remains to be understood whether bivalent histone modifications are stable throughout developmental stages. In this study, by systematically analyzing ChIP-seq data of H3K4me3 and H3K27me3, the first panoramic view is provided of bivalent histone modifications in Drosophila from embryonic 0-4 to 20-24 hr. In this study, it was found that bivalent histone modifications occur at other locations in the genome in addition to the promoter region. Additionally, the different genomic regions occupied by bivalent histone modifications exhibit spatiotemporal specificity at each stage. Furthermore, gene ontology and motif analysis reflected continuous and gradual changes of target genes during different developmental process. In summary, it is suggested that bivalent histone modifications have potential regulatory functions throughout Drosophila embryonic stage.
Ranjan, R., Snedeker, J., Wooten, M., Chu, C., Bracero, S., Mouton, T. and Chen, X. (2022). Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage. Dev Cell 57(9): 1102-1118.e1107. PubMed ID: 35483360
During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. this study tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. Unique features were found that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination.
Gandara, L., Tsai, A., Ekelof, M., Galupa, R., Preger-Ben Noon, E., Alexandrov, T. and Crocker, J. (2022). Developmental phenomics suggests that H3K4 monomethylation confers multi-level phenotypic robustness. Cell Rep 41(11): 111832. PubMed ID: 36516782
How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. This study genetically reduce H3K4me1 deposition in Drosophila melanogaster and found that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness. Using a developmental phenomics approach, changes were found in morphology, metabolism, behavior, and offspring production. However, many phenotypic changes are only detected when hypomethylated flies develop outside of standard laboratory environments or with specific genetic backgrounds. Therefore, quantitative phenomics measurements can unravel how pleiotropic modulators of gene expression affect developmental robustness under conditions resembling the natural environments of a species.
Shapiro-Kulnane, L., Selengut, M. and Salz, H. K. (2022). Safeguarding Drosophila female germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein. PLoS Genet 18(12): e1010568. PubMed ID: 36548300
H3K9me3-based gene silencing is a conserved strategy for securing cell fate, but the mechanisms controlling lineage-specific installation of this epigenetic mark remain unclear. In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate phf7 transcription. Thus, phf7 regulation in the female germline provides a powerful system to dissect the molecular mechanism underlying H3K9me3 deposition onto protein coding genes. Genetic studies were used to identify the essential cis-regulatory elements, finding that the sequences required for H3K9me3 deposition are conserved across Drosophila species. Transposable elements are also silenced by an H3K9me3-mediated mechanism. But the finding that phf7 regulation does not require the dedicated piRNA pathway components, piwi, aub, rhino, panx, and nxf2, indicates that the mechanisms of H3K9me3 recruitment are distinct. Lastly, it was discovered that an uncharacterized member of the zinc finger associated domain (ZAD) containing C2H2 zinc finger protein family, IDENTITY CRISIS (IDC; CG4936), is necessary for H3K9me3 deposition onto phf7. Loss of idc in germ cells interferes with phf7 transcriptional regulation and H3K9me3 deposition, resulting in ectopic PHF7 protein expression. IDC's role is likely to be direct, as it localizes to a conserved domain within the phf7 gene. Collectively, these findings support a model in which IDC guides sequence-specific establishment of an H3K9me3 mini domain, thereby preventing accidental female-to-male programming.
Jangam, S., Briere, L. C., Jay, K., Andrews, J. C., Walker, M. A., Rodan, L. H., High, F. A., Yamamoto, S., Sweetser, D. A. and Wangler, M. (2023). A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster. medRxiv. PubMed ID: 36778246
EZH1 (Enhancer of Zeste, homolog 1), a Polycomb Repressive Complex-2 (PRC2) component, is involved in a myriad of cellular processes through modifying histone 3 lysine27 (H3K27) residues. EZH1 represses transcription of downstream target genes through H3K27 trimethylation (H3K27me3). Genetic mutations in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. This study reports a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo variant in EZH1, p.Ala678Gly, through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and was the best candidate variant found in the exome. Human EZH1 / 2 are homologous to fly Enhancer of zeste E(z), an essential gene in flies, and the residue (A678 in humans, A691 in Drosophila) is conserved. To further study this variant, Drosophila null alleles were obtained and transgenic flies expressing wild-type (E(z) (WT)) and the variant (E(z) (A691G)) were generated. The E(z) (A691G) variant led to hyper H3K27me3 while the E(z) (WT) did not, suggesting this is as a gain-of-function allele. When expressed under the tubulin promotor in vivo the variant rescued null-lethality similar to wild-type but the E(z) (A691G) flies exhibit bang sensitivity and shortened lifespan. In conclusion, this study presents a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, it was found that this variant has a functional impact in Drosophila. Biochemically this allele leads to increased H3K27me3 suggesting gain-of-function, but when expressed in adult flies the E(z) (A691G) has some characteristics of partial loss-of-function which may suggest it is a more complex allele in vivo.
Ciabrelli, F., Rabbani, L., Cardamone, F., Zenk, F., Loser, E., Schachtle, M. A., Mazina, M., Loubiere, V. and Iovino, N. (2023). CBP and Gcn5 drive zygotic genome activation independently of their catalytic activity. Sci Adv 9(16): eadf2687. PubMed ID: 37083536
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. This study used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. The screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. It was demonstrated that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, these data identify Nejire and Gcn5 as key regulators of ZGA.
McPherson, J. E., Grossmann, L. C., Armstrong, R. L., Kwon, E., Salzler, H. R., Matera, A. G., McKay, D. J. and Duronio, R. J. (2023). Reduced histone gene copy number disrupts Drosophila Polycomb function. bioRxiv. PubMed ID: 37034607
The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. This study demonstrates that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. Two regions of chromosome 3 were identified that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. It was further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is also reduced. It is concluded that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.
Zhao, T., Wang, M., Li, Z., Li, H., Yuan, D., Zhang, X., Guo, M., Qian, W. and Cheng, D. (2023). Wds-Mediated H3K4me3 Modification Regulates Lipid Synthesis and Transport in Drosophila. Int J Mol Sci 24(7). PubMed ID: 37047100
Lipid homeostasis is essential for insect growth and development. The complex of proteins associated with Set 1 (COMPASS)-catalyzed Histone 3 lysine 4 trimethylation (H3K4me3) epigenetically activates gene transcription and is involved in various biological processes, but the role and molecular mechanism of H3K4me3 modification in lipid homeostasis remains largely unknown. The present study showed in Drosophila that fat body-specific knockdown of will die slowly (Wds) as one of the COMPASS complex components caused a decrease in lipid droplet (LD) size and triglyceride (TG) levels. Mechanistically, Wds-mediated H3K4me3 modification in the fat body targeted several lipogenic genes involved in lipid synthesis and the Lpp gene associated with lipid transport to promote their expressions; the transcription factor heat shock factor (Hsf) could interact with Wds to modulate H3K4me3 modification within the promoters of these targets; and fat body-specific knockdown of Hsf phenocopied the effects of Wds knockdown on lipid homeostasis in the fat body. Moreover, fat body-specific knockdown of Wds or Hsf reduced high-fat diet (HFD)-induced oversized LDs and high TG levels. Altogether, this study reveals that Wds-mediated H3K4me3 modification is required for lipid homeostasis during Drosophila development and provides novel insights into the epigenetic regulation of insect lipid metabolism.
Amiad Pavlov, D., Unnikannan, C. P., Lorber, D., Bajpai, G., Olender, T., Stoops, E., Reuveny, A., Safran, S. and Volk, T. (2023). The LINC Complex Inhibits Excessive Chromatin Repression. Cells 12(6). PubMed ID: 36980273
The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. This study has shown that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, it is suggested that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.
Jauregui-Lozano, J., McGovern, S. E., Bakhle, K. M., Hagins, A. C. and Weake, V. M. (2023). Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors. Sci Rep 13(1): 5105. PubMed ID: 36991154
Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. This study profiled chromatin marks associated with active transcription in the aging Drosophila eye to understand how chromatin modulates transcriptional outputs. Both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, no correlation was found with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because aging Drosophila undergo a decrease in visual function, these data suggest that H3K36me3 could play a role in maintaining visual function in the aging eye through regulating alternative splicing.
Salzler, H. R., Vandadi, V., McMichael, B. D., Brown, J. C., Boerma, S. A., Leatham-Jensen, M. P., Adams, K. M., Meers, M. P., Simon, J. M., Duronio, R. J., McKay, D. J. and Matera, A. G. (2023). Distinct roles for canonical and variant histone H3 lysine-36 in Polycomb silencing. Sci Adv 9(9): eadf2451. PubMed ID: 36857457
Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. This study showa that mutants of Drosophila replication-dependent (H3.2(K36R)) or replication-independent (H3.3(K36R)) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3(K36R)H3.2(K36R)) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2(K36R) mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3(K36R) animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. It is concluded that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.
Jin, Z., Yu, B. and Huang, Y. (2023). Structural insights into the chromodomain of Oxpecker in complex with histone H3 lysine 9 trimethylation reveal a transposon silencing mechanism by heterodimerization. Biochem Biophys Res Commun 652: 95-102. PubMed ID: 36841100
Oxpecker, the homolog of Rhino/HP1D, exclusively expressed in Drosophila ovaries, belongs to the Heterochromatin Protein 1 family, as does Rhino. Rhi recognizes piRNA clusters enriched with the heterochromatin marker H3K9me3 via its N-terminal chromodomain and recruits Deadlock via its C-terminal chromoshadow domain, further recruits Moonshiner, a paralog of the TATA box-binding protein-related factor 2 large subunits, to promote transcription of piRNA precursors, thereby protecting the genome. Despite Oxp possessing only the chromodomain, its loss leads to the upregulation of transposons in the female germline. This study ∂solved the crystal structure of the Oxp chromodomain in complex with the histone H3K9me3 peptide. As the Oxp chromodomain dimerizes, two H3K9me3 peptides bind to the Oxp chromodomain in an antiparallel manner. ITC experiments and site-directed mutagenesis experiments showed that E44 determines Oxp's five-fold stronger binding ability to H3K9me3 than that of Rhi. In addition, it was found that Oxp and Rhi can form a heterodimer, which may shed light on the molecular mechanism by which Oxp regulates transposon silencing in the absence of CSD.
Lizana, L., Nahali, N. and Schwartz, Y. B. (2023). Polycomb proteins translate histone methylation to chromatin folding. J Biol Chem 299(9): 105080. PubMed ID: 37499944
Epigenetic repression often involves covalent histone modifications. Yet, how the presence of a histone mark translates into changes in chromatin structure that ultimately benefits the repression is largely unclear. Polycomb group proteins comprise a family of evolutionarily conserved epigenetic repressors. They act as multi-subunit complexes one of which tri-methylates histone H3 at Lysine 27 (H3K27). This study describes a novel Monte Carlo-Molecular Dynamics simulation framework, which we employed to discover that stochastic interaction of Polycomb Repressive Complex 1 (PRC1) with tri-methylated H3K27 that is sufficient to fold the methylated chromatin. Unexpectedly, such chromatin folding leads to spatial clustering of the DNA elements bound by PRC1. Our results provide further insight into mechanisms of epigenetic repression and the process of chromatin folding in response to histone methylation.
Kushwaha, A. and Thakur, M. K. (2023). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Mol Neurobiol. PubMed ID: 37626270
Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It was previously reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. The current study made an attempt to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia.
Tortora, M. M. C., Brennan, L. D., Karpen, G. and Jost, D. (2023). HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation. Proc Natl Acad Sci U S A 120(33): e2211855120. PubMed ID: 37549295
The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Using a minimal theoretical framework, this study systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. The specific affinity of HP1 for H3K9me2/3 loci was shown to facilitate coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.
Yang, J., Tang, R., Chen, S., Chen, Y., Yuan, K., Huang, R., Wang, L. (2023). Exposure to high-sugar diet induces transgenerational changes in sweet sensitivity and feeding behavior via H3K27me3 reprogramming. Elife, 12 PubMed ID: 37698486
Human health is facing a host of new threats linked to unbalanced diets, including high-sugar diet (HSD), which contributes to the development of both metabolic and behavioral disorders. Studies have shown that diet-induced metabolic dysfunctions can be transmitted to multiple generations of offspring and exert long-lasting health burden. Meanwhile, whether and how diet-induced behavioral abnormalities can be transmitted to the offspring remains largely unclear. This study showed that ancestral HSD exposure suppressed sweet sensitivity and feeding behavior in the offspring in Drosophila. These behavioral deficits were transmitted through the maternal germline and companied by the enhancement of H3K27me3 modifications. PCL-PRC2 complex, a major driver of H3K27 trimethylation, was upregulated by ancestral HSD exposure, and disrupting its activity eliminated the transgenerational inheritance of sweet sensitivity and feeding behavior deficits. Elevated H3K27me3 inhibited the expression of a transcriptional factor Cad and suppressed sweet sensitivity of the sweet-sensing gustatory neurons, reshaping the sweet perception and feeding behavior of the offspring. Taken together, this study uncovered a novel molecular mechanism underlying behavioral abnormalities spanning multiple generations of offspring upon ancestral HSD exposure, which would contribute to the further understanding of long-term health risk of unbalanced diet.
Sarkar, K., Kotb, N. M., Lemus, A., Martin, E. T., McCarthy, A., Camacho, J., Iqbal, A., Valm, A. M., Sammons, M. A., Rangan, P. (2023). A feedback loop between heterochromatin and the nucleopore complex controls germ-cell-to-oocyte transition during Drosophila oogenesise. Dev Cell 58(22):2580-2596 PubMed ID: 37673064
Germ cells differentiate into oocytes that launch the next generation upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrently with oocyte specification. This study discovered that nuclear SETDB1 is required for silencing a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for upregulating 18 of the ∼30 nucleoporins (Nups) that compose the nucleopore complex (NPC), promoting NPC formation. NPCs anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to the loss of SETDB1 or Nups results in the loss of oocyte identity, cell death, and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification, which is critical for establishing oocyte identity.
Zhu, J. Y., Liu, C., Huang, X., van de Leemput, J., Lee, H. and Han, Z. (2023). H3K36 Di-Methylation Marks, Mediated by Ash1 in Complex with Caf1-55 and MRG15, Are Required during Drosophila Heart Development. J Cardiovasc Dev Dis 10(7). PubMed ID: 37504562
Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. This study used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.
Zion, E. H., Ringwalt, D., Rinaldi, K., Kahney, E. W., Li, Y. and Chen, X. (2023). Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions. EMBO Rep 24(7): e56404. PubMed ID: 37255015
This study reports that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, a mutant H3T3A was expressed to compromise asymmetric histone inheritance. Under this condition, an increase was observed in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, these results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells.
Cai, Y., Cui, T., Yin, P., Paganelli, P., Vicini, S., Wang, T. (2023). Dysregulated glial genes in Alzheimer's disease are essential for homeostatic plasticity: Evidence from integrative epigenetic and single cell analyses. Aging Cell, 22(11):e13989 PubMed ID: 37712202
Synaptic homeostatic plasticity is a foundational regulatory mechanism that maintains the stability of synaptic and neural functions within the nervous system. Impairment of homeostatic regulation has been linked to synapse destabilization during the progression of Alzheimer's disease (AD). Various glial cell types play critical roles in modulating synaptic functions both during the aging process and in the context of AD. This study investigated the impact of glial dysregulation of histone acetylation and transcriptome in AD on synaptic homeostatic plasticity, using computational analysis combined with electrophysiological methods in Drosophila. By integrating snRNA-seq and H3K9ac ChIP-seq data from the same AD patient cohort, this study pinpointed cell type-specific signature genes that were transcriptionally altered by histone acetylation. The role of these glial genes in regulating presynaptic homeostatic potentiation was subsequently investigated in Drosophila. Remarkably, nine glial-specific genes, which were identified through computational methods as targets of H3K9ac and transcriptional dysregulation, were found to be crucial for the regulation of synaptic homeostatic plasticity at the NMJ in Drosophila. This genetic evidence connects abnormal glial transcriptomic changes in AD with the impairment of homeostatic plasticity in the nervous system. In summary, these integrative computational and genetic studies highlight specific glial genes as potential key players in the homeostatic imbalance observed in AD.
Fablet, M., Salces-Ortiz, J., Jacquet, A., Menezes, B. F., Dechaud, C., Veber, P., Rebollo, R., Vieira, C. (2023). A Quantitative, Genome-Wide Analysis in Drosophila Reveals Transposable Elements' Influence on Gene Expression is Species-Specific. Genome biology and evolution, 15(9) PubMed ID: 37652057
Transposable elements (TEs) are parasite DNA sequences that are able to move and multiply along the chromosomes of all genomes. They can be controlled by the host through the targeting of silencing epigenetic marks, which may affect the chromatin structure of neighboring sequences, including genes. This study used transcriptomic and epigenomic high-throughput data produced from ovarian samples of several Drosophila melanogaster and Drosophila simulans wild-type strains, in order to finely quantify the influence of TE insertions on gene RNA levels and histone marks (H3K9me3 and H3K4me3). Thw results reveal a stronger epigenetic effect of TEs on ortholog genes in D. simulans compared with D. melanogaster. At the same time, a larger contribution of TEs to gene H3K9me3 variance within genomes in D. melanogaster, which is evidenced by a stronger correlation of TE numbers around genes with the levels of this chromatin mark in D. melanogaster. Overall, this work contributes to the understanding of species-specific influence of TEs within genomes. It provides a new light on the considerable natural variability provided by TEs, which may be associated with contrasted adaptive and evolutionary potentials.
Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology 61(1):487-497 PubMed ID: 37626270
Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. The current study made an attempt to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia.
DiVito Evans, A., Fairbanks, R. A., Schmidt, P., Levine, M. T. (2023). Histone methylation regulates reproductive diapause in Drosophila melanogaster. PLoS Genet, 19(9):e1010906 PubMed ID: 37703303
Fluctuating environments threaten fertility and viability. To better match the immediate, local environment, many organisms adopt alternative phenotypic states, a phenomenon called "phenotypic plasticity." Natural populations that predictably encounter fluctuating environments tend to be more plastic than conspecific populations that encounter a constant environment, suggesting that phenotypic plasticity can be adaptive. Despite pervasive evidence of such "adaptive phenotypic plasticity," gene regulatory mechanisms underlying plasticity remains poorly understood. This study tested the hypothesis that environment-dependent phenotypic plasticity is mediated by epigenetic factors. To test this hypothesis, the adaptive reproductive arrest of Drosophila melanogaster females, called diapause, was exploited. Using an inbred line from a natural population with high diapause plasticity, it was demonstrated that diapause is determined epigenetically: only a subset of genetically identical individuals enter diapause and this diapause plasticity is epigenetically transmitted for at least three generations. Upon screening a suite of epigenetic marks, it was discovered that the active histone marks H3K4me3 and H3K36me1 are depleted in diapausing ovaries. Using ovary-specific knockdown of histone mark writers and erasers, it was demonstrated that H3K4me3 and H3K36me1 depletion promotes diapause. Given that diapause is highly polygenic, that is, distinct suites of alleles mediate diapause plasticity across distinct genotypes, the potential for genetic variation in diapause-determining epigenetic marks was also investigated. Specifically, it was asked if these histone marks were similarly depleted in diapause of a genotypically distinct line. Evidence was found of divergence in both the gene expression program and histone mark abundance. This study reveals chromatin determinants of phenotypic plasticity and suggests that these determinants may be genotype-dependent, offering new insight into how organisms may exploit and evolve epigenetic mechanisms to persist in fluctuating environments.
Godneeva, B., Ninova, M., Fejes Toth, K., Aravin, A. A. (2023). SUMOylation of Bonus, the Drosophila homolog of Transcription Intermediary Factor 1, safeguards germline identity by recruiting repressive chromatin complexes to silence tissue-specific genes. bioRxiv, PubMed ID: 37645991
The conserved family of Transcription Intermediary Factors (TIF1) proteins consists of key transcriptional regulators that control transcription of target genes by modulating chromatin state. Unlike mammals that have four TIF1 members, Drosophila only encodes one member of the family, Bonus. Bonus has been implicated in embryonic development and organogenesis and shown to regulate several signaling pathways, however, its targets and mechanism of action remained poorly understood. This study found that knockdown of Bonus in early oogenesis results in severe defects in ovarian development and in ectopic expression of genes that are normally repressed in the germline, demonstrating its essential function in the ovary. Recruitment of Bonus to chromatin leads to silencing associated with accumulation of the repressive H3K9me3 mark. Bonus associates with the histone methyltransferase SetDB1 and the chromatin remodeler NuRD and depletion of either component releases Bonus-induced repression. This study further established that Bonus is SUMOylated at a single site at its N-terminus that is conserved among insects and this modification is indispensable for Bonus's repressive activity. SUMOylation influences Bonus's subnuclear localization, its association with chromatin and interaction with SetDB1. Finally, Bonus SUMOylation was shown to be mediated by the SUMO E3-ligase Su(var)2-10, revealing that although SUMOylation of TIF1 proteins is conserved between insects and mammals, both the mechanism and specific site of modification is different in the two taxa. Together, this work identified Bonus as a regulator of tissue-specific gene expression and revealed the importance of SUMOylation as a regulator of complex formation in the context of transcriptional repression.
Soukar, I., Mitra, A., Pile, L. A. (2023). Analysis of the chromatin landscape and RNA polymerase II binding at SIN3-regulated genes. Biol Open, 12(11) PubMed ID: 37850739
The chromatin environment has a significant impact on gene expression. Chromatin structure is highly regulated by histone modifications and RNA polymerase II binding dynamics. The SIN3 histone modifying complex regulates the chromatin environment leading to changes in gene expression. In Drosophila melanogaster, the Sin3A gene is alternatively spliced to produce different protein isoforms, two of which include SIN3 220 and SIN3 187. Both SIN3 isoforms are scaffolding proteins that interact with several other factors to regulate the chromatin landscape. The mechanism through which the SIN3 isoforms regulate chromatin is not well understood. This study analyze publicly available data sets to facilitate specific questions on how SIN3 isoforms regulate chromatin and gene activity. It was determined that genes repressed by the SIN3 isoforms exhibited enrichment in histone H3K4me2, H3K4me3, H3K14ac and H3K27ac near the transcription start site. An increase was observed in the amount of paused RNA polymerase II on the promoter of genes repressed by the isoforms as compared to genes that require SIN3 for maximum activation. Furthermore, a subset of genes was analyzed regulated by SIN3 187 that suggest a mechanism in which SIN3 187 might exhibit hard regulation as well as soft regulation. Data presented in this study expand knowledge of how the SIN3 isoforms regulate the chromatin environment and RNA polymerase II binding dynamics.
Nil, Z., Deshwar, A. R., Huang, Y., ..., Mao, X., Wegner, D. J., Sisco, K., Shinawi, M., Wangler, M. F., Weksberg, R., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. These results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. Histone lysine methylation occurs on lysines 4, 9, 27, 36, and 79 of Histone H3 and on lysine 20 of Histone H4. Biochemical and genetic studies indicate that methylation of different lysine residues, with the exception of H3-K79, is catalyzed by different SET domain-containing proteins. Histone H3 methylated at lysine 9 (H3-mLys9) is characteristic of the heterochromatic (genetically silent) state. Immunofluorescent staining of Drosophila polytene chromosomes shows that the bulk of the H3-mLys9 is present in the pericentric heterochromatin and in a banded pattern on the fourth chromosome, known sites of repetitive DNA. Similarly, chromatin immunoprecipitation experiments demonstrate that H3-mLys9 is a prominent component of the silent mating type locus in fission yeast (Schizosaccharomyces pombe), while essentially absent from flanking regions containing inducible genes. Methylation of histone H3-Lys9 has also been associated with the silencing of euchromatic genes (Richards, 2002 and references therein).

A key role for gene silencing and specification of heterochromatin is shown by the demonstration that mammalian homologs of Drosophila Su(var)3-9, including human SUV39H1 and murine Suv39h1, encode enzymes that specifically methylate histone H3 on lysine 9. Su(var)3-9 was originally identified as a suppressor of PEV in Drosophila, indicating that the wild-type gene product is involved in heterochromatin formation. A homolog in S. pombe, Clr4, is also a specific histone H3-Lys9 methyltransferase, suggesting that this activity is widely distributed and well conserved. clr4 mutants exhibit reduced heterochromatin formation at centromeres, with elevated mitotic chromosome loss and reduced silencing within both pericentromeric heterochromatin and the silent mating type locus. Similarly, mammalian Su(var)3-9-like proteins have been implicated in both centromere activity and gene silencing. Disruption of the murine Suv39h1 and Suv39h2 paralogs causes genome instability, chromosome missegregation, and male meiotic defects (Richards, 2002 and references therein).

Drosophila Polycomb Group (PcG) complexes are responsible for the maintenance of the repressed state of genes subject to their control. The best-known PcG targets are the homeotic genes, which are activated in the early embryo by the products of segmentation genes. At this stage, transient, localized activators and repressors determine the segmental domains of expression of each homeotic gene but, after gastrulation, epigenetic (genetically based non-heritable) mechanisms take over to maintain the segmental pattern of expression for the rest of development. These mechanisms are mediated by the Polycomb Response Elements (PREs), regulatory regions of several hundred base pairs, where two kinds of chromatin complexes are assembled. One kind, the PcG complexes, is repressive and can maintain a silent state. The other kind involves the Trithorax protein (Trx) and mediates the persistence of the active state. Which of the two predominates depends on the state of activity of the target promoter at the blastoderm stage of development. If the genes had been repressed by early regulators, the PcG silencing mechanisms maintain the repressed state throughout the rest of development. If the gene was active in the early embryo, the Trx function stimulates its expression and prevents later silencing by the PcG complexes. The gene remains then unrepressed and potentially active for the rest of development. Thus, early transcriptional activity of a target gene sets a mark that maintains transcriptional competence and prevents the establishment of PcG silencing, while early repression results in an antagonistic mark that ensures the maintenance of the silenced state in subsequent cell cycles (Czermin, 2002 and references therein).

Formally, therefore, the PRE mediates both the memory of the silent state and the memory of the derepressed state. The repressive memory is illustrated by the fact that, although PcG proteins are nearly ubiquitous, at every cell division they restore the repressed chromatin state only in cells in which their target genes had been previously repressed. The memory of the derepressed state is shown by the fact that if the target gene is active in the early embryo, or if derepression is forced by massive doses of activator, the derepressed state is inherited by the progeny cells. This memory is affected by trx mutations. In the absence of trx function, cells in which a PRE-containing construct had been activated in the early embryo may lose the derepressed state and become silenced again (Czermin, 2002 and references therein).

In the preblastoderm embryo, Polycomb complexes are assembled at the PREs, which contain consensus sequences for DNA binding proteins such as GAGA factor and Pleiohomeotic (Pho), the fly homolog of the mammalian YY1 factor. These, together with other, unidentified DNA binding proteins, recruit cooperatively a PcG complex that includes Pc, Ph, and GAGA factor but also Esc, E(z), Pho, and Rpd3. The E(z)/Esc/Pho complex dissociates from the Pc-containing complex after the blastoderm stage, and Esc ceases to be produced by the end of embryogenesis. However, the E(z) protein continues to be needed, at least intermittently, to maintain the silent state and is most likely recruited to the PRE by the Pho DNA binding protein. Experiments with Pc targeted to a reporter gene by the LexA DNA binding domain show that, while it can recruit the Esc/E(z) component to establish silencing in the early embryo, it can no longer recruit E(z) at later stages, when the early complex has dissociated. LexA-Pc repression continues in the embryo but the memory of the repressed state is then lost and the reporter gene becomes derepressed during larval stages. These results suggest that E(z) might mediate the creation of a chromatin mark necessary for repression and responsible for maintaining the memory of the silent state (Czermin, 2002 and references therein).

Trx and E(z) are therefore good candidates for the functions required for the positive and negative memories, respectively. Structurally, these two proteins share with Su(var)3-9 the SET domain, named after the three founding members Su(var)3-9, E(z), and Trx. Advances in the past years have shown that the SET domain in many proteins is responsible for a histone H3 methyltransferase (MTase) activity. With one exception, all reported histone MTases that methylate lysine residues contain a SET domain, which harbors the amino acids important for MTase function. Su(var)3-9 and its homologs, in particular, are necessary for the formation of heterochromatic complexes in mammals, flies, and fission yeast. Their activities methylate lysine 9 of histone H3, which becomes a binding site for the chromodomain of heterochromatin proteins such as Hp1 (Czermin, 2002 and references therein).

It was reasoned that Trithorax and Enhancer of zeste might reside in complexes that possess MTase activities. Pc contains a chromodomain whose structure and essential residues are homologous to those found in Hp1 and related methyl lysine binding proteins and might therefore recognize a nucleosomal methylation mark. Both Trx and E(z) complexes have now been shown to contain an H3 MTase activity. To study the Esc/E(z) complex, Drosophila nuclear extract was fractionated and it was asked if a MTase activity copurifies with E(z) and Esc. A complex containing E(z) and Esc trimethylates lysine 9 and methylates lysine 27 of histone H3 and the trimethylated lysine 9 mark is closely correlated with PcG binding sites on polytene chromosomes. The conjecture that the Esc/E(z) complex contains a histone MTase activity has been confirmed by the finding that the complex immunoprecipitated by anti-Esc or purified biochemically methylates in vitro histone H3 whether assembled in a nucleosome, as a free histone, or in the form of oligopeptides. The purified complex contains several components as predicted from previous studies: in addition to E(z), Su(z)12, Esc, p55, and Rpd3 are found. An additional component of approximately 168 kDa remains to be identified (Czermin, 2002).

The activity of the Esc/E(z) complex leads to trimethylation of lysine 9 and probably also of lysine 27 of H3. In vivo, an antibody directed against dimethylated H3 lysine (9me2K9 antibody) does not detectably stain chromosomal PcG sites while a me3K9 antibody directed against trimethylated H3 lysine decorates all chromosomal PcG sites. Whether three methyl groups are added processively or by independent events and whether lysine 9 and lysine 27 are targeted simultaneously remains to be elucidated. The partial ability of the complex to methylate the peptide acetylated at lysine 9 is accounted for by the presence of the Rpd3 deacetylase (Czermin, 2002).

The Su(var)3-9 product has been reported to dimethylate H3 lysine 9, constituting a mark for heterochromatic complexes (Rea, 2000; Lachner, 2001). However, antibody staining of polytene chromosomes detects abundant trimethyl lysine 9 in heterochromatin as well as most of chromosome 4, sites that do not contain PcG complexes. At least some of the heterochromatic me3K9 is lost in Su(var)3-9 mutants. A participation of E(z) in heterochromatic H3 methylation cannot be excluded. This function would be consistent with the report that E(z) mutations are also suppressors of heterochromatic position-effect variegation. However, a more distinct difference between the Su(var)3-9 mark and the E(z) mark is the methylation of H3 K27. In vitro, Su(var)3-9 does not methylate the K27 peptide. It will be important to test whether K27 methylation is present in heterochromatin, but it is likely that K27 methylation differentiates heterochromatic from PcG sites (Czermin, 2002).

Suvar3-9 methylation of lysine 9 of histone H3 is thought to stabilize or even target Hp1-containing heterochromatic complexes through binding of the Hp1 chromodomain to the methylated lysine 9 (Bannister, 2001; Lachner, 2001). The structure of the Hp1 chromodomain bound to histone H3 di- or tri-methylated at lysine 9 shows that either peptide fits in a groove and lodges the methyllysine in a hydrophobic pocket (Nielsen, 2002; Jacobs, 2002). This was confirmed by peptide binding experiments and makes it unlikely that H3 K9 methylation would be sufficient to discriminate between the heterochromatic methylation imprint and the PcG methylation imprint. The parallelism suggests that the chromodomain of Pc would recognize trimethyl lysine 9 H3. Pc has been shown to bind to histone H3 and to nucleosomes in vitro; however, the domain involved does not appear to be the chromodomain but the C-terminal region (Breiling, 1999). Binding experiments detected little increased affinity of Pc for the trimethyl K9 peptide compared to the unmethylated peptide. Instead, methyl K27 appears to make the major contribution to Pc affinity for methylated H3. The amino acid context of K27 (KAARKS) resembles that of K9 (QTARKS) but Hp1 binds weakly to a methylated K27 peptide (Nielsen, 2002; Jacobs, 2002). It remains to be seen which domain of Pc interacts with methylated H3 and whether Hp1 can bind to meK9 meK27 H3. Nevertheless, the presence of me3K9 at PcG sites, as well as in heterochromatin and chromosome 4, suggests that the meK27 or other factors must contribute to discriminate between heterochromatic and PcG sites (Czermin, 2002).

Specific recognition of the methylated histone H3 by Polycomb complexes might be facilitated by other PcG components or by other modifications of the histones. E(z)-dependent methylation might contribute to the stability of the PcG complex, particularly in the early stages of assembly at the PRE, for example, by permitting complex formation to spread to neighboring sequences for a distance of 2- 3 kb. However, the fact that in the E(z)S2 mutant chromosomes the trimethylation mark is lost well before the binding of PcG proteins indicates that methylation is not essential for the binding of the complex. Alternatively, the trimethyl mark might signify the difference between the mere recruitment of a PcG complex and its repressive function. Chromatin immunoprecipitation shows that PcG complexes are present at some PREs whether or not the corresponding gene is repressed, implying that, while recruitment of the complexes may be constitutive, the decision to repress or not depends on other features transmitted epigenetically. The methylation might then constitute the epigenetic mark triggering the silent state. It is interesting to note, therefore, that two polytenic sites that are strongly stained with anti-Psc antibody but very weakly with anti-me3K9 antibody are 2D and 49F, respectively, the sites of the PcG genes ph and Psc. These PcG genes are downregulated but not silenced by PcG mechanisms. If trimethylation of H3 lysine 9 signals strong silencing, these sites might be expected to be occupied by PcG complexes but only partly repressed (Czermin, 2002).

A role of H3 methylation in the assembly of stable PcG complexes is suggested by another observation. When the chromodomain in Hp1 is substituted by the chromodomain of Pc, the chimeric Hp1 is recruited to PcG binding sites on polytene chromosomes. This implies either that the chromodomain is sufficient to recognize and bind to the meK9 meK27 H3 or that the Pc chromodomain specifies critical interactions with other PcG components that are recruited to PcG sites. That the latter interpretation is correct is shown by the fact that the chimeric Hp1 also recruits PcG proteins to heterochromatin, where they are not normally found, and that this recruitment is dependent on E(z) function. Strong staining is observed with the anti-me3K9 antibody in the chromocenter and chromosome 4, implying that me3K9 H3 is widespread in heterochromatin. If this is, in fact, due to E(z) activity, it would explain the intervention of E(z) in the heterochromatic recruitment of PcG proteins mediated by the chimeric Hp1 (Czermin, 2002).

It would be important, therefore, to determine whether E(z) contributes to heterochromatic me3K9. Anti-E(z) antibody does not stain the chromocenter of salivary polytenic chromosomes. In E(z)S2 mutant larvae raised at nonpermissive temperature, me3K9 staining is still seen in heterochromatin although with very variable intensity. One possible explanation for these two observations is that since heterochromatin is very little replicated in polytene chromosomes, the methylated H3 produced before the temperature shift might perdure a long time at nonpermissive temperature (Czermin, 2002).

It is interesting to note that anti-me3K9 staining at telomeres is not affected in Su(var)3-9 null polytene chromosomes but is lost in the absence of E(z) function. Telomeres, particularly those of chromosome 2R and 2L, stain prominently with antibodies against PcG proteins but also against Hp1. A critical role has been attributed to telomeric Hp1 in 'capping' chromosome termini and preventing telomeric fusions, failure to segregate chromosomes, and chromosome breakage. The fact that Drosophila Su(var)3-9 null mutants are perfectly viable implies that this role of Hp1 is not dependent on Su(var)3-9. In contrast, embryos lacking both maternal and zygotic E(z) function are reported to have mitotic phenotypes similar to those attributed to Hp1 mutants. It is likely, therefore, that the recruitment of Hp1 to telomeric sites depends at least in part on E(z) and not on Su(var)3-9. Unfortunately, the evidence obtained with the E(z)S2 mutant is inconclusive (Czermin, 2002).

It is concluded that a combination of histone methylation marks could be a major factor in the establishment of stable patterns of homeotic gene expression and constitutes the molecular basis of a cellular memory system. The analysis of the existing methylation patterns in cells expressing different homeotic genes by chromatin immunoprecipitation will give further insight into how the MTases such as E(z) are targeted to different sites and how they are regulated at different developmental stages (Czermin, 2002).

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).

The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).

The ability of E(Z) overexpression to suppress the small rough eye phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007b; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).

This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro. The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest that CBP is the major H3K27 acetylase in Drosophila. The conservation of H3K27 acetylation by human p300, together with the reported association of CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).

The genome-wide distribution of H3K27ac, as estimated from human ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests that H3K27ac is much more widely distributed than just at Polycomb target genes, which are estimated to number several thousand in mammalian cells and hundreds in Drosophila. Although these numbers could grow with the identification of additional Polycomb-silenced genes in additional cell types, the recently reported strong correlation of H3K27ac with active genes suggests that it plays an additional role(s) in promoting the transcription of active genes, including those that are never targets of Polycomb silencing. (Note that the H3K27ac at non-Polycomb target genes will not be directly affected by global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on the transcribed regions of Polycomb target genes, which might reflect a role for H3K27ac in facilitating transcriptional elongation, and, conversely, a role for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for recruitment of other proteins with additional enzyme activities that alter local chromatin structure further to facilitate or promote transcription. Prime candidates are those containing a bromodomain, a conserved acetyl-lysine-binding module present in several dozen chromatin-associated proteins, including a number of TrxG proteins that also antagonize Polycomb silencing (Tie, 2009).

The results presented in this study provide new insight into how TRX and CBP function together to antagonize Polycomb silencing. Robust H3K27 acetylation by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the anti-silencing activity of TRX. Consistent with this, preliminary genetic evidence suggests that the Polycomb phenotypes caused by TRX overexpression are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).

The physical association of TRX and CBP and the widespread coincidence of H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the two modifications might be coordinately executed by TRX and CBP. However, the results also raise the possibility that H3K4 trimethylation by TRX itself might be less important for antagonizing Polycomb silencing than H3K27 acetylation. This possibility is also suggested by the discovery of Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks (both H3K4me3 and H3K27me3) in their promoter regions (although the H3K4me3 levels at these inactive genes are typically lower, on average, than they are at active genes, hinting at the possible importance of quantitative effects of the two marks) (Tie, 2009).

A speculative model is proposed for the regulation of Polycomb silencing that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation in mammals) requires RPD3 (and possibly other histone deacetylases) to deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain silencing through 'downstream' events, including those promoted by the H2AK119 mono-ubiquitylation mediated by its RING subunit. Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27 acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as suggested by the observation that H3K27me3 levels are significantly increased on salivary gland polytene chromosomes from trx, ash1 and kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for developmentally regulated reversal of Polycomb silencing at the onset of differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as a signal for recruitment of other TrxG proteins with additional chromatin-modifying activities that may protect the H3K27ac modification and also alter local chromatin structure to promote transcription and further inhibit Polycomb silencing (Tie, 2009).

Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).

This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).

Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).

In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok1 and enok2 ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).

Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300 . However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).

The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).

In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).

Histone H3 Serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).

This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).

Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).

Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).

The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).

Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).

The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells

The preservation of germ cell sexual identity is essential for gametogenesis. This study shows that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes (Smolko, 2018).

In metazoans, germ cell development begins early in embryogenesis when the primordial germ cells are specified as distinct from somatic cells. Specified primordial germ cells then migrate into the embryonic gonad, where they begin to exhibit sex-specific division rates and gene expression programs, ultimately leading to meiosis and differentiation into either eggs or sperm. Defects in sex-specific programming interferes with germ cell differentiation leading to infertility and germ cell tumors. Successful reproduction, therefore, depends on the capacity of germ cells to maintain their sexual identity in the form of sex-specific regulation of gene expression (Smolko, 2018).

In Drosophila melanogaster, germ cell sexual identity is specified in embryogenesis by the sex of the developing somatic gonad. However, extrinsic control is lost after embryogenesis and sexual identity is preserved by a cell-intrinsic mechanism. The Sex-lethal (Sxl) female-specific RNA binding protein is an integral component of the cell-intrinsic mechanism, as loss of Sxl specifically in germ cells leads to a global upregulation of spermatogenesis genes and a germ cell tumor phenotype. Remarkably, sex-inappropriate transcription of a single gene, PHD finger protein 7 (phf7), a key regulator of male identity, is largely responsible for the tumor phenotype. Depletion of phf7 in mutants lacking germline Sxl suppresses the tumor phenotype and restores oogenesis. Moreover, forcing PHF7 protein expression in ovarian germ cells is sufficient to disrupt female fate and give rise to a germ cell tumor. Interestingly, sex-specific regulation of phf7 is achieved by a mechanism that relies primarily on alternative promoter choice and transcription start site (TSS) selection. Sex-specific transcription produces mRNA isoforms with different 5' untranslated regions that affect translation efficiency, such that PHF7 protein is only detectable in the male germline. Although the Sxl protein is known to control expression post-transcriptionally in other contexts the observation that germ cells lacking Sxl protein show defects in phf7 transcription argues that Sxl is likely to indirectly control phf7 promoter choice. Thus, how this sex-specific gene expression program is stably maintained remains to be determined (Smolko, 2018).

This study reports the discovery that female germ cell fate is maintained by an epigenetic regulatory pathway in which SETDB1 (aka EGGLESS, KMT1E, and ESET) is the required chromatin writer and phf7 is one of the critical SETDB1 target genes. SETDB1 trimethylates H3K9 (H3K9me3), a feature of heterochromatin. Using tissue-specific knockdown approaches this study established that germ cell specific loss of SETDB1, its protein partner WINDEI [WDE, aka ATF7IP, MCAF1 and hAM10], and the H3K9me3 reader, HP1a, encoded by the Su(var)205 locus, leads to ectopic expression of euchromatic protein-encoding genes, many of which are normally expressed only in testis. It was further found that H3K9me3 repressive marks accumulate in a SETDB1 dependent manner at 21 of these ectopically expressed genes, including phf7. Remarkably, SETDB1 dependent H3K9me3 deposition is highly localized and does not spread into neighboring loci. Regional deposition is especially striking at the phf7 locus, where H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific TSS. Lastly, this study found that H3K9me3 accumulation at many of these genes, including phf7, is dependent on Sxl. Collectively these findings support a model in which female fate is preserved by deposition of H3K9me3 repressive marks on key spermatogenesis genes (Smolko, 2018).

This study reveals a role for H3K9me3 chromatin, operationally defined as facultative heterochromatin, in securing female identity by silencing lineage-inappropriate transcription. H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, its binding partner WDE, and the H3K9 binding protein HP1a, are required for silencing testis gene transcription in female germ cells. These studies further suggest a mechanism in which SETDB1, in conjunction with the female fate determinant Sxl, controls transcription through deposition of highly localized H3K9me3 islands on a select subset of these genes. The male germ cell sexual identity gene phf7 is one of the key downstream SETDB1 target genes. H3K9me3 deposition on the region surrounding the testis-specific TSS guaranties that no PHF7 protein is produced in female germ cells. In this model, failure to establish silencing leads to ectopic PHF7 protein expression, which in turn drives aberrant expression of testis genes and a tumor phenotype (Smolko, 2018).

Prior studies have established a role for SETDB1 in germline Piwi-interacting small RNA (piRNA) biogenesis and TE silencing. However, piRNAs are unlikely to contribute to sexual identity maintenance as mutations that specifically interfere with piRNA production, such as rhino, do not cause defects in germ cell differentiation. These findings, together with the observation that rhino does not control sex-specific phf7 transcription, suggests that the means by which SETDB1 methylates chromatin at testis genes is likely to be mechanistically different from what has been described for piRNA-guided H3K9me3 deposition on TEs.

The accumulation of H3K9me3 at many of these genes, including phf7, is dependent on the presence of Sxl protein. Thus, these studies suggest that Sxl is required for female-specific SETDB1 function. Sxl encodes an RNA binding protein known to regulate its target genes at the posttranscriptional levels. Sxl control may therefore be indirect. However, studies in mammalian cells suggest that proteins with RNA binding motifs are important for H3K9me3 repression, raising the tantalizing possibility that Sxl might play a more direct role in governing testis gene silencing. Further studies will be necessary to clarify how the sex determination pathway feeds into the heterochromatin pathway (Smolko, 2018).

phf7 stands out among the cohort of genes regulated by facultative heterochromatin because of its pivotal role in controlling germ cell sexual identity. Because ectopic protein expression is sufficient to disrupt female fate, tight control of phf7 expression is essential. phf7 regulation is complex, employing a mechanism that includes alternative promoter usage and TSS selection. This study reports that SETDB1/H3K9me3 plays a critical role in controlling phf7 transcription. In female germ cells, H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific transcription start site. Dissolution of the H3K9me3 marks via loss of Sxl or SETDB1 protein is correlated with transcription from the upstream testis-specific site and ectopic protein expression, demonstrating the functional importance of this histone modification. Together, these studies suggest that maintaining the testis phf7 promoter region in an inaccessible state is integral to securing female germ cell fate (Smolko, 2018).

Although the loss of H3K9me3 pathway members in female germ cells leads to the ectopic, lineage-inappropriate transcription of hundreds of genes, integrative analysis identified only 21 SETDB1/H3K9me3 regulated genes. Given that one of these genes is phf7 and that ectopic PHF7 is sufficient to destabilize female fate, it is likely that inappropriate activation of a substantial number of testis genes is a direct consequence of ectopic PHF7 protein expression. How PHF7 is able to promote testis gene transcription is not yet clear. PHF7 is a PHD-finger protein that preferentially binds to H3K4me2, a mark associated with poised, but inactive genes and linked to epigenetic memory. Thus, one simple model is that ectopic PHF7 binds to H3K4me2 marked testis genes to tag them for transcriptional activation (Smolko, 2018).

It will be interesting to explore whether any of the other 20 SETDB1/H3K9me3 regulated genes also have reprogramming activity. In fact, ectopic fate-changing activity has already been described for the homeobox transcription factor Lim1 in the eye-antenna imaginal disc. However, whether Lim1 has a similar function in germ cells is not yet known. Intriguingly, protein prediction programs identify three of the uncharacterized testis-specific genes as E3 ligases. SkpE is a member of the SKP1 gene family, which are components of the Skp1-Cullin-F-box type ubiquitin ligase. CG12477 is a RING finger domain protein, most of which are believed to have ubiquitin E3 ligases activity. CG42299 is closely related to the human small ubiquitin-like modifier (SUMO) E3 ligase NSMCE2. Given studies that have linked E3 ligases to the regulation of chromatin remodeling, it is tempting to speculate that ectopic expression of one or more of these E3 ligases will be sufficient to alter cell fate. Future studies focused on this diverse group of SETDB1/H3K9me3 regulated genes and their role in reprogramming may reveal the multiple layers of regulation required to secure cell fate (Smolko, 2018).

The SETDB1-mediated mechanism for maintaining sexual identity uncovered in this study may not be restricted to germ cells. Recent studies have established that the preservation of sexual identity is essential in the adult somatic gut and gonadal cells for tissue homeostasis. Furthermore, the finding that loss of HP1a in adult neurons leads to masculinization of the neural circuitry and male specific behaviors suggests a connection between female identity maintenance and H3K9me3 chromatin. Thus, it is speculated that SETDB1 is more broadly involved in maintaining female identity (Smolko, 2018).

These studies highlight an emerging role for H3K9me3 chromatin in cell fate maintenance. In the fission yeast S. pombe, discrete facultative heterochromatin islands assemble at meiotic genes that are maintained in a silent state during vegetative growth. Although less well understood, examples in mammalian cells indicate a role for SETDB1 in lineage-specific gene silencing. Thus, silencing by SETDB1 controlled H3K9 methylation may be a widespread strategy for securing cell fate. Interestingly, H3K9me3 chromatin impedes the reprogramming of somatic cells into pluripotent stem cells (iPSCs). Conversion efficiency is improved by depletion of SETDB1. If erasure of H3K9me3 via depletion of SETDB1 alters the sexually dimorphic gene expression profile in reprogrammed cells, as it does in Drosophila germ cells, the resulting gene expression differences may cause stem cell dysfunction, limiting their therapeutic utility (Smolko, 2018).

Probing the function of metazoan histones with a systematic library of H3 and H4 mutants

Replication-dependent histone genes often reside in tandemly arrayed gene clusters, hindering systematic loss-of-function analyses. This study used CRISPR/Cas9 and the attP/attB double-integration system to alter numbers and sequences of histone genes in their original genomic context in Drosophila melanogaster. As few as 8 copies of the histone gene unit supported embryo development and adult viability, whereas flies with 20 copies were indistinguishable from wild-types. By hierarchical assembly, 40 alanine-substitution mutations (covering all known modified residues in histones H3 and H4) were introduced and characterized. Mutations at multiple residues compromised viability, fertility, and DNA-damage responses. In particular, H4K16 was necessary for expression of male X-linked genes, male viability, and maintenance of ovarian germline stem cells, whereas H3K27 was essential for late embryogenesis. Simplified mosaic analysis showed that H3R26 is required for H3K27 trimethylation. This study has developed a powerful strategy and valuable reagents to systematically probe histone functions in D. melanogaster (Zhang, 2018).

Chromatin is essential for genome packaging and regulation. The basic unit of chromatin is the nucleosome, consisting of 147 base pairs of DNA wrapped around a histone octamer comprising two copies each of histone proteins H3, H4, H2A, and H2B. A fifth 'linker histone,' H1, dynamically binds DNA residing between histone octamers at a subset of nucleosomes. Histones do not merely provide a binding platform for DNA; they also actively participate in DNA-related processes, such as transcription. One mechanism for histones to carry out these functions is though post-translational modifications (PTMs) (Zhang, 2018).

In the past two decades, over 20 types of PTMs have been identified on histones, including acetylation, methylation, phosphorylation, ubiquitination, and crotonylation. Among these PTMs, 12 are added to lysine residues. The N-terminal, flexible 'tail' domains are the most heavily modified portions of histones, presumably because they are more easily accessible to histone-modifying enzymes than other domains. However, PTMs have also been detected within the globular core domains of histones. Histone PTMs are thought to modulate chromatin structure and gene expression either directly or via recruitment of specific chromatin-associated proteins (Zhang, 2018).

Whether PTMs are always involved in chromatin structure remains controversial. Studies involving genetic or chemical interventions targeting histone-modifying enzymes have provided substantial evidence for biological functions of specific PTMs. For example, H3K27 methylation by the polycomb repressive complex 2 (PRC2) is involved in maintenance of cellular identity. Unfortunately, because these modifying enzymes generally have other protein substrates in addition to histones, and chromatin-regulating enzymes might also have functions unrelated to their enzymatic activities, these experimental data must be interpreted cautiously (Zhang, 2018).

The roles of PTMs can be directly queried by systematic mutation of histone residues. Such studies have been carried out in Saccharomyces cerevisiae, but experiments in higher organisms pose additional challenges. For example, there are 64 histone genes within the human genome, distributed at three major loci on different chromosomes, making it difficult to substantially alter levels of particular histone proteins inside human cells (Zhang, 2018).

Currently, the only multicellular organism in which histone mutagenesis has been performed is Drosophila melanogaster, in which all core-histone genes reside at a single locus on the left arm of chromosome 2, with ~100 copies of histone gene-repeat units (His-GUs) per chromosome. Each His-GU (~5 kb in length) contains the four core-histone genes in two pairs (His2A-His2B and His3-His4), each under the control of a divergent promoter, plus the linker-histone gene, His1, which is regulated independently (Zhang, 2018).

Histone residue function in D. melanogaster has been explored by removing the His-GU cluster (Df(2L)HisC, referred to as HisC hereafter) and complementing it with transgenes from plasmids or bacterial artificial chromosomes (BACs). These methods are labor intensive partly because four plasmids are needed for transgenic complementation and complex crossing procedures. Therefore, only limited sites within histone H3 and H4 have been analyzed. In addition, since the transgenes are randomly integrated, positional effects could confound data interpretation (Zhang, 2018).

This study generated an efficient histone-mutagenesis platform, enabling the functional study of each residue in all five histones with much higher throughput than with previous techniques. As a proof-of-concept study, H3 and H4 were targetted, revealing several interesting insights that would have been difficult to obtain by other means (Zhang, 2018).

This study has developed an efficient histone-mutagenesis system with several advantages over previous approaches. The histone-deletion line facilitates histone rescue in situ. A single plasmid is sufficient for complementation, and the plasmid is targeted to the original histone locus, which eliminates consideration of positional effects associated with random integration of plasmids and BACs. This high-throughput strategy to assemble multiple copies of His-GUs is fast and efficient and enables introduction of not only singular but also compound histone mutations (Zhang, 2018).

The results demonstrated that a low His-GU copy number causes developmental defects in both testes and ovaries, with more severe effects in ovary development. The ovarian defect was not the result of a loss of GSCs, and, instead, the budding processes were impaired), which leads to reduced fecundity or to sterility and which explains the severe fertility defects in females. The number of GSCs was only slightly reduced in testes from adult males with low histone copy numbers (compared with wild-type). Because histone copy numbers are altered globally in these flies, mosaic analysis could reveal whether reduced histone copies reflects an autonomous or non-autonomous effect on GSCs (Zhang, 2018).

The finding that H4K16 was critical for sex-dosage compensation and male development is consistent with the fact that MOF-MSL, which acetylates H4K16, contributes to male X-linked transcriptional activation. Notably, some H4K16A male adults were recovered and a weak homozygous mutant stock was generated under normal culture conditions, whereas the mof RNAi and mutant each lead to 100% male lethality. It is proposed that MOF has functions in male development beyond H4K16 acetylation (Zhang, 2018).

H4K16A mutation caused a severe sex bias (1:10 male:female) in homozygotes, reminiscent of that resulting from inactivation of the non-coding roX gene (another dosage compensation component) in D. melanogaster. Given that MOF-MSL-mediated H4K16 acetylation is roX-dependent, roX might act by stimulating H4K16 acetylation, directly or indirectly, which merits further exploration (Zhang, 2018).

H4K16A mutation severely depleted GSCs in the ovary, which presumably contributed to the infertility in the mutants. This finding is not surprising, given that MOF is involved in maintaining pluripotency and self-renewal of embryonic stem cells, and mof mutations lead to failure in the reprogramming of stem cells. The H4K16A mutation might additionally compromise follicle-cell development, as suggested by the fact that Chameau, another H4K16 acetyltransferase, regulates the developmental transition of follicle cells into the amplification stages of oogenesis (Zhang, 2018).

H3K27me3 is essential for gene repression involving polycomb-group (PcG) proteins, but it is not clear which other histone residues are also involved. Traditional mosaic cloning analysis has identified H3S28 as one such residue. This method requires the generation of fly mutants with a complex genotype, which is laborious as it involves multistep crosses. The current strategy for mosaic analysis is much faster and simpler, enabling readily screening of mutations of 19 essential histone residues. This study confirmed the previous finding about H3S28 and further demonstrated that H3R26 is also essential for PcG function, thereby validating this strategy (Zhang, 2018).

This study has shown that H3R26 is required for H3K27 trimethylation, which contributes to PcG-mediated gene repression. Additionally, H3R26 might stimulate PRC2 catalytic activity, as suggested by in vitro data showing that human PRC2 catalytic activity is partially dependent on H3R26. H3R26 may also facilitate PcG protein recognition, with the positive side chain of H3R26 contacting the SET domain of the E(z) methyltransferase. Whether H3R26 is modified remains unclear, although H3R26 methylation has been reported in mouse embryos. Further studies are needed to clarify these issues (Zhang, 2018).

Functional dissection of Drosophila melanogaster SUUR protein influence on H3K27me3 profile

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. This study performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm SuUR-sensitive chromosomal regions were revealed that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. This study tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. SUUR did not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. The possible effect of SUUR on histone genes expression and its involvement in DSB repair were also ruled out. These results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication (Posukh, 2017).

SuUR mutation affects two processes in the repressed regions of polytene chromosomes-their polytenization and repressed histone modifications maintenance. In SuUR mutants, the replication in these regions becomes more efficient; however, the levels of H3K27me3 and H3K9me3 decrease significantly. A 'differential diagnosis' was performed for the effects of SuUR mutation on H3K27me3 level in polytene chromosomes. Successive conclusions allowed exclusion of SUUR involvement in certain mechanisms that, to this point, obscured the assessment of its function. Fur potential explanations were tested for the remarkable, but insufficiently studied, effects that SUUR has on chromosome replication and chromatin. The study revealed that H3K27 methylation in SuUR-sensitive regions (SSRs) of wild type chromosomes does not happen in response to DSB formation during under-replication as was shown in other model systems. Indeed, many regions that are 100% polytenized in wild type contain H3K27me3 that is sensitive to SuUR mutation. It was also shown that SuUR mutation does not affect Polycomb DamID profile in salivary gland and is not involved in the initial placement of H3K27me3 mark early in embryogenesis. These results indicate that the effect of SuUR mutation on H3K27me3 level develops during the ontogenesis. Finally, the possibility of SUUR protein regulating the expression of histone genes was excluded (Posukh, 2017).

In a previous work, a hypothesis was proposed that SUUR protein is involved in the maintenance of repressive histone modifications during replication in Drosophila. It was suggested that SUUR protein could function in the replication-coupled re-establishment of repressed histone modifications in polytene chromosomes. According to this model, SUUR impedes the progression of the replication complex through heterochromatin regions until the pattern of repressed histone marks is properly re-established on the newly synthesized DNA strands (or until the context for future chromatin maturation is properly formed). In the absence of this regulation, replication forks progress through heterochromatin regions more efficiently, but at the expense of the significant depletion of H3K27me3 and H3K9me3. This model combines all major effects of SUUR protein and provides a causal link between them. In this study, necessary experiments were performed to test this model in context of SUUR effect on H3K27me3 (Posukh, 2017).

New data obtained in this study add fascinating details to the well-known effects of SUUR protein. Analysis of the published H3K27me3 profile in salivary gland chromosomes revealed two distinct types of H3K27me3-containing regions-SSRs and SNRs-that differ in H3K27me3 levels, sensitivity to SuUR mutation and the presence of Pc. Intriguingly, the reduction in H3K27me3 levels upon SuUR mutation is observed only in regions that are moderately enriched with H3K27me3 and lack Pc, whereas highly enriched regions remain unaffected. Although, SUUR DamID signal in SNRs is even higher than in SSRs, which is consistent with early cytological studies. Thus, SUUR function is required to preserve H3K27me3 levels at the regions, which are devoid of Pc (Posukh, 2017).

The majority of SSRs detected in this study overlap with the BLACK chromatin type described in Kc167 cells. Genomic regions corresponding to BLACK chromatin were recently shown to contain H3K27me2 in Sg4 cells. Given the known cross-reactivity of the antibodies, which were used in ChIP experiments that detected SuUR effect on H3K27 methylation level, it could be suggested that SSRs mainly contain di-methylated H3K27 and SuUR mutation affects the level of this modification. This suggestion contradicts with the previous immunostaining results that showed no effect of SuUR mutation on H3K27me2 level; however, the effect may be too subtle to be detected with the cytological methods. The present study proved that the previously observed effect of SuUR mutation is specifically directed on tri-methylated H3K27; however, to further address the mechanism of SUUR action in chromatin it would be useful to study the effects of this protein considering a wider spectrum of histone modifications (Posukh, 2017).

It is highly plausible that Pc-G proteins maintain H3K27 methylation at their target regions by temporarily over-producing H3K27me3-marked histones prior to replication, as reported earlier. Hence, the regions of high H3K27me3 enrichment resist the effect of SuUR mutation (Posukh, 2017).

Although the presence of Polycomb protein in SNRs apparently compensates the effect of SuUR mutation on H3K27me3 level, it fails to neutralize the effect of SUUR protein on the replication in these regions. Indeed, the very first characterized under-replicated region (89DE) contains Bithorax complex, which is densely covered with Pc, but is still under-replicated in wild type and fully polytenized in SuUR mutants. Similar situation is observed in the Antennapedia complex. Both Bithorax complex and Antennapedia complex are as large as 200–300 kb, so it seems that under-replication normally occurs at H3K27me3-enriched regions that exceed a certain length and lack internal replication origins. This suggestion is in line with recently discovered negative correlation between the length of the under-replicated regions and their polytenization levels. Hence, the selectivity of SuUR mutation effect on H3K27me3 level turns out to be associated with the presence of Pc protein. However, the effect of SuUR mutation on under-replication apparently is largely dependent on the size of the repressed domain, rather than its overall level of H3K27me3. These conclusions are consistent with earlier cytological observations based on immunostaining. Discovered SUUR protein effects on replication and chromatin in polytene chromosomes are schematically summarized in a Scheme explaining the effects of SUUR on H3K27me3 maintenance and under-replication in polytene chromosomes. (Posukh, 2017).

