MacroH2A histone variants have a major role in nuclear organization and tether heterochromatinic repeats to lamin B1
ABSTRACT: Genetic loss-of-function studies in development, cancer and somatic cell reprogramming suggested that the group of macroH2A histone variants could function through stabilizing the differentiated state by a yet unknown mechanism. Here, we now present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences in an H3K9me3-dependent manner. We further identify macroH2A on sites of interstitial heterochromatin. Loss of macroH2A leads to major defects in nuclear organization including loss of nuclear circularity, disaggregation of nucleoli and a global loss of dense heterochromatin. Domains formed by repeat sequences when depleted of macroH2A are disorganized, expanded and fragmented, and partially re-expressed. This can be explained by our finding that macroH2A tethers these repeats to the nucleostructural protein Lamin B1. ChIPseq to determine the distribution of histone variants macroH2A1 and macroH2A2 were realized in the HepG2 cell line
Project description:Genetic loss-of-function studies in development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by H3K9me3. Loss of macroH2A leads to major defects in nuclear organization including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by repeat sequences when depleted of macroH2A are disorganized, expanded and fragmented and mildly re-expressed. On the molecular level we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein Lamin B1. Taken together our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher order chromatin architecture. Overall design: ChIPseq of macroH2A1 and macroH2A2 on HepG2 Cell-line
Project description:Transcription factor-induced reprogramming of somatic cells to pluripotency is a very inefficient process, probably due to the existence of important epigenetic barriers that are imposed during differentiation and that contribute to preserve cell identity. In an effort to decipher the molecular nature of these barriers, we followed a genome-wide approach, in which we identified macro histone variants (macroH2A) as highly expressed in human somatic cells but downregulated after reprogramming to pluripotency, as well as strongly induced during differentiation. Knock down of macro histone variants in human keratinocytes increased the efficiency of reprogramming to pluripotency, while overexpression had opposite effects. Genome-wide occupancy profiles show that in human keratinocytes macroH2A.1 preferentially occupies genes that are expressed at low levels and are marked with H3K27me3, including pluripotency-related genes and bivalent developmental regulators, at which its presence prevents the regain of H3K4me2 during reprogramming, over imposing an additional layer of repression that preserves cell identity. Gemone wide occupancy of HA:macroH2A.1 in human keratinocytes
Project description:MacroH2As core histone variants have a unique structure that includes C-terminal nonhistone domain. MacroH2As are highly conserved in vertebrates, and are thought to regulate gene expression. However the nature of genes regulated by macroH2As and the biological significance of macroH2As for the organism remain unclear. Our gene expression studies indicate that macroH2A.1 and macroH2A.2 work together to regulate specific genes. In these studies we examine the distributions of macroH2A.1 and macroH2A.2 nucleosomes to determine if they are localized to the genes that show altered expression in macroH2A knockout mouse liver. MacroH2A.1 and macroH2A.2 nucleosomes prepared from ~ 50 fetal mouse livers were purified by thio-affinity chromatography. Five samples were sequenced: Thiopropyl Sepharose, Normal Liver - contains mononucleosomal DNA from macroH2A.1-containing nucleosomes; Activate Thiol Sepharose, Normal Liver - contains mononucleosomal DNA primarily from macroH2A.2-containing nucleosomes. Starting Material, Normal Liver - this is a reference samplefor the first two samples. It contains mononucleosomal DNA from bulk fetal liver chromatin. Activated Thiol Sepharose, Knockout Livers - this is a control sample that contains mononucleosomal DNA from non-macroH2A nucleosomes that contaminate the macroH2A.2 nucleosomes. This fraction was prepared from macroH2A1/2 double knockