Project description:A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin [ChIP]
Project description:Nucleosome composition actively contribute to the chromatin structure and accessibility. To preserve chromatin state during replication, transcription and DNA repair, cells have evolved mechanisms to evict or recycle histones, generating a landscape of differentially aged nucleosomes. To map the stability of nucleosomes, we have adapted the recombination induced tag exchange (RITE) method to Schizosaccharomyces pombe histone H3. The RITE method allows us to study replication-independent protein turnover both through the occurrence of, in our case, new histone H3 and the disappearance or preservation of old histone H3. We contrast the RITE system to nucleosome turnover measured by chromatin incorporation of an epitope-tagged H3 under an inducible promoter. We confirm previous findings that stable nucleosomes are found at heterochromatin, but also at coding regions of actively transcribed genes. Genome-wide comparisons with several chromatin marks showed that high turnover nucleosomes correlate with H2A.Z, acetylated H4 and H3K4me2. The histones with high turnover are primarily found at the nucleosomes on the 5´and/or 3´ edges of the transcribed unit. In addition, in this study we have determined genome-wide maps of all three methylation marks at H4K20. All methylation of H4K20 appeared in low turnover nucleosomes and particularly in euchromatic regions. H4K20me1 marks stable nucleosomes at loci proximal to nucleosome depleted regions (NDR) and H4K20me2/3 were found further inside of transcribed units, especially at coding regions of long genes expressed at low levels. Further, this transcription-dependent accumulation of histone methylations was dependent on the histone chaperone complex FACT (Facilitates Chromatin Transcription), as predicited from its role in recycling nucleosomes during transcription.
Project description:Nucleosome composition actively contribute to the chromatin structure and accessibility. To preserve chromatin state during replication, transcription and DNA repair, cells have evolved mechanisms to evict or recycle histones, generating a landscape of differentially aged nucleosomes. To map the stability of nucleosomes, we have adapted the recombination induced tag exchange (RITE) method to Schizosaccharomyces pombe histone H3. The RITE method allows us to study replication-independent protein turnover both through the occurrence of, in our case, new histone H3 and the disappearance or preservation of old histone H3. We contrast the RITE system to nucleosome turnover measured by chromatin incorporation of an epitope-tagged H3 under an inducible promoter. We confirm previous findings that stable nucleosomes are found at heterochromatin, but also at coding regions of actively transcribed genes. Genome-wide comparisons with several chromatin marks showed that high turnover nucleosomes correlate with H2A.Z, acetylated H4 and H3K4me2. The histones with high turnover are primarily found at the nucleosomes on the 5´and/or 3´ edges of the transcribed unit. In addition, in this study we have determined genome-wide maps of all three methylation marks at H4K20. All methylation of H4K20 appeared in low turnover nucleosomes and particularly in euchromatic regions. H4K20me1 marks stable nucleosomes at loci proximal to nucleosome depleted regions (NDR) and H4K20me2/3 were found further inside of transcribed units, especially at coding regions of long genes expressed at low levels. Further, this transcription-dependent accumulation of histone methylations was dependent on the histone chaperone complex FACT (Facilitates Chromatin Transcription), as predicited from its role in recycling nucleosomes during transcription.
Project description:Goal of this project was the identification of chromatin interacting proteins whose binding is differentially regulated by di-methylation of lysine 20 on histone H4 (H4K20me2). To achieve this unodified and H4K20me2-modified histone H4 were generated by native chemical ligation and assembled into recombinant di-nucleosomes. The di-nucleosomes were immobilized on streptavidin-coated beads via the biotinylated di-nucleosomal DNA and used for nucleosome affinity purifications to identify proteins regulated by H4K20me2 from SILAC-labelled HeLaS3 nuclear extracts (Arg10 and Lys8). SILAC affinity purifications were carried out in "forward" (heavy extract on modified nucleosome and light extract on unmodified nucleosome) and "reverse" (light extract on modified nucleosome and heavy extract on unmodified nucleosome) label-swap experiments and protein abundances were quantified by MaxQuant.