The fact that SSRs do not bind Pc suggests two plausible mechanisms of how SUUR could maintain the level of H3K27me3 in these regions. On the one hand, SUUR could mediate the interaction between the replication complex and H3K27-specific methylase PRC2 and possibly other histone-modifying enzymes. On the other hand, SUUR could regulate the incorporation of parental modified histones (or those over-produced by PRCs at their binding sites) into nascent chromatin of SSRs. Notably, a recent study suggests that linker histone H1 may be involved in this process. Future studies will elucidate the exact mechanism of this process (Posukh, 2017).

Nuclear lamina integrity is required for proper spatial organization of chromatin in Drosophila

How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. This study shows that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, these findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes (Ulianov, 2019).

The nuclear lamina (NL) is a meshwork of lamins and lamin-associated proteins lining the nuclear envelope (NE). Several lines of evidence support the idea that the NL is a platform for the assembly of the repressive compartment in the nucleus. In mammals, nematode and Drosophila, the lamina-associated chromatin domains (LADs) contain mostly silent or weakly expressed genes. Activation of tissue-specific gene transcription during cell differentiation is frequently associated with translocation of loci from the NL to the nuclear interior. The expression level of a reporter gene is ~5-fold lower when it is inserted into LADs compared to inter-LADs. Artificial tethering of weakly expressed reporter genes to the NL results in their downregulation thus indicating that contact with the NL may cause their repression. Accordingly, many transcriptional repressors, including histone deacetylases (HDACs) are linked to the NL. The high throughput chromosome conformation capture (Hi-C) technique has revealed the spatial segregation of open (DNase I-sensitive) and closed (DNase I-resistant) chromatin into two well-defined compartments. Importantly, in mammalian cells, the DNase I-resistant compartment is strongly enriched with NL contacts. Moreover, a whole-genome DNase I-sensitivity assay in Drosophila S2 cells indicated that LADs constitute the densely packed chromatin. Additionally, super-resolution microscopy studies in Kc167 cells show that inactive chromatin domains (including Polycomb (Pc)-enriched regions) are more compact than active ones (Ulianov, 2019).

The newly developed single-cell techniques demonstrate that LADs, operationally determined in a cell population, may be located either at the NL or in the nuclear interior in individual cells. Surprisingly, the positioning of LADs in the nuclear interior barely affects the inactive state of their chromatin. This raises the question as to whether contact with the NL makes the chromatin in LADs compact and inactive. However, few studies directly address this issue. It has been shown that lamin Dm0 knock-down (Lam-KD) in Drosophila S2 cells decreases the compactness of a particular inactive chromatin domain. Accordingly, the accessibility of heterochromatic and promoter regions has been shown to increase upon Lam-KD in Drosophila S2R+ cells. However, the impact of the NL on the maintenance of the overall chromatin architecture remains mostly unexplored (Ulianov, 2019).

This study shows that upon loss of all lamins, the density of peripheral chromatin is decreased in Drosophila S2 cells leading to the slight overall chromatin compaction. At the same time, chromatin in LADs becomes less tightly packed which correlates with the enhancement of initially weak level of histone H3 acetylation and background transcription in these regions (Ulianov, 2019).

Using immunostaining and FISH experiments, Lam-KD in Drosophila S2 cells was shown to lead to a slight reduction in total chromatin volume and, as a result, an increase in chromatin packaging density. However, the stronger compaction of chromatin is not homogeneous and depends on the epigenetic state and scale. Hi-C analysis clearly indicates two opposite trends in chromatin behaviour. The contact frequency in the active chromatin increases over short distances (i.e., within the 'active' TADs and the inter-TADs) and decreases over long distances (i.e., within the A compartment). Whereas in the inactive chromatin it, inversely, decreases over short distances (i.e. within the TADs mostly corresponding to LADs), but increases at the chromosomal scale (Ulianov, 2019).

A model is suggested explaining general chromatin stretching as well as the condensation of inactive chromatin in TADs mediated by the NL. If chromatin mobility is constrained by its tethering to the NL, then the release from this tethering will lead to chromatin shrinkage due to macromolecular crowding and inter-nucleosomal interactions. Therefore counterintuitively, the NL appears not to restrict chromatin expansion but provides an anchoring surface necessary to keep interphase chromosomes slightly stretched. At the same time, inactive chromatin may become additionally condensed due to the deacetylation by HDACs, linked to the NL, and/or mechanically, due to chromatin binding with the NL (Ulianov, 2019).

A recently published study analysed the 3D genome organization upon NL disruption in mouse embryonic stem cells (mESCs). It is interesting to compare the current results from Drosophila with those from mice. Upon loss of all lamins, the general TAD profile is still preserved in both species, however, intra- and inter-TAD interactions are altered. Strikingly, upon loss of all lamins, a fraction of NL-attached TADs becomes less condensed in both species. However, in contrast to Drosophila S2 cells, this is not accompanied by a general detachment of chromatin from the NE in mESCs. Additionally, distant interactions within the inactive chromatin are mostly increased in both species upon lamin loss. Finally, while some genes located at the nuclear periphery and in the nuclear interior have changed their expression both in mESCs and in Drosophila S2 cells, an increase in the background transcription upon lamin loss is detected specifically in Drosophila LADs, and this was not reported for mESCs44. Taken together, these findings indicate that both in mammals and Drosophila the NL not only makes nearby chromatin more compact and repressed, but also affects chromatin interactions and gene expression in the nuclear interior (Ulianov, 2019).

The diversity of mechanisms of chromatin attachment to the NL in Drosophila and mammals may explain the differences in chromatin behaviour in response to the lack of all lamins. For example, it was shown that LBR and PRR14 proteins participate in the tethering of the H3K9-methylated chromatin to the NE in mammals. Whereas in mammalian ESCs LADs are strongly enriched with the H3K9me2/32, in Drosophila Kc167 and, likely, in S2 cells this modification is not present in LADs. Accordingly, the results indicate that LBR is not required to keep chromatin at the nuclear periphery in S2 cells. Therefore, the removal of all lamins may not be sufficient to detach all LADs from the NE in mESCs, but can release LADs in Drosophila S2 cells (Ulianov, 2019).

In conclusion, using different approaches this study revealed that NL disruption in Drosophila S2 cells leads to general chromatin compaction, accompanied by the impaired spatial segregation of total chromatin into active and inactive types, and the decompaction of a fraction of NL-attached TADs linked to partial derepression of their chromatin. Importantly, the observed phenomena may be related to the abnormal expression of genes in lamin-associated diseases (Ulianov, 2019).

Structure and function of an ectopic Polycomb chromatin domain

Polycomb group proteins (PcGs) drive target gene repression and form large chromatin domains. In Drosophila, DNA elements known as Polycomb group response elements (PREs) recruit PcGs to the DNA. This study shows that, within the invected-engrailed (inv-en) Polycomb domain, strong, constitutive PREs are dispensable for Polycomb domain structure and function. It is suggested that the endogenous chromosomal location imparts stability to this Polycomb domain. To test this possibility, a 79-kb en transgene was inserted into other chromosomal locations. This transgene is functional and forms a Polycomb domain. The spreading of the H3K27me3 repressive mark, characteristic of PcG domains, varies depending on the chromatin context of the transgene. Unlike at the endogenous locus, deletion of the strong, constitutive PREs from the transgene leads to both loss- and gain-of function phenotypes, demonstrating the important role of these regulatory elements. These data show that chromatin context plays an important role in Polycomb domain structure and function (De, 2019).

Polycomb group proteins (PcGs) are critical for organismal development and stem cell maintenance. PcGs were first found in Drosophila as repressors of homeotic genes, and PcG repression is one of the earliest epigenetic regulatory mechanisms to be identified. In Drosophila, nearly all PcG proteins are subunits of one of four principal protein complexes: Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), Pho repressive complex (PhoRC), and Polycomb repressive deubiquitinase (PR-DUB). PcG protein complexes bind to DNA elements known as Polycomb group response elements (PREs), deposit the repressive chromatin modification mark H3K27me3, and drive chromatin compaction leading to gene repression. Genes repressed by PcG are covered with H3K27me3 and are thought to form their own topologically associating domains (TADs). In Drosophila, TADs vary from a few kilobases to several hundred kilobases in size. Regulatory DNAs present within TADs preferentially interact with genes located within the same domain, with limited contacts outside of the TAD boundaries. In Drosophila, genome-wide data suggest that mini-domains formed by actively transcribed regions form the boundaries of some TADs, while other TAD boundaries are demarcated by insulator elements. In mammals, the insulator protein CTCF colocalizes with a subset of TAD boundaries (De, 2019).

The current understanding in chromatin biology hypothesizes that the folding of chromatin into domains assists in the packaging of long stretches of DNA inside the eukaryotic nucleus. Further, it is the organization of these domains that facilitates spatial and temporal regulation of genes within them. Thus, understanding how the domains are formed is of prime importance. How large PcG domains/TADs are formed is a central question. To date, researchers have extensively studied how PcG proteins are recruited to specific DNA sequences and which proteins are present in PcG complexes. PREs are required for the recruitment of PcG proteins in Drosophila and are thought to initiate the formation of a Polycomb domain. The 113-kb PcG domain that encompasses the invected (inv) and engrailed (en) genes of Drosophila. Unexpectedly, deletion of strong, constitutive PREs from the endogenous inv-en domain had little effect on inv-en PcG domain organization. Weak PREs present in the inv-en domain were sufficient to establish and maintain the overall domain organization, and some weak PREs overlap with enhancers present within the inv-en domain. Similarly, deletion of the bxd PRE had a mild effect on Ubx expression, whereas deletion of the iab7 PRE resulted in misexpression of Abd-B in very specific parasegments. A recent report showed that deletion of two PREs from the dac locus causes prominent structural and functional changes in the locus (De, 2019).

The primary aim in this work was to study the effect of "chromatin context" on a PcG domain; the following main conclusions can be drawn from this study. First, the en PcG domain at ectopic genomic sites rescues the null mutants of inv-en. This shows that the information to form the chromatin domain is primarily present within the domain itself. Second, spreading of the chromatin state at different sites of the genome is very much dependent on the local "context" itself. Third, a repressive chromatin domain can interact with null chromatin (has no chromatin mark) but stays segregated from the active chromatin. Fourth, a fragment of DNA containing the strong, constitutive PREs is required at an ectopic location to ensure PcG silencing in all tissues. Strikingly, the endogenous locus is resilient to loss of the same DNA fragment, indicating the importance of context on PcG chromatin domain formation and function. Fifth, ubiquitously expressed flanking genes may act as boundaries for enhancers within PcG domains. These data provide experimental evidence for the hypothesis that chromosomal neighborhood plays an important role in regulating gene expression (De, 2019).

Transgenic assays to test 'functional PREs' have highlighted the effect of chromatin context on reporter gene expression. For example, the strong PREs upstream of en, present in the 1.5-kb fragment examined in this study, only act to repress reporter transgene expression in about 50% of chromosomal insertion sites. Previous work has shown that these PREs are dispensable from the endogenous inv-en locus. The normal phenotype of en80Δ1.5 flies also supports these data. However, this study shows that the HAen79@attP40 transgene has a higher level of H3K27me3 accumulation than HAen79Δ1.5@attP40. This reduced level of H3K27me3 is sufficient to maintain correct en expression in embryos and imaginal discs. Notably, the absence of strong PREs in HAen79Δ1.5@attP40 caused misexpression of en during adult abdomen development. These data emphasize the importance of context itself on the stability and resiliency of the PcG domain toward modification (mutation or deletion) of regulatory sequences present within the domain. The data also show that deletion of the 1.5-kb DNA fragment that includes the strong en PREs rendered the HAen79 transgene unable to rescue inv en mutants. Strikingly, HA-en expression from HAen79Δ1.5 was very low in the embryonic nervous system, a result not seen with HAen79 or in en80Δ1.5 flies. These data suggest that the embryonic nervous system enhancers are not able to interact well with the HA-en promoter in the absence of the 1.5-kb DNA fragment. This same deletion, when present at the endogenous locus, does not cause a loss of en expression in the nervous system. This suggests that the structure of the endogenous locus is resilient to the loss of this DNA. This study suggests that the chromosomal locationof the endogenous locus aids in the proper folding of en to facilitate enhancer-promoter communication in the embryonic nervous system. Another explanation for this observation is that the 1.5-kb PRE could be acting as a Trithorax response element (TRE) and bind to Trithorax group proteins for proper en expression. In any case, the essential role of 1.5-kb PRE/TRE at the ectopic locus is alleviated by the 'local context' effect at the endogenous locus (De, 2019).

It has been proposed that establishment and inheritance of H3K27me3 are dependent on two factors: (i) sequence-specific recruitment of the chromatin modifiers and (ii) the ability of H3K27me3 to act as a template for PRC2 to bind and modify other nucleosomes present in the vicinity. The current experiments show that the spreading of H3K27me3 to the flanking chromatin surrounding the insertion site is not very efficient, although no active chromatin mark was present in the vicinity (particularly at attP3). However, 4C-seq analysis showed that the ectopic en PcG domain interacted with flanking chromatin significantly until it encountered the active domain. These observations indicate that mere interactions between the PcG domain and flanking chromatin were not able to spread the repressive mark efficiently. This observation highlights the importance of PcG recruitment via PREs for PcG domain formation (De, 2019).

One of the surprising observations in this study was the deposition of the repressive mark over the exons of Msp300 in larvae that contain HAen79@attP40. This accumulation is also observed in Kc167 cells but not in larval samples that did not have HAen79 inserted at attP40. It is assumed that some regions in the genome are more susceptible to the PcG regulation and that the weak binding peaks of PcGs near the attP40 site might be acting as PREs to facilitate spreading of the mark. It is posited that, when a PcG domain is inserted into attP40, it renders the adjacent chromatin more likely to form an H3K27me3 domain. It is noted that the Msp300 gene is covered by both H3K27me3 and H3K36me3. This is likely due to the mixed cell population in larval brains and discs. What about the spreading of the H3K27me3 mark over the exons? This is unusual; however, pre-mRNA and cotranscriptional activity have been linked to the local chromatin structure. In a genomic study of many histone modifications in human and Caenorhabditis elegans DNA, the H3K27me3 mark was enriched over exons. In addition, physical interaction between mammalian splicing factors (U2snRNP and Sf3b1) and PcG proteins (Zfp144 and Rnf2) was reported to be required for proper repression of Hox genes. It is speculated that cotranscriptional recruitment of PcG proteins over the Msp300 gene in the transgenic line might have established the exon-specific deposition of the H3K27me3 mark. It is proposed that the exon-specific H3K27me3 accumulation can act as an intermediate step to repress transcriptionally active target genes and establish PcG domains during development (De, 2019).

Two interesting questions for chromatin biologists are the following: How does a meter-long genome fit into a nucleus and how does this folding influence genome function? High-resolution Hi-C experiments have given structural insights into interphase chromosomes in eukaryotic nuclei. According to the Hi-C data, chromatin is organized into TADs or 'contact domains', and the TADs form compartments A (enriched with active domains) and B (enriched with inactive domains). While the Drosophila genome has 'compartments,' the existence of TADs in Drosophila is disputed, and despite the evidence that TADs and compartments are important for chromatin organization and function, basic information about how these structures are formed and maintained has been incomplete. The current data shows that it is the intrinsic property of chromatin to segregate based on histone modifications and gene activity. This property of chromatin is not locus specific; in different chromatin contexts, repressed chromatin tends to segregate from active domains. Biochemical and molecular evidence on the antagonistic behavior between the H3K27me3 and H3K36me3 modifications also support this claim and provide evidence toward H3K36me3 as a chromatin component that restricts the PcG-mediated spread of H3K27me3. How are large PcG domains formed and maintained? The establishment of the PcG domain and the spreading of H3K27me3 start from the strong PREs present in the PcG domain during cell cycle 14. Repressive loops between PREs within PcG domains are also formed during cell cycle 14. These loops are proposed to play a synergistic role in establishing PcG domains. Surprisingly, deletion of some strong PREs in situ resulted in weak phenotypes, suggesting redundancy of PcG recruitment. This study provides evidence that apart from the minor PREs present in the large PcG domains, chromatin context itself is a critical factor that determines robustness and function of PcG domains (De, 2019).

Cis-acting variation is common across regulatory layers but is often buffered during embryonic development

Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequence, and chromatin. How DNA mutations affecting any one of these regulatory 'layers' is buffered or propagated to gene expression remains unclear. To address this, allele-specific changes were quantified in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses at three embryonic stages, yielding a comprehensive dataset of 240 samples spanning multiple regulatory layers. Genetic variation (allelic imbalance) impacts gene expression more frequently than chromatin features, with metabolic and environmental response genes being most often affected. Allelic imbalance in cis-regulatory elements (enhancers) is common and highly heritable, yet its functional impact doesn't generally propagate to gene expression. When it does, genetic variation impacts RNA levels through H3K4me3 or independently through chromatin accessibility and H3K27ac. Changes in RNA are more predictive of variation in H3K4me3 than vice versa, suggesting a role for H3K4me3 downstream of transcription. The impact of a substantial proportion of genetic variation is consistent across embryonic stages, with 50% of allelic imbalanced features at one stage being also imbalanced at subsequent developmental stages. Crucially, buffering, as well as the magnitude and evolutionary impact of genetic variants, are influenced by regulatory complexity (i.e., number of enhancers regulating a gene), with transcription factors being most robust to cis-acting, but most influenced by trans-acting variation (Floc'hlay, 2020).

This study generated an extensive F1 data set to better understand the functional impact of genetic variation in regulatory DNA on embryonic gene expression and to shed light on how these effects are propagated or buffered through different regulatory layers. This analysis revealed several new insights into the impact of regulatory mutations on transcriptional phenotypes (Floc'hlay, 2020).

First, although cis-acting genetic variation is common in development, its effects are not equally distributed across the genome. Allelic variation both is more frequent and has greater magnitude at distal regulatory elements (putative enhancers) compared with promoters, despite genetic variation itself being more common at promoters. This may in part be owing to differences in the relative importance of sequence content at promoters and enhancers: Many promoters, particularly for broadly expressed genes, have high tolerance to genetic variation (Schor, 2017). But despite having a greater magnitude, allelic imbalance (AI) at distal elements is less likely to be propagated to other regulatory layers, suggesting that enhancer mutations are often effectively buffered, a hypothesis that fits well with the observed robustness of gene expression to deletions that remove distal regulatory elements (Floc'hlay, 2020).

Second, although all data types (open chromatin, histone modifications, RNA levels) are highly correlated, their explanatory values (potential causal relationships) as revealed by partial correlation analysis are not equal. By using cis-acting variation as perturbations to development, a strong, likely direct, relationship was observed between variants affecting open chromatin (TF binding) at proximal and distal sites to a degree that was not observed at the total count level. A strong, potentially causal, link was uncovered between allelic imbalance in H3K4me3 signal and AI in the expression of the corresponding genes, which was largely independent of imbalance in H3K27ac signal. Copula analysis placed H3K4me3 downstream from RNA, suggesting that although AI at the RNA level is predictive of AI in H3K4me3, mutations impacting H3K4me3 often do not directly impact RNA. This placement of RNA upstream of H3K4me3, inferred from the analysis of the functional impact of genetic variation, is supported by recent genetic ablation studies showing that RNA transcription does not require H3K4me3 and is consistent with suggestions that H3K4me3 is deposited as a consequence of transcription and may be required in more downstream post-transcriptional events (Howe et al. 2017) (Floc'hlay, 2020).

Third, the impact of cis-acting variation on gene expression is influenced by regulatory complexity, with genes having more regulatory elements being less likely to show AI. This may reflect selection against variation in regulatory elements associated with these genes, and indeed, less AI is observed in regulatory elements associated with developmental regulators, extending previous findings. But even accounting for reduced overall AI, TFs and other genes with complex regulation show more independence from AI in associated regulatory layers. Although, a role for allele-specific post-transcriptional processes cannot be ruled OUT, the results suggest an active buffering process resulting from the presence of multiple regulatory inputs. That said, as the number of regulatory elements with AI near a gene increases, so does the probability that the gene will show AI, suggesting that such buffering is not absolute (Floc'hlay, 2020).

Finally, trans-acting variation is more common for RNA than other regulatory layers, particularly for genes with complex regulatory landscapes such as TFs. This later observation, likely owing to the buffering effects of complex cis regulatory landscapes, has potentially counterintuitive evolutionary consequences: Although reduced in genetic variation overall, predominantly trans-influenced genes are more likely to show nonadditive, and thus less selectable, patterns of inheritance. As a result, trans-acting variation in genes such as TFs may remain in populations even as negative selection and buffering act to reduce the influence of cis-acting mutations. Why trans-acting variation is more common for RNA is not immediately clear but may reflect the accumulation of variation impacting both transcriptional and post-transcriptional processes (Floc'hlay, 2020).

In summary, allelic variation in chromatin accessibility and histone modifications at regulatory elements is prevalent and capable of propagating across regulatory layers. The extent of this information flow, or propagation, depends on the type of regulatory element and appears mitigated at developmental regulators (Floc'hlay, 2020).

The Dm-Myb oncoprotein contributes to insulator function and stabilizes repressive H3K27me3 PcG domains

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. This study further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. This study now reveals a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation (Santana, 2020).

This study identified four novel roles for the Drosophila Myb oncoprotein. First, it was shown that Myb acts as a potentiator of target gene transcription, not merely as an epigenetic factor that maintains activated expression. Potentiation is associated with increased H3K4me3 as well as recruitment and pause release/elongation of RNA Pol II. Next, it was shown that in addition to binding promoter potentiator sites, Myb binds to insulator sites genome wide, is necessary to promote the binding of BEAF-32 and CP190 at Myb-shared sites, and is required for the function of at least a subset of insulators. Third, it was shown that Myb plays a critical role in formation and/or stabilization of H3K27me3 domains, preventing silent genes within these domains from becoming derepressed, in stark contrast to Myb's well-known role as an activator of gene expression. Finally, this study showed that Myb is enriched at TAD boundaries and chromatin loop anchors (Santana, 2020).

Similar to what has previously been reported, an enrichment was identified of cell cycle genes directly regulated by Myb, with the majority being G2/M but also several S phase genes (consistent with studies on B-Myb, supporting its role in S phase). Notably, a recent study reported that Cyclin A (the cyclin that regulates aspects of both S phase and G2/M) directly binds to Myb and is required for expression of many of its targets (Rotelli, 2019). Further supporting a role for Myb in transcriptional regulation, this study found that Myb is required for optimal levels of H3K4 promoter methylation, with Myb playing a key role in RNA Pol II dynamics. Analysis of the promoter regions of Myb-potentiated targets in Myb mutants revealed two distinct classes of targets: one that requires Myb for efficient recruitment of RNA Pol II and both that use Myb for efficient RNA Pol II pause release/elongation. Myb may use NURF to establish a nucleosomal-free region that allows recruitment of RNA Pol II, similar to what has been observed with GAF at several targets. Further suggesting a critical role for Myb in transcriptional regulation, the failure to potentiate key targets in Myb mutants (e.g., okr and Rad9 for S phase; Cap-D2 for chromosome condensation; mad2, Mps1, Pen, dgt6, and msd5 for M phase) likely explains the varied phenotypes observed in Myb mutants (including increased mitotic index, partially condensed chromosomes, aneuploidy, and S phase defects) (Santana, 2020).

Recent studies have shown that several components of dREAM can bind insulator sites, and RNAi depletion of Mip40, Mip130, and E2f2 resulted in impairment of enhancer-blocking function at specific sites. Furthermore, components of dREAM (Myb, Mip120, Mip130, Rbf1, E2f2, and DP) were shown to directly interact with dCTCF and/or CP190. Interestingly, double knockdown of dCTCF and CP190 resulted in loss of dREAM (Mip40, Mip120, Mip130, and E2f2) at some shared sites. The results presented in this study further extend these studies and demonstrate that Myb directly contributes to boundary insulator function (including sites located at divergently paired genes). Intriguingly, in many cases where one gene of a divergently paired genes (DPGs) is transcribed and the other is silent, it was found that Myb has no role in expression of the activated gene; rather, Myb prevents inappropriate activation of the silent gene. This cannot be explained by loss of the primary repressive arm of the Myb complex (E2f2/Rbfs) because knockdown of E2f2 or Rbf1/2 in Kc167 cells led to derepression of only 17 out of the 96 genes upregulated when Myb is knocked down. Furthermore, out of the 46 genes upregulated in SL2 cells upon knockdown of E2f2 or Rbf1/2, only 3 are upregulated in Myb mutant wing discs. Finally, the absence of E2f2 in 3rd instar larvae led to upregulation of genes from four DPGs; yet, these genes are not upregulated in Myb mutant wing discs. Of the insulator sites shown in this study to require Myb, the vast majority (89%) contain Myb but not E2f2 consensus sites, similar to what was observed in Kc167 cells for genes requiring Myb for repression (Santana, 2020).

Consistent with Myb playing a role in insulators that separate different chromatin neighborhoods, this study found a striking correlation between Myb occupancy and active, null, PcG H3K27me3 (all 99%), and APBS TAD boundaries (>93%). TAD boundaries have been associated with chromatin looping, and studies on CTCF and CP190 in vertebrates and invertebrates, respectively, have proposed or confirmed a role for these insulator factors in tethering the chromatin loops. Further work has shown that chromatin loop anchors can also exist within TADs. It is particularly noteworthy that Myb occupies the majority of chromatin loop anchor points identified in another study Eagen (2017), in addition to significant overlaps with several other chromatin factors (including dCTCF, Rad21, and Pc) at anchor points. Further work is needed to determine whether Myb plays a role in tethering chromatin loops, although it is intriguing to speculate that the loss of the H3K27me3 mark in Myb or dCTCF mutants could result from disrupting H3K27me3 loops, which, in turn, might diminish that region's ability to associate with an H3K27me3 nuclear subcompartment. The observation that dCTCF has been shown to directly interact with Myb further suggests that these two chromatin factors can collaborate, and a remarkable 73% of the anchors bound by dCTCF are also bound by Myb (similar to the Rad21-bound anchors, for which 75% are also occupied by Myb). It is postulated that B-Myb (the most closely related vertebrate Myb family member to Dm-Myb) and CTCF might be playing similar collaborative roles in humans (Santana, 2020).

Although a role for Myb in transcriptional activation has been recognized for some time, its role in insulator function is particularly intriguing, as this activity appears to be separate from its transcriptional role. Indeed, the chromatin changes observed at many DPGs and other insulator elements strongly support the direct role of Myb in promoting a barrier function between chromatin states (in part by promoting binding of BEAF-32/ CP190). However, unlike previously described examples of barrier insulator loss, which results in heterochromatin spreading into euchromatin, the opposite scenario were observed in Myb mutants, namely the spreading of H3K4me3, which may be enabled by the significant loss of the H3K27me3 mark. This is the very definition of barrier insulator function, namely, maintaining a discrete separation of chromatin environments. Whether Myb is playing a role in chromatin loop formation is unclear at this point, but its overlap with the majority of loop anchor points identified in at least one study in flies is intriguing. Further work will be needed to address Myb's role at the loop anchors and whether this role relates to stabilization of H3K27me3 domains and/or barrier insulator function. For example, directed mutagenesis of Myb-dependent insulator sites by using tools, such as CRISPR-Cas9, to specifically target Myb, dCTCF, or BEAF-32 sites can provide additional insight into the respective roles of these insulator factors. In addition, assay for transposase-accessible chromatin using sequencing (ATAC-seq) or chromosome conformation capture (3C)-type techniques performed in both controls and Myb mutants can be used to determine whether important chromatin/ 3D structure changes might underlie the altered transcriptional (Santana, 2020).

Drosophila Prp40 localizes to the histone locus body and regulates gene transcription and development

In eukaryotes, a large amount of histones must be synthesized during the S phase of the cell cycle to package newly synthesized DNA into chromatin. The transcription and 3' end processing of histone pre-mRNA are controlled by the histone locus body (HLB), which is assembled in the H3/H4 promoter. This study identified the Drosophila Prp40 pre-mRNA processing factor (dPrp40) as a novel HLB component. dPrp40 is essential for Drosophila development, with functionally conserved activity in vertebrates and invertebrates. It was observed that dPrp40 is fundamental in endocycling cells, highlighting a role for this factor in mediating replication efficiency in vivo. The depletion of dPrp40 from fly cells inhibited the transcription but not the 3' end processing of histone mRNA. These results establish that dPrp40 is an essential gene for Drosophila development that can localize to the HLB and may participate in histone mRNA biosynthesis (Prieto-Sanchez, 2019).

In eukaryotic cells, the nucleus is compartmentalized and contains several dynamic nonmembrane-bound structures referred to as nuclear bodies or nuclear compartments, which are essential for the correct maintenance of nuclear architecture and the gene-regulatory processes that occur within the nucleus. The study of the constituents and the spatial and dynamic properties of these nuclear bodies is essential for understanding the regulation of gene expression programs, which are critical for cell stability and survival. Given the importance of nuclear bodies in controlling how gene expression is exerted, alterations in the regulation or biosynthesis of these structures can lead to pathological consequences (Prieto-Sanchez, 2019).

Because of the absence of a delineated membrane, the structural integrity of nuclear bodies is mediated by protein-protein and/or protein-RNA interactions. This property and the rapid dynamics of nuclear bodies are consistent with a self-organization model in which the structure of a body is determined by the global interactions among its constituents. Although significant progress has been made regarding the role of these nuclear bodies in gene expression, understanding how they are assembled in the cell is still far from being understood. Many studies have led to two main distinct but not exclusive assembly models. While one model posits that assembly occurs through an ordered, hierarchical process through which constituents are assembled around a primordial scaffolding factor or RNA, the other model considers that self-organization is accomplished randomly without any particular ordered or hierarchical nuclear body assembly. More recently, a third model for nuclear body formation involves intracellular phase separation to promote the assembly of droplets of nuclear protein/RNA has been proposed. This model posits the existence of different nucleoplasmic phases with distinct physical properties through which proteins may transition to gain favorable thermodynamic states so that nuclear body assembly is mediated by this phase transition (Prieto-Sanchez, 2019).

