http://repub.eur.nl/res/aut/15813/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/38304/ 2012-02-01T00:00:00Z The nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of Drosophila NURD, (P)BAP, INO80, and ISWI, representing the four major remodeler families. Each remodeler has a unique set of genomic targets and generates distinct chromatin signatures. Remodeler loci have characteristic DNA sequence features, predicted to influence nucleosome formation. Strikingly, remodelers counteract DNA sequence-driven nucleosome distribution in two distinct ways. NURD, (P)BAP, and INO80 increase histone density at their target sequences, which intrinsically disfavor positioned nucleosome formation. In contrast, ISWI promotes open chromatin at sites that are propitious for precise nucleosome placement. Remodelers influence nucleosome organization genome-wide, reflecting their high genomic density and the propagation of nucleosome redistribution beyond remodeler binding sites. In transcriptionally silent early embryos, nucleosome organization correlates with intrinsic histone-DNA sequence preferences. Following differential expression of the genome, however, this relationship diminishes and eventually disappears. We conclude that the cellular nucleosome landscape is the result of the balance between DNA sequence-driven nucleosome placement and active nucleosome repositioning by remodelers and the transcription machinery. http://repub.eur.nl/res/pub/25547/ 2011-04-01T00:00:00Z http://repub.eur.nl/res/pub/25577/ 2011-04-01T00:00:00Z Protein ubiquitylation is involved in the regulation of virtually all aspects of eukaryotic cell biology, including gene expression. The central function of E3 ubiquitin ligases in target selection is well established. More recently, it has become appreciated that deubiquitylating enzymes (DUBs) are crucial components of ubiquitin-regulated cellular switches. Here, we discuss advances in our understanding of how DUBs regulate chromatin dynamics and gene expression. DUBs are integral components of the transcription machinery, involved in both gene activation and repression. They modulate the ubiquitylation status of histones H2A and H2B, which play pivotal roles in a cascade of molecular events that determine chromatin status. A DUB module in the SAGA coactivator complex is required for gene activation, whereas other DUBs are part of the Polycomb gene-silencing machinery. DUBs also control the level or subcellular compartmentalization of selective transcription factors, including the tumour suppressor p53. Typically, DUB specificity and activity are defined by its partner proteins, enabling remarkably versatile and sophisticated regulation. Recent findings not only underscore the pervasive and pivotal role of DUBs in gene expression control, but also raise paradoxical questions concerning the molecular mechanisms involved. http://repub.eur.nl/res/pub/21317/ 2010-11-01T00:00:00Z ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment. http://repub.eur.nl/res/pub/27846/ 2010-02-01T00:00:00Z Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/ USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors. Copyright http://repub.eur.nl/res/pub/25193/ 2009-12-08T00:00:00Z An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that ∼500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development. http://repub.eur.nl/res/pub/20193/ 2009-12-01T00:00:00Z Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK- mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases. http://repub.eur.nl/res/pub/17388/ 2009-09-24T00:00:00Z Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing. http://repub.eur.nl/res/pub/16431/ 2009-04-01T00:00:00Z Polycomb group (PcG) proteins are transcriptional repressors that control expression of developmental regulator genes in animals and plants. Recent advances in our understanding of the PcG system include biochemical purifications that revealed a substantial variety in PcG complex composition. These different complexes contain distinct chromatin-modifying activities and engage in cross-talk with other chromatin modifications. Complementing these biochemical analyses, structural studies have begun to provide insight into how PcG proteins interact with each other and with chromatin. Finally, genome-wide binding profiling and the ensuing functional analysis of target gene regulation revealed that the PcG system is not only used for the permanent silencing of developmental regulator genes. Rather, PcG mediated repression also constitutes a mechanism for dynamic control of gene transcription. http://repub.eur.nl/res/pub/14491/ 2008-10-15T00:00:00Z Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression. http://repub.eur.nl/res/pub/29503/ 2008-05-01T00:00:00Z Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16INK4ainduction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15INK4band p16INK4a, but not of p14ARF. Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control. Copyright http://repub.eur.nl/res/pub/29557/ 2008-05-01T00:00:00Z SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme. Copyright http://repub.eur.nl/res/pub/29488/ 2008-03-01T00:00:00Z Protein ubiquitylation plays a central role in multiple signal transduction pathways. However, the substrate specificity and potential developmental roles of deubiquitylating enzymes remain poorly understood. Here, we show that the Drosophila ubiquitin protease UBP64 controls cell fate in the developing eye. UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Using a proteomics approach, we identified the transcriptional repressor Tramtrack (TTK) as a primary UBP64 substrate. In common with TTK, reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Previously, it was demonstrated that the blockade of neuronal cell fate was relieved by SINA-dependent ubiquitylation and degradation of TTK. We found that UBP64 counteracts SINA function by deubiquitylating TTK, leading to its stabilization and thereby promoting a nonneuronal cell fate. Mass spectrometric mapping revealed that SINA ubiquitylates multiple sites dispersed throughout TTK, which are duly deubiquitylated by UBP64. This observation suggests that both E3 SINA and UBP64 use a scanning mechanism to (de)ubiquitylate TTK. We conclude that the balance of TTK ubiquitylation by SINA and deubiquitylation by UBP64 constitutes a specific posttranslational switch controlling cell fate. Copyright http://repub.eur.nl/res/pub/36969/ 2007-10-09T00:00:00Z The histone chaperone Asf1 assists in chromatin assembly and remodeling during replication, transcription activation, and gene silencing. However, it has been unclear to what extent Asf1 could be targeted to specific loci via interactions with sequence-specific DNA-binding proteins. Here, we show that Asf1 contributes to the repression of Notch target genes, as depletion of Asf1 in cells by RNAi causes derepression of the E(spl) Notch-inducible genes. Conversely, overexpression of Asf1 in vivo results in decreased expression of target genes and produces phenotypes that are strongly modified (enhanced and suppressed) by mutations affecting the Notch pathway, but not by mutations in other signaling pathways. Asf1 can be coprecipitated with the DNA-binding protein Su(H) and the corepressor Hairless and interacts directly with two components of this complex, Hairless and SKIP. Thus, in addition to playing more general roles in chromatin dynamics, Asf1 is directed via interactions with sequence-specific complexes to mediate silencing of specific target genes. http://repub.eur.nl/res/pub/35984/ 2007-01-01T00:00:00Z Drosophila BAP and PBAP represent two evolutionarily conserved subclasses of SWI/SNF chromatin remodelers. The two complexes share the same core subunits, including the BRM ATPase, but differ in a few signature subunits: OSA defines BAP, whereas Polybromo (PB) and BAP170 specify PBAP. Here, we present a comprehensive structure-function analysis of BAP and PBAP. An RNA interference knockdown survey revealed that the core subunits BRM and MOR are critical for the structural integrity of both complexes. Whole-genome expression profiling suggested that the SWI/SNF core complex is largely dysfunctional in cells. Regulation of the majority of target genes required the signature subunit OSA, PB, or BAP170, suggesting that SWI/SNF remodelers function mostly as holoenzymes. BAP and PBAP execute similar, independent, or antagonistic functions in transcription control and appear to direct mostly distinct biological processes. BAP, but not PBAP, is required for cell cycle progression through mitosis. Because in yeast the PBAP-homologous complex, RSC, controls cell cycle progression, our Finding reveals a functional switch during evolution. BAP mediates G2/M transition through direct regulation of string/cdc25. Its signature subunit, OSA, is required for directing BAP to the string/cdc25 promoter. Our results suggest that the core subunits play architectural and enzymatic roles but that the signature subunits determine most of the functional specificity of SWI/SNF holoenzymes in general gene control. Copyright http://repub.eur.nl/res/pub/13882/ 2005-08-01T00:00:00Z Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Here, we report that Pleiohomeotic (PHO) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. We identified a conserved sequence motif, named PCC-binding element (PBE), which is required for PcG silencing in vivo. PHO sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive PHO/PCC complex. We termed this nucleoprotein complex silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome.