Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone H2A variants and histone modifications in WT Col-0 seedlings.
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ABSTRACT: How histone intrinsic sequence variation or regulatory modifications regulate nucleosome interactions with transcription remain unclear. To clarify this question, we examined how histone variants and histone modifications assemble in the Arabidopsis thaliana genome, identifying a limited number of chromatin states that divide euchromatin and heterochromatin in biologically significant subdomains. We showed that histone variants were as significant as histone modifications to determine the composition of chromatin states. The loss of function of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) prevented the exchange between the histone variants H2A.Z and H2A.W over transposons resulting in their enrichment in chromatin states found only on proteins coding genes in the wild type. Hence, the dynamics of histone H2A variants exchange impacted the definition and distribution of chromatin states. We propose that dynamics of histone variants control the organization of histone modifications into chromatin states to achieve landmarks that signify the ability for transcription. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone H2A variants and histone modifications in seedlings .
Project description:How histone intrinsic sequence variation or regulatory modifications regulate nucleosome interactions with transcription remain unclear. To clarify this question, we examined how histone variants and histone modifications assemble in the Arabidopsis thaliana genome, identifying a limited number of chromatin states that divide euchromatin and heterochromatin in biologically significant subdomains. We showed that histone variants were as significant as histone modifications to determine the composition of chromatin states. The loss of function of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) prevented the exchange between the histone variants H2A.Z and H2A.W over transposons resulting in their enrichment in chromatin states found only on proteins coding genes in the wild type. Hence, the dynamics of histone H2A variants exchange impacted the definition and distribution of chromatin states. We propose that dynamics of histone variants control the organization of histone modifications into chromatin states to achieve landmarks that signify the ability for transcription. Chromatin immunoprecipitation sequencing (ChIP-seq) for histone H2A variants and histone modifications in Col0 and ddm1(-/-) .
Project description:Histone variants play crucial roles in gene expression, genome integrity and chromosome segregation. However, to what extent histone variants control chromatin architecture remains largely unknown. Here, we show that the previously uncharacterized histone variant H2A.W plays a crucial role in condensation of heterochromatin. Genome-wide profiling of all four types of H2A variants in Arabidopsis shows that H2A.W specifically associates with heterochromatin. H2A.W recruitment is independent of heterochromatic marks H3K9me2 and DNA methylation. Genetic interactions show that H2A.W acts in synergy with CMT3 mediated methylation to maintain genome integrity. In vitro, H2A.W enhances chromatin condensation through a higher propensity to make fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, elimination of H2A.W causes decondensation of heterochromatin and conversely, ectopic expression of H2A.W promotes heterochromatin condensation. These results demonstrate that H2A.W plays critical roles in heterochromatin by promoting higher order chromatin condensation. Since similar H2A.W C-terminal motifs are present in other variant found in mammals and other organisms our findings impact our understanding of heterochromatin condensation in a wide variety of eukaryotic organisms. Two mRNA-seq samples, two bisulfite-seq samples, six ChIP-seq samples.
Project description:Diversification of histone variants is marked by the acquisition of distinct motifs and features through convergent evolution. H2A variants tend to be associated with defined domains of the genome. Specific features distinguish H2A variants in eukaryotes but whether evolution of these features predated the evolution of deposition mechanisms or vice-versa has remained unclear.In flowering plants, the variant H2A.W is tightly associated with heterochromatin. H2A.W evolved in land plants through acquisition of an extended C-terminal tail enriched with basic residues and a KSPK motif. Here, we used a synthetic approach in fission yeast, which lacks H2A.W and its dedicated deposition mechanism, to recapitulate the evolutionary steps that led to H2A.W and to assess the impact of the KSPK motif on heterochromatin composition and its properties. In conclusion, the acquisition of the KSPK motif in yeast promotes chromatin properties that are comparable to the properties and function of H2A.W in plant heterochromatin. Hence, the KSPK motif could have been selected before the evolution of direct heterochromatin deposition mechanisms. We propose that the acquisition of functional histone variant motifs can confer properties which affect only specific chromatin states, thereby driving the evolution of specific deposition mechanisms.
Project description:Diversification of histone variants is marked by the acquisition of distinct motifs and features through convergent evolution. H2A variants tend to be associated with defined domains of the genome. Specific features distinguish H2A variants in eukaryotes but whether evolution of these features predated the evolution of deposition mechanisms or vice-versa has remained unclear.In flowering plants, the variant H2A.W is tightly associated with heterochromatin. H2A.W evolved in land plants through acquisition of an extended C-terminal tail enriched with basic residues and a KSPK motif. Here, we used a synthetic approach in fission yeast, which lacks H2A.W and its dedicated deposition mechanism, to recapitulate the evolutionary steps that led to H2A.W and to assess the impact of the KSPK motif on heterochromatin composition and its properties. In conclusion, the acquisition of the KSPK motif in yeast promotes chromatin properties that are comparable to the properties and function of H2A.W in plant heterochromatin. Hence, the KSPK motif could have been selected before the evolution of direct heterochromatin deposition mechanisms. We propose that the acquisition of functional histone variant motifs can confer properties which affect only specific chromatin states, thereby driving the evolution of specific deposition mechanisms.
Project description:Diversification of histone variants is marked by the acquisition of distinct motifs and features through convergent evolution. H2A variants tend to be associated with defined domains of the genome. Specific features distinguish H2A variants in eukaryotes but whether evolution of these features predated the evolution of deposition mechanisms or vice-versa has remained unclear.In flowering plants, the variant H2A.W is tightly associated with heterochromatin. H2A.W evolved in land plants through acquisition of an extended C-terminal tail enriched with basic residues and a KSPK motif. Here, we used a synthetic approach in fission yeast, which lacks H2A.W and its dedicated deposition mechanism, to recapitulate the evolutionary steps that led to H2A.W and to assess the impact of the KSPK motif on heterochromatin composition and its properties. In conclusion, the acquisition of the KSPK motif in yeast promotes chromatin properties that are comparable to the properties and function of H2A.W in plant heterochromatin. Hence, the KSPK motif could have been selected before the evolution of direct heterochromatin deposition mechanisms. We propose that the acquisition of functional histone variant motifs can confer properties which affect only specific chromatin states, thereby driving the evolution of specific deposition mechanisms.
Project description:Histone variants play crucial roles in gene expression, genome integrity and chromosome segregation. However, to what extent histone variants control chromatin architecture remains largely unknown. Here, we show that the previously uncharacterized histone variant H2A.W plays a crucial role in condensation of heterochromatin. Genome-wide profiling of all four types of H2A variants in Arabidopsis shows that H2A.W specifically associates with heterochromatin. H2A.W recruitment is independent of heterochromatic marks H3K9me2 and DNA methylation. Genetic interactions show that H2A.W acts in synergy with CMT3 mediated methylation to maintain genome integrity. In vitro, H2A.W enhances chromatin condensation through a higher propensity to make fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, elimination of H2A.W causes decondensation of heterochromatin and conversely, ectopic expression of H2A.W promotes heterochromatin condensation. These results demonstrate that H2A.W plays critical roles in heterochromatin by promoting higher order chromatin condensation. Since similar H2A.W C-terminal motifs are present in other variant found in mammals and other organisms our findings impact our understanding of heterochromatin condensation in a wide variety of eukaryotic organisms.
Project description:Silencing of transposons by the chromatin remodeler DDM1 is mediated by the deposition of heterochromatic H2A variants. Transposon mobility and silencing participates in genome evolution but also threaten genome integrity. DECREASED DNA METHYLATION 1 (DDM1) belongs to a conserved family of chromatin remodelers that are required to silence transposons, yet the underlying molecular mechanism has remained unknown. Here we show that DDM1 binds the histone variant H2A.W through two conserved domains that are required to deposit H2A.W and maintain transposon silencing. The mechanism of transcriptional silencing described here is likely shared among chromatin remodelers of the DDM1 family and heterochromatic H2A variants that have evolved in mammals.
Project description:Silencing of transposons by the chromatin remodeler DDM1 is mediated by the deposition of heterochromatic H2A variants. Transposon mobility and silencing participates in genome evolution but also threaten genome integrity. DECREASED DNA METHYLATION 1 (DDM1) belongs to a conserved family of chromatin remodelers that are required to silence transposons, yet the underlying molecular mechanism has remained unknown. Here we show that DDM1 binds the histone variant H2A.W through two conserved domains that are required to deposit H2A.W and maintain transposon silencing. The mechanism of transcriptional silencing described here is likely shared among chromatin remodelers of the DDM1 family and heterochromatic H2A variants that have evolved in mammals.
Project description:Silencing of transposons by the chromatin remodeler DDM1 is mediated by the deposition of heterochromatic H2A variants. Transposon mobility and silencing participates in genome evolution but also threaten genome integrity. DECREASED DNA METHYLATION 1 (DDM1) belongs to a conserved family of chromatin remodelers that are required to silence transposons, yet the underlying molecular mechanism has remained unknown. Here we show that DDM1 binds the histone variant H2A.W through two conserved domains that are required to deposit H2A.W and maintain transposon silencing. The mechanism of transcriptional silencing described here is likely shared among chromatin remodelers of the DDM1 family and heterochromatic H2A variants that have evolved in mammals.
Project description:The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange where either H2A.Z or H2A can be exchanged from nucleosomes. This result is confirmed in vivo, where genome-wide analysis demonstrates widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a M-bM-^@M-^\lockM-bM-^@M-^] that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators. H2A.Z ChIP seq experiments in mutants with constitutive H3K56ac