Some nuclear bodies are also associated with specific gene loci, and this association with a specific nuclear function or activity may be important for their formation and function. The histone locus body (HLB) is a chromatin-associated nuclear body that specifically associates with replication-dependent histone gene clusters to coordinate the transcription and 3' end processing of histone pre-mRNA. In Drosophila, the histone gene cluster is composed of ~100 copies of tandemly arranged histone H1, H2a, H2b, H3 and H4 gene cassettes. Histones play a crucial role in the packaging of DNA into chromatin. Consistent with this role, histone expression is restricted to the early S phase of the cell cycle, which is tightly coupled to DNA synthesis. Defects in histone biosynthesis result in genomic instability, which may promote oncogenesis. Since the initial characterization of the HLB by the Gall laboratory, many factors have been identified as components of this nuclear body. Some of these factors are constitutively present in these nuclear bodies throughout the cell cycle, whereas others are recruited to the HLB only during the S phase when histone transcription is active. The first group of factors includes Multi sex combs (Mxc), FLASH, the U7 snRNP and Mute, whereas general and elongation transcription factors, such as RNA polymerase II (RNAPII), TBP, Spt6 and Myc, and factors regulating histone pre-mRNA processing, such as Symplekin and other proteins, associate with the HLB upon the activation of histone gene transcription. The emerging picture is that the Drosophila HLB assembles through the hierarchical recruitment of components; Mxc and FLASH form the foundational HLB that is detected in the early embryo at cycle 10, and U7 snRNP and Mute are recruited at cycle 11 in the absence of histone mRNA transcription. A sequence located between the histone H3 and H4 genes contains the shared H3 and H4 promoter (hereafter, denoted as the H3/H4 promoter), which is essential for histone gene expression, and is required for the recruitment of Mxc and FLASH. A significant number of proteins are subsequently joined to the HLB in a manner coupled to active histone gene transcription. How the initial interaction of Mxc and FLASH with the histone loci occurs and what the actual composition of a fully formed HLB is remain to be resolved (Prieto-Sanchez, 2019).

Prp40 was initially identified as an essential yeast factor that participates as a scaffold in the early steps of spliceosome complex formation. Prp40 has a characteristic domain organization, with two WW domains in the N-terminus and five FF domains in the C-terminus, which is a structure shared by a relatively small number of proteins. Strikingly, most of these structurally related proteins have been implicated in transcription and splicing regulation. There are two putative mammalian orthologs of Prp40, PRPF40A and PRPF40B. Based on phylogenomic data, PRPF40A appears to be more closely related to Prp40 than does PRPF40B, which emerged much later in evolutionary history probably due to a gene duplication event from an ancestral PRPF40A. PRPF40A and PRPF40B interact with the transcription and splicing machineries, and at least for PRPF40B, the modulation of alternative splice site selection in apoptosis-related genes has been shown. The Drosophila ortholog of Prp40, herein denoted dPrp40, encoded by the CG3542 gene, shares 23% and 41% sequence identity with the yeast Prp40 and the human PRPF40A proteins, which suggests that the function of these proteins in forming bridges between the 5' and 3' splice sites in the first spliceosomal complex might be conserved. In fact, the regulation of alternative pre-mRNA splicing of the glial-specific cell-adhesion molecule Neurexin IV by dPrp40 has been reported (Prieto-Sanchez, 2019 and references therein).

This study characterize dPrp40 and identifies a putative new role for this protein in histone mRNA transcription. dPrp40 localizes to the Drosophila HLB during prophase after the incorporation of the HLB primary protein components. dPrp40 is essential for Drosophila development. Moreover, dPrp40 and its human orthologs can rescue the phenotype of dPrp40 mutant flies, demonstrating a functional conservation of eukaryotic Prp40 activities in vivo. An essential requirement is shown for for dPrp40 in endocycling cells, highlighting a role for this factor in the replication efficiency in vivo. In a molecular context, this study shows that the depletion of dPrp40 from fly cells inhibits histone mRNA transcription without affecting the 3' maturation of histone mRNA. Furthermore, H3/H4-dependent transcription, which is essential for HLB assembly and high-level histone gene expression, is rescued by overexpressing dPrp40 in the depleted cells. Together, these results establish that dPrp40 is required for normal embryonic development and that dPrp40 can localize to the HLB and might regulate histone gene transcription, which could have important consequences for the cell cycle and maturation, development and viability (Prieto-Sanchez, 2019).

This study performed experiments to characterize the function of Prp40 (dPrp40) in Drosophila. dPrp40 was shown to be essential for Drosophila viability and development via siRNA-mediated depletion and single P element-mediated gene disruption approaches. Conditional knockdown of dPrp40 using different drivers resulted in abnormal phenotypes and increased apoptotic cells in certain regions of wing imaginal discs. These abnormal phenotypes were rescued by expression of the human orthologs of Prp40, indicating that the fly and human proteins have shared functions that affect cell viability. In agreement with this result, it was found previously that PRPF40B depletion increased both the number of Fas/CD95 receptors and cell apoptosis in mammalian cells, thus suggesting a role for this protein in programmed cell death. The pleiotropic effects caused by the lack of dPrp40 expression, however, may indicate that dPrp40 also regulates other genes. In fact, transgenic PRPF40A expression resulted in a faint Notch-like phenotype with wing margin 'notches', which may suggest either an effect of the overexpressed protein on Notch function or an involvement of dPrp40 in the Notch signaling pathway. Recently a transcriptome analysis of dPrp40 fly mutants was performed, and the preliminary results support the involvement of dPrp40 in the Notch signaling pathway. Further study is required to determine the molecular targets and signaling pathways regulated by dPrp40 (Prieto-Sanchez, 2019).

This study also identified a putative function for dPrp40 in the regulation of histone gene transcription. The localization of dPrp40 to the HLB pointed to a possible role of dPrp40 in the regulation of histone gene expression. This hypothesis is supported by the observation that dPrp40 loss-of-function mutants exhibited altered S phase progression and decreased histone gene mRNA expression. The 3' end processing of pre-mRNAs plays an important role in the regulation of histone mRNAs, and HLB components are required for the 3' end maturation of histone mRNAs. The results showed that dPrp40 depletion in Drosophila cells does not result in the polyadenylation of histone mRNAs, indicating that dPrp40 is not required for the 3' end processing of histone pre-mRNAs in vivo. These experiments, however, suggest that dPrp40 regulates histone mRNA expression by modulating transcription. An effect of dPrp40 on transcription synthesis is favored based on data using histone promoters and ChIP analysis. A possible effect of dPrp40 on RNA stability remains to be studied. The regulation of histone gene expression at the level of transcription by dPrp40 was an unexpected finding. The only function described for Prp40 in Drosophila is the regulation of alternative pre-mRNA splicing of the glial-specific cell adhesion molecule Neurexin IV This role of dPrp40 in the splicing process agrees with the proposed role for yeast Prp40 in the early steps of spliceosome formation and with previous data in Drosophila supporting a role for the mammalian Prp40 ortholog PRPF40B in pre-mRNA splicing. Other data challenge this view and suggest alternative mechanisms of action, including a role for this protein in the later steps of spliceosome assembly and in transcriptional regulation, which would be consistent with the unexpected data suggesting a role for dPrp40 in histone mRNA transcription. However, the possibility that dPrp40 is regulating other cellular processes and causing phenotypic defects by modulating the pre-mRNA alternative splicing of important Drosophila genes cannot be excluded. In fact, the FF domains that are critical for dPrp40 function are responsible for binding to Luc7 and Snu71, two proteins within the U1 snRNP complex. The effect of dPrp40 on splicing, however, seems not to be prominent in Drosophila tissues, according to the results of the current genome-wide analysis of transcript- and exon-level changes in siRNA flies. Further studies will be required to fully characterize the function of the dPrp40 protein in mRNA synthesis and processing (Prieto-Sanchez, 2019).

The data demonstrate that dPrp40 depletion results in growth defects and that dPrp40 localizes to the HLB and regulates histone gene transcription. Although it is tempting to link the phenotypic defects resulting from dPrp40 loss of function with histone gene expression, it is believed that dPrp40 may regulate cell growth and proliferation by mechanism(s) other than the regulation of histone genes. The current data support this notion. First, this study showed that dPrp40 associates with the HLB during interphase and early mitosis. Despite the colocalization of dPrp40 with MPM2, which is associated with the HLB only during S phase when the bulk of histone protein synthesis occurs, dPrp40 staining was also detected during the starting phase of cell division, when DNA replication is over. These data are consistent with dPrp40 being present in the HLB throughout interphase and early mitosis and therefore disengaged from the activation of histone gene transcription. The small increase in dPrp40 binding at the promoter sequences in cells in late S phase compared to at the G1/S transition is not in disagreement with this hypothesis. Second, whereas the expression of the ΔWWdPrp40 construct resulted in the deficient accumulation of dPrp40 in the HLB, indicating a less-important role of the FF domains in the localization of dPrp40 to this nuclear compartment, the FF domains of dPrp40 were essential for rescuing the phenotype resulting from the loss of dPrp40 and were also important in the activation of the histone H3/H4 promoter. Therefore, and in the absence of convincing evidence of dPrp40 having a direct role in histone mRNA metabolism, these observations suggest that the growth defects resulting from dPrp40 loss of function were not linked to the localization of dPrp40 at the HLB and the regulation of histone gene expression (Prieto-Sanchez, 2019).

An interesting question that arises from this study regards the means by which dPrp40 might be targeted to the HLB. Seminal work by Duronio provided evidence for an ordered process in Drosophila HLB assembly. Mxc and FLASH are first recruited to the HLB, whereas the other components, including U7 snRNP, Mute and other transcription and mRNA factors, are subsequently recruited in a histone gene transcription-dependent fashion. Because of the reported association of the WW and FF domains of Prp40 with the phosphorylated C-terminal domain (phospho-CTD) of RNAPII, an exciting possibility is that dPrp40 might be recruited to the HLB via a mechanism involving the phospho-CTD. Importantly, phosphorylated RNAPII is highly associated with the HLB during the S phase, when histone mRNA transcription activation occurs. Several other interpretations are also possible. Interactions among HLB components are necessary for the ordered recruitment of additional HLB factors. For example, the C-terminal region of FLASH is necessary for the recruitment of U7 snRNP to the HLB. Similarly, dPRP40 might be recruited to the HLB complex through interactions of its WW domains with other components of the complex. Another mechanism potentially collaborating in the formation of the HLB complex involves phosphorylation by Cyclin E-Cdk2, which is essential for histone mRNA expression. Although Mxc is a target of this kinase, Mxc localization to the HLB does not require Cyclin E-Cdk2 activity. The Spt6 HLB component is specifically immunoprecipitated by the phosphoprotein epitope-specific MPM2 monoclonal antibody, and phosphate treatment of the extract disrupts the interaction of Spt6 with the HLB complex, thus suggesting a role of Cyclin E-Cdk2 activity in Spt6 localization to the HLB (White, 2011). Because of the cyclin-dependent kinase consensus motif at position 739 of dPrp40, assessing the localization of dPrp40 to the HLB with respect to Cyclin E-Cdk2 activity would be informative (Prieto-Sanchez, 2019).

In summary, this study has characterized the function of Prp40 in Drosophila and has identified dPrp40 as a new component of the HLB. dPrp40 was also shown to be required for normal embryonic development and might participate in histone mRNA biosynthesis. Further study of dPrp40 will clearly be useful to define the detailed mechanism of its function (Prieto-Sanchez, 2019).

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. This study investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, this study reveals a hierarchical organization of the PCH domains into distinct 'territories.' Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. These findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin (Lee, 2020).

An appreciable fraction of most eukaryotic genomes comprises constitutive heterochromatin, which is enriched for megabases of repetitive DNA localized predominantly around centromeres (PCH). However, because of technical difficulties associated with repetitive DNA, a global and in-depth understanding of the 3D organization of the PCH domain, which encompasses at least a fifth of the human and a third of the D. melanogaster genomes, is lacking. This study provides a comprehensive and detailed picture of the 3D organization of PCH domains in D. melanogaster by combining genome-wide Hi-C analyses and cytological FISH studies. A novel analysis approach was developed that overcomes the challenges posed by repeated DNAs when determining 3D contact frequencies from Hi-C reads. Specifically, the single-locus mapping restriction was relaxed to include reads originating from the abundant repetitive DNA in PCH and different combinations of PCH reads (single-locus mapping or not) were used depending on the question being addressed. These investigations reveal significant, new insights into the interactions between different PCH regions and their 3D contacts with the euchromatic genome (Lee, 2020).

The coalescence of PCHs on different D. melanogaster chromosomes contributes to the formation of a large PCH domain in 3D nuclear space. However, DNA contacts within the PCH domain are far from homogeneous. Hi-C analysis reveals the strongest interactions (~98%) involve PCH regions on the same chromosome arm (e.g., 2L), suggesting PCH regions from each arm are organized into distinct 'territories'. This is similar to identified chromosome territories for the euchromatic genome. It is clear from both the fusion of multiple PCH domains from different chromosomes and from Hi-C and FISH analyses presented in this study that PCH regions from all the chromosomes do interact. However, some interactions occur more often than random, in particular the inter-arm (2L-2R, 3L-3R) and specific inter-chromosomal (3L/3R-4) 3D associations. Most strikingly, ~14% of identified H3K9me2-enriched regions in epigenomically defined euchromatin display preferential 3D contacts with the central PCH domains. Quantitative FISH analysis further provides cytogenetic support for the Hi-C results. The bimodal distributions of PCH-PCH or EU-PCH distances in nuclei demonstrate that these 3D contacts are dynamic and can vary among cells, similar to what has been previously shown for the euchromatic Hox loci in mouse. Importantly, polymorphic TE insertions in euchromatin allowed direct comparison of homologous sequences with and without H3K9me2 enrichment, which strongly supports the conclusion that H3K9me2 enrichment is required for EU-PCH 3D contacts (Lee, 2020).

Overall, the Hi-C and FISH analyses reveal a previously unknown picture of the 3D architecture of the PCH domains: the spatial interactions within the domains, instead of being random, are hierarchical. In addition, despite the separation of euchromatic and PCH territories on the same chromosome arm, short stretches of H3K9me2/3 enrichment in the euchromatic genome (with and without TEs) also dynamically interact with the main PCH domains. Both PCH-PCH and EU-PCH interactions happen most often within chromosome arms, which is consistent with the predictions of polymer physics on chromosome folding. Importantly, the tendency of H3K9me2 islands to interact with PCH strongly depends on their distance to PCH on a linear chromosome. This suggests that euchromatic regions and PCH could be in spatial proximities transiently with a frequency that largely follows the polymer physics of chromosome folding. The enrichment of H3K9me2/3 and the reader protein HP1a at specific euchromatic loci would then inevitably lead to their liquid-like fusion with HP1a-enriched PCH, resulting in frequent and/or maintained EU-PCH 3D interactions. Alternatively, this association with PCH may be an active process, regulating gene expression in specific subsets of cells. Indeed, in mice, the spatial clustering of olfactory receptor genes into heterochromatin domains silences all except for one receptor gene that spatially loops out from the cluster (Lee, 2020).