Project description:Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5’ promoter and 3’ termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.
Project description:A striking unusual genome architecture characterizes the two related human parasitic pathogens Plasmodium falciparum and Toxoplasma gondii. A major fraction of the bulk parasite genome is packaged as transcriptionally permissive euchromatin with few loci embedded in silenced heterochromatin. Primary chromatin shapers include histone modifications at the nucleosome lateral surface close to the DNA but their mode of action remains unclear. We identify versatile modifications at Lys31 within the globular domain of histone H4 as key determinants of genome organization and expression in Apicomplexa. H4K31 acetylation promotes a relaxed chromatin state at the promoter of active genes through nucleosome disassembly in both parasites. In contrast, monomethylated H4K31 is enriched in the core body of Toxoplasma active genes but inversely correlates with transcription while being astonishingly enriched at transcriptionally inactive pericentromeric heterochromatin in Plasmodium. This is the first evidence for a methylated residue of H4 associating with transcriptional regulation likely by reducing histone turnover hence slowing RNA polymerase progression across transcribed loci.
Project description:Histone acetylation is important for the activation of gene transcription but little is known about its direct ‘read/write’ mechanisms. Here, we report cryo-electron microscopy structures in which a p300/CBP multidomain monomer recognizes histone H4 N-terminal tail (NT) acetylation (ac) in a nucleosome and acetylates non-H4 histone NTs within the same nucleosome. p300/CBP not only recognized H4NTac via the bromodomain pocket responsible for ‘reading’, but also interacted with the DNA minor grooves via the outside of that pocket. This directed the catalytic center of p300/CBP to one of the non-H4 histone NTs. The primary target that p300 ‘writes’ by ‘reading’ H4NTac was H2BNT, and H2BNTac promoted H2A-H2B dissociation from the nucleosome. We propose a model in which p300/CBP ‘replicates’ histone NT acetylation within the H3-H4 tetramer to inherit epigenetic storage, and ‘transcribes’ it from the H3-H4 tetramer to the H2B-H2A dimers to activate context-dependent gene transcription through local nucleosome destabilization.
Project description:Plants often experience recurrent stressful events, for example during heat waves. They can adapt to such recurrent heat stress (HS), allowing subsequent survival of more severe HS conditions. At certain genes HS induces sustained expression for several days beyond the actual HS. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), however, how this is maintained for extended periods of time is unclear. Here, we determined histone turnover by measuring chromatin association of a HS-induced histone H3.3. Genome-wide Histone turnover was not homogenous, in particular, H3.3 was retained longer at HS memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, its loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation (and not vice versa). Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally-mediated epigenetic modifications.
Project description:Compared to the initiation and elongation stages of transcription, the role of chromatin in transcription termination is poorly understood. Through a yeast genetic screen, we identified histone H3 and H4 substitutions that cause transcription to read through the terminator of a small noncoding gene. The substitutions map to the nucleosome DNA entry-exit site, a region that controls nucleosome stability and certain histone modifications. Genome-wide studies on the strongest mutants revealed evidence of transcription read-through of noncoding and coding genes and reduced nucleosome occupancy. Replacement of the native sequence downstream of a gene with a “superbinder” sequence that increases nucleosome occupancy in vivo increased termination efficiency and suppressed the effect of a DNA entry-exit site substitution at this locus. Our results highlight the importance of the DNA entry-exit site in maintaining the integrity of the transcriptome and suggest that nucleosomes can facilitate termination by serving as a barrier to RNA polymerase.
Project description:A multitude of histone chaperones is required to protect histones from their biosynthesis to DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways is unclear. Using explorative interactomics approaches, we map the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperones systems. We identify and validate a panel of novel histone (PTM) dependent complexes. We show DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylated new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly.