BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dual-core binding sites.

Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element-associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core-associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle

Polycomb Controls Gliogenesis by Regulating the Transient Expression of the Gcm/Glide Fate Determinant

The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system. © 2012 Popkova et al.Fondation pour la Recherche Médicale and by Centre Européen de Recherche en Biologie et en Médecine; Association pour la Recherche sur le Cancer; Institut National de la Santé et de la Recherche Médicale; Centre National de la Recherche Scientifique; Université de Strasbourg; Hôpital de Strasbourg; Institut National du Cancer; the Agence Nationale de la Recherche; Alma Mater Studiorum; Università di Bologna; European Research Council (ERC-2008-AdG No 232947); Institut National de la Santé et de la Recherche Médicale; Centre National de la Recherche Scientifique; European Network of Excellence EpiGeneSys; Fundacion Mutua Madrileña (FMM-2006) and Ministerio de Ciencia y Tecnología (BFU-2008-5404)Peer Reviewe

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS

Functional Anatomy of Polycomb and Trithorax Chromatin Landscapes in Drosophila Embryos

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation

Polycomb Domain Formation Depends on Short and Long Distance Regulatory Cues

<div><p>Background</p><p>Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In <i>Drosophila, cis</i>-regulatory regions termed PcG response elements (PREs) act as nucleation sites for PcG proteins to create large repressive PcG domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3). In addition to an action in <i>cis,</i> PREs can interact over long distances, thereby enhancing PcG dependent silencing. How PcG domains are established, which factors limit their propagation <i>in cis</i>, and how long range interactions of PREs <i>in trans</i> affect the chromatin structure is largely unknown.</p> <p>Principal Findings</p><p>We demonstrate that the insertion of a PRE-containing transgene in the <i>Drosophila</i> genome generates an artificial PcG domain and we analyze its organization by quantitative ChIP and ChIP-on-chip experiments. Intriguingly, a boundary element and known insulator proteins do not necessarily interfere with spreading of H3K27me3. Instead, domain borders correlate with the presence of promoter regions bound by RNA Polymerase II and active chromatin marks. In contrast, genes that are silent during early fly development get included within the PcG domain and this incorporation interferes with gene activation at later developmental stages. Moreover, <i>trans-</i>interaction of the transgenic PRE with its homologous endogenous PRE results in increased PcG binding, correlating with reinforced silencing of genes within the domain borders.</p> <p>Conclusions</p><p>Our results suggest that higher-order organization of PcG-bound chromatin can stabilize gene silencing within PcG domains. Further we propose that multi-protein complexes associated with active promoters are able to define the limits of PcG domains. Future work aimed to pinpoint the factors providing this barrier function will be required to understand the precise molecular mechanism by which active promoter regions can act as boundaries to stop spreading of H3K27me3.</p> </div

Chromatin state of the <i>Fab-7</i> PRE and the <i>sd</i> gene locus as a function of <i>Fab-7</i> long-range interactions in embryos.

<p>(A) Insertion of the a transgene containing a <i>Fab-7</i> element (shown as yellow cylinder) at the <i>scalloped</i> (<i>sd</i>) gene locus leads to pairing with the endogenous <i>Fab-7</i> element at the BX-C and increased silencing of the <i>mini-white</i> reporter gene (Fab-X). Long range interaction of <i>Fab-7</i> elements is dependent on sequence homology: Deletion of the endogenous <i>Fab-7</i> sequence (represented as black box), giving rise to the Fab-X, <i>Fab-7</i>¹ fly line results in loss of pairing and reduced silencing of the <i>mini-white</i> reporter gene. (B) qChIP analysis on 4–12 hours old embryos. Fly genotypes and antibodies used for IP are indicated at the bottom of the graph. Immunoprecipitated DNA was analysed by quantitative PCR with the primers amplifying the <i>Fab-7</i> element, or the <i>Rp49</i> gene. ChIP signal levels are represented as percentage of input chromatin precipitated for each region. The standard deviation was calculated from at least two independent experiments. Note that <i>Fab-7</i> primers amplify both, the endogenous <i>Fab-7</i> sequence and the transgenic element. (*P<0.05 as calculated from a two-tailed t-test). (C) and (D) ChIP analysis on 4–12 hours old embryos using H3K27me3 antibodies (C) or H3K4me3 antibodies (D). Immunoprecipitated DNA was analysed by quantitative PCR using primers amplifying genomic regions at the <i>sd</i> gene locus or the <i>Rp49</i> gene. Annotated genes are shown below the graph and are drawn in scale. ChIP signal levels are represented as percentage of input chromatin precipitated for each region. The standard deviation was calculated from two independent experiments. (*P<0.05 as calculated from a two-tailed t-test).</p

RNA Polymerase II binding and expression of genes at the <i>sd</i> gene locus during <i>Drosophila</i> development.

<p>(A) qChIP analysis on 4–12 hours old wild type embryos using RNA Pol II antibodies. Immunoprecipitated DNA was analysed by quantitative PCR using primers amplifying genomic regions at the <i>sd</i> gene locus or the <i>Rp49</i> gene. Annotated genes are shown below the graph and are drawn in scale. ChIP signal levels are represented as percentage of input chromatin precipitated for each region. The standard deviation was calculated from at least two independent experiments. Orange asterisk represents the transgene insertion site. (B) qRT PCR analysis of <i>PGRP</i>-LE, <i>sd</i>-RD, <i>CG8509</i> and <i>sd</i>-RE gene expression during <i>Drosophila</i> development. RNA was extracted from staged embryos at different times after egg laying or from female adult flies. RNA levels were normalized to the housekeeping gene <i>Rp49</i>. The standard deviation was calculated from at least two independent experiments. Primer pairs amplifying exon/intron junctions were used to determine levels of nascent transcripts.</p

Spreading of H3K27me3 into flanking genomic regions after insertion of the <i>Fab-7</i> transgene at the <i>sd</i> gene locus.

<p>(A) Schematic representation of the <i>Fab-7</i> containing transgene and the <i>scalloped</i> (<i>sd</i>) gene locus on the X chromosome. Orange asterisk represents the transgene insertion site. Note that the boundary region of the <i>Fab-7</i> element is located between the PRE and the reporter genes. (B) and (C) ChIP-on-chip analysis of the <i>sd</i> gene locus from 4–12 hours old embryos of indicated fly lines. Fold changes between IP with H3K27me3 antibodies (B) or H3K4me3 antibodies (C) and mock IP are plotted on the Y axis. Annotated genes are shown on the top of the panel. Orange asterisk indicates the transgene insertion site. (D) and (E) ChIP-on-chip analysis of <i>sd</i> gene locus from female adult flies in the indicated fly lines. Fold changes between IP with H3K27me3 antibodies (D) or H3K4me3 antibodies (E) and mock IP are plotted on the Y axis. Annotated genes are shown on the top of the panel. Orange asterisk indicates the transgene insertion site. Note that H3K27me3 can spread into downstream regions of the transgene insertion site until the <i>sd</i>-RE promoter region, which is marked by H3K4me3. No significant levels of H3K27me3 can be observed upstream the transgene insertion site. Levels of H3K4me3 do not change significantly after insertion of the <i>Fab-7</i> containing transgene.</p

RNA Polymerase II binding and expression of genes at the <i>sd</i> gene locus during <i>Drosophila</i> development before and after insertion of the <i>Fab-7</i> containing transgene.

<p>(A–C) qRT PCR analysis of <i>sd</i>-RD, <i>CG8509</i> and <i>sd</i>-RE gene expression during <i>Drosophila</i> development in WT, Fab-X and Fab-X,<i>Fab-7</i>¹ flies. RNA was extracted from staged embryos 2 hours (A) 12 hours (B) after egg laying or from female adult flies (C). RNA levels were normalized to the housekeeping gene <i>Rp49</i>. The standard deviation was calculated from at least two independent experiments. Note that primers amplifying the <i>sd</i>-RE transcript also amplify the <i>sd</i>-RD transcript. (D) qChIP analysis of female adult flies in the indicated fly lines using RNA Pol II antibodies. Immunoprecipitated DNA was analyzed by quantitative PCR using primers amplifying genomic regions at the <i>sd</i> gene locus or the <i>Rp49</i> gene. Annotated genes are shown below the graph and are drawn to scale. ChIP signal levels are represented as percentage of input chromatin precipitated for each region. The standard deviation was calculated from at least two independent experiments. (*P<0.05 as calculated from a two-tailed t-test).</p

Autoregulation of Mouse Histone Deacetylase 1 Expression

Histone deacetylase 1 (HDAC1) is a major regulator of chromatin structure and gene expression. Tight control of HDAC1 expression is essential for development and normal cell cycle progression. In this report, we analyzed the regulation of the mouse HDAC1 gene by deacetylases and acetyltransferases. The murine HDAC1 promoter lacks a TATA box consensus sequence but contains several putative SP1 binding sites and a CCAAT box, which is recognized by the transcription factor NF-Y. HDAC1 promoter-reporter studies revealed that the distal SP1 site and the CCAAT box are crucial for HDAC1 promoter activity and act synergistically to constitute HDAC1 promoter activity. Furthermore, these sites are essential for activation of the HDAC1 promoter by the deacetylase inhibitor trichostatin A (TSA). Chromatin immunoprecipitation assays showed that HDAC1 is recruited to the promoter by SP1 and NF-Y, thereby regulating its own expression. Coexpression of acetyltransferases elevates HDAC1 promoter activity when the SP1 site and the CCAAT box are intact. Increased histone acetylation at the HDAC1 promoter region in response to TSA treatment is dependent on binding sites for SP1 and NF-Y. Taken together, our results demonstrate for the first time the autoregulation of a histone-modifying enzyme in mammalian cells