The observed specific spatial contacts between PCH regions located on different chromosomes are surprising, but nevertheless consistent with the coalescence of PCH of all chromosomes into chromocenters. The varying frequencies of inter-chromosomal interactions could result from non-random positioning of PCH regions upon mitotic exit or constraints imposed by other nuclear structures. For example, nucleoli, whose formation is driven by the transcription of rDNA arrays on the X chromosome, may impose structural constraints that lead to less frequent than expected spatial contacts involving X PCH. In addition, variation in biophysical properties (e.g., viscosity or varying protein compositions) among PCH domains arising from specific chromosomes could result in different frequencies of liquid-liquid fusion. Indeed, the 4th chromosome has a unique composition of histone modifications and chromatin proteins and depends on a specific suite of genes for its regulation (e.g., requirement of Egg for histone methylation), both of which could result in biophysical properties that promote frequent 3D contacts between 4th chromosome and specific PCH regions (Lee, 2020).

Importantly, the population genetic analysis reveals that euchromatic TEs with PCH interactions have lower population frequencies than TEs lacking frequent PCH contacts, suggesting that EU-PCH 3D interactions may influence individual fitness. What are the potential functional consequences of TE-PCH interactions that could influence individual fitness? TE-PCH interactions could lead to increased TE-induced enrichment of repressive epigenetic marks on neighboring sequences/genes. However, this study found no difference in the extent or the magnitude of H3K9me2 spread around TEs with and without PCH interactions, suggesting that TE-PCH interactions influence other aspects of nuclear organization critical for gene regulation and/or other genome functions. For instance, 3D interactions between PCH and TEs could bring neighboring euchromatic genes into the PCH domains and result in aberrant or enhanced silencing. On the other hand, the enrichment of HP1a, and likely spatial localization in the PCH domains, can promote the expression of genes in both PCH and the euchromatic genome. Still another possibility is that the spatial contact with PCH on one chromosome may 'drag' its homolog to the same nuclear compartment due to somatic homolog pairing, resulting in trans-silencing. A preliminary analysis found that ~15% of heterozygous TEs induced H3K9me2 enrichment not only in cis, but also in trans on the homologous chromosome without the TE insertion (i.e., trans-epigenetic effects). Accordingly, the fitness consequences of TE-PCH spatial interactions could potentially result from their positive or negative impacts on the expression of genes in cis or in trans to TEs, or from influencing other genome functions, such as replication and repair. Further studies are needed to test these hypotheses (Lee, 2020).

It is important to note that TEs comprise an appreciable fraction of the euchromatic genomes of virtually all eukaryotes. For instance, more than 50% of assembled human euchromatin contains TEs or TE-derived sequences, many of which are interspersed with actively transcribed genes and can influence gene expression through H3K9me2/3 spreading. Moreover, the presence of many TE insertions at specific locations are polymorphic between individuals in natural populations (e.g., human, Caenorhabditis, Drosophila, and Arabidopsis). Spatial interactions between euchromatic TEs and PCH can thus generate polymorphic 3D organization of the euchromatic genomes, leading to variation in critical biological functions that depend on chromosome conformations and even varying fitness between individuals. This investigation of the spatial architecture of PCH domains could thus have strong implications for how such 3D organizations could influence gene regulation, genome function, and even genome evolution of both heterochromatin and the gene-rich euchromatin (Lee, 2020).

Acetyltransferase Enok regulates transposon silencing and piRNA cluster transcription

The piRNA pathway is a highly conserved mechanism to repress transposon activation in the germline in Drosophila and mammals. This pathway starts from transcribing piRNA clusters to generate long piRNA precursors. The majority of piRNA clusters lack conventional promoters, and utilize heterochromatin- and HP1D/Rhino-dependent noncanonical mechanisms for transcription. However, information regarding the transcriptional regulation of piRNA clusters is limited. This study reports that the Drosophila acetyltransferase Enok, which can activate transcription by acetylating H3K23, is critical for piRNA production from 54% of piRNA clusters including 42AB, the major piRNA source. Surprisingly, it was found that Enok not only promotes rhino expression by acetylating H3K23, but also directly enhances transcription of piRNA clusters by facilitating Rhino recruitment. Taken together, this study provides novel insights into the regulation of noncanonical transcription at piRNA clusters and transposon silencing (Tsai, 2021).

In a wide range of organisms, repressing the activation of transposon insertions is essential for maintenance of genome stability. Small RNA-mediated heterochromatin formation plays important roles in silencing transposons in eukaryotic genomes. Mammals and Drosophila utilize the PIWI-interacting RNA (piRNA) pathway to achieve transcriptional and post-transcriptional silencing of transposons in the germline. In Drosophila, the piRNA pathway starts from transcription of the 142 piRNA clusters, usually ranging from 50 to a few hundred kilobases and containing multiple copies of truncated or full-length transposons, which produce long piRNA precursors. The long RNA precursors would then be processed through slicer- and Zucchini (Zuc)-dependent mechanisms into mature 23–29 nt piRNAs that get loaded to the Piwi protein. In addition, another two PIWI-clade Argonaute proteins, Ago3 and Aubergine (Aub), function in the ping-pong cycle to specifically amplify piRNAs against active transposons. Guided by complementary piRNAs, Ago3 and Aub can mediate degradation of transposon transcripts, and the Piwi-piRNA complex can also direct heterochromatin formation at the loci of transposons and piRNA clusters by recruiting epigenetic factors, resulting in effective repression of transposons both transcriptionally and post-transcriptionally (Tsai, 2021).

The Drosophila piRNA clusters can be divided into two classes: uni-strand, which produces piRNAs mainly from one genomic strand, and dual-strand, which produces piRNAs from both genomic strands. Transcription of the uni-strand clusters is proposed to be similar to the canonical transcription of protein-coding genes, as they contain clear promoter structures with enriched H3K4me2 and peaks of RNA polymerase II (Pol II). In contrast, dual-strand clusters lack clear Pol II promoter regions and are enriched for the heterochromatic H3K9me3 mark. Therefore, these clusters undergo noncanonical transcription that utilizes multiple internal initiation sites via heterochromatin- and Rhino (Rhi)-dependent mechanisms (Tsai, 2021).

Rhi is the germline-specific heterochromatin protein 1D (HP1D), and it associates with Deadlock (Del) and Cutoff (Cuff) to form the RDC complex. The RDC complex is recruited to dual-strand clusters by the interaction between H3K9me3 and the chromodomain of Rhi. At dual-strand clusters, the RDC complex licenses and promotes their transcription through four mechanisms. First, Del interacts with the germline-specific paralog of transcription factor IIA (TFIIA)-L, Moonshiner (Moon), and in turn recruits TFIIA and the TATA-box binding protein (TBP)-related factor TRF2 for transcription initiation. Second, the RDC complex has been shown to suppress the splicing of piRNA cluster transcripts, which is proposed to facilitate piRNA production. Third, Cuff recruits the transcription-export (TREX) complex to nascent transcripts to promote efficient transcription at piRNA clusters. Fourth, Cuff interferes with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex, and therefore prevents premature termination during transcription of piRNA precursors. While the positive roles of the RDC complex in noncanonical transcription of piRNA clusters were studied extensively, further transcriptional regulation upstream to the recruitment of this complex to piRNA clusters is still unclear (Tsai, 2021).

The KAT6 acetyltransferases are highly conserved from budding yeast to mammals, and preferentially acetylate histone H3 among the four core histones. The fly KAT6, Enok, has been shown to function as the major acetyltransferase for establishing the H3K23ac mark, which plays activating roles in transcription of genes. H3K23ac has been suggested to destabilize the interaction between H3K27me3 and the chromodomain of Polycomb, and therefore may contribute to transcription activation. In the ovarian germline, Enok is important for the maintenance of germline stem cells, and is required for proper polarization of oocytes by promoting expression of the actin nucleator spir. This study reports a novel role for Enok in the piRNA pathway. Mutating or knocking-down enok in the ovarian germline led to derepression of transposons and reduction in levels of piRNAs produced from a subset of piRNA clusters including the major piRNA source 42AB. Enok binds to and acetylates H3K23 in the 5' region of rhi, and is required for its normal expression levels in the ovary. It was further shown that Enok is also required for proper Rhi recruitment to a subset of piRNA clusters to promote their transcription. Therefore, Enok contributes to proper transposon silencing in the germline by promoting transcription of rhi and piRNA clusters (Tsai, 2021).

This paper reports a novel role for Enok in suppressing the activation of transposons in the germline. Loss of functional Enok in the ovarian germline resulted in activation of 7 transposon families. This amount of activated transposon families in enok mutant ovaries is comparable to the 17 families activated in the rhi mutant. RNA-seq analysis showed a ~75% reduction in the mRNA levels of rhi in enok mutant germline clone ovaries as compared with the WT control. Knocking down enok in the ovarian germline using two different UAS-shRNA-enok constructs also reduced the mRNA levels of rhi as compared with two different control fly lines. In addition, Enok ChIP-seq analysis revealed that Enok is localized to the 5' region of rhi, and the Enok-dependent enrichment of H3K23ac at the 5' end of rhi suggests that the Enok-mediated H3K23ac mark promotes rhi expression, contributing to proper piRNA production. Indeed, enok mutant germline clone ovaries showed decreased levels of piRNAs that mapped to Rhi-dependent source loci (RD-SL). However, not all RD-SL showed decreased piRNA levels in enok mutants. About 20% of the 6426 RD-SL showed reduced piRNA levels in enok mutants. Therefore, the remaining 25% of rhi levels in enok mutant ovaries may be sufficient to support transcription of the RD-SL that were not affected by loss of Enok. More strikingly, knocking down enok in the germline, without affecting the global protein levels of Rhi, reduced Rhi occupancies at Enok-dependent source loci (ED-SL) but not at Enok-independent source loci (EI-SL). This result suggests that Enok regulates Rhi recruitment specifically at ED-SL. The enok and rhi mutants show similar effects on the fold changes in transposon family expression and in antisense piRNAs. However, among the top 24 most highly overexpressed families in rhi, loss of Enok in the germline specifically activates 7 families. This specificity suggests that these 7 families may be more sensitive to reductions in Rhi recruitment to a subset of piRNA source loci. Taken together, Enok may contribute to fine-tuning transcription of piRNA clusters by modulating rhi expression and by regulating Rhi recruitment to Enok-dependent piRNA source loci (Tsai, 2021).

Three genome-wide RNAi screens have been reported before, but two of them were specifically performed in ovarian somatic cells. Knocking down enok in ovarian somatic cells using the tj-Gal4 driver did not activate the soma-dominant transposon, Gtwin, suggesting that Enok may be dispensable for transposon silencing in the soma. In the genome-wide screen in the germline, the enok RNAi construct (KK108400) is a long hairpin RNA. The efficiency of knocking down enok by long hairpin RNAs is lower than by short hairpin RNAs in the germline, even in the presence of additional Dicer-2. It has been shown that knocking down enok weakly activated the blood and Burdock transposons (z-scores of -0.5 and -0.74, respectively). However, this activation effect did not reach the threshold (z-score of -1.5 or lower) applied in the screen. This study used two different short hairpin RNA constructs against enok to deplete Enok in the germline, and therefore it was possible to detect stronger activation of transposons, possibly due to better knockdown efficiencies (Tsai, 2021).

Enok is the major enzyme responsible for the abundant H3K23ac mark. It was previously demonstrated that Enok is localized to the 5' end of its target genes, spir and mael, and promotes their expression by acetylating H3K23. This study further reports rhi and a subset of RD-SL (defined as ED-SL) as novel targets that are transcriptionally regulated by Enok. Intriguingly, while the 5' region of rhi is enriched with Enok and H3K23ac, Enok is not enriched at ED-SL relative to EI-SL. Also, knocking down enok in ovaries reduced the H3K23ac levels at the 5' end of rhi but not at piRNA clusters. These results suggest that Enok facilitates rhi expression by acetylating H3K23, but regulates the transcription of ED-SL through other mechanisms. Notably, knocking down enok in the ovarian germline severely reduced the Rhi occupancy to sites in 42AB, while global protein levels of Rhi and the H3K9me3 levels at 42AB were largely unaffected. Therefore, Enok is likely to promote transcription of ED-SL by regulating Rhi recruitment (Tsai, 2021).

The transcription of dual-strand piRNA clusters utilizes noncanonical heterochromatin-dependent internal initiation. Transcription initiation at these clusters was proposed to take place by the H3K9me3-bound RDC complex recruiting the germline-specific paralog of TFIIA-L. This study shows that Enok is important for both Rhi and Pol II occupancies at a subset of RD-SL (defined as ED-SL), suggesting that Enok can facilitate transcription of these piRNA source loci. As Rhi is highly enriched across the entire 42AB cluster and no Enok peaks were detected within 42AB, Enok is unlikely to regulate the Rhi occupancy at 42AB by directly recruiting it. Also, the Co-IP assay failed to detect interaction between Enok and the overexpressed Rhi in ovaries. Interestingly, the HAT activity of Enok is critical for transcription of 42AB even when rhi is overexpressed. Therefore, it is possible that Enok may play a role in acetylating some factors that are required for Rhi recruitment, or it may have an indirect role in Rhi recruitment by promoting expression of other genes with yet unidentified functions in the piRNA pathway. Notably, while knocking down enok in the germline decreased the RNA levels transcribed from both genomic strands at cl1-A and from the sense strand at cl1-32, RNA levels transcribed from the antisense strand at cl1-32 was not affected by depletion of Enok. Thus, within dual-strand clusters, Enok may regulate the internal initiation in specific regions. Taken together, this study provides novel information regarding noncanonical transcription and transposon silencing in the germline (Tsai, 2021).


4.8kb and 5.0kb repeats containing the histone genes His1, His2A, His2B, His3 and His4 were present in all of the more than 20 D. melanogaster strains studied. The strains differ in the relative amounts of the two repeat types, with the 5.0kb repeat always present in equal or greater amounts than the 4.8kb repeat. The strains also differ in a number of far less abundant fragments containing histone gene sequences. The expression of HIS-C genes, including His3, during oogenesis has been studied, and compared to periods of DNA synthesis and actin expression during this developmental stage. The D. virilis core histone genes (Dvir\His2B, Dvir\His3, Dvir\His4 and Dvir\His2A), are arranged in the same order and orientation as the D. melanogaster core histone genes (His2B, His3, His4 and His2A). However, the His1 gene that is located between His2B and His3 in D. melanogaster is not found between Dvir\His2B and Dvir\His3 in D. virilis. The genomic organization of the histone genes in D. hydei closely resembles that of D. melanogaster. The position of the homologous histone gene repeats within the nuclei of early embryo cells has been investigated. The two homologous histone gene clusters are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. During interphase of cycle 14, the two clusters colocalize with high frequency, and move from near the midline of the nucleus towards the apical side. The codon bias of the histone genes from D. melanogaster and D. hydei illustrates that the generalization -- that abundantly expressed genes have a high codon bias and low rates of silent substitution -- does not hold for the histone genes. DNA replication of the 5kb histone gene repeating unit in tissue culture cells (Drosophila Kc cells) initiates at multiple sites located within the repeating unit. Several replication pause sites are located at 5' upstream regions of some histone genes. The TFIID complex interacts with the promoter of His3 making contacts at the TATA element, initiator, +18 and +28 regions. Distinct specific subsets of lysines are utilized during deposition-related His4 diacetylation (FlyBase record for His3 and references therein).


Amino Acids - 135

For information on H3 structure see Structures of Histone Proteins and Proteins Containing the Histone Fold Motif and associated links.

Histone H3: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 25 April 2024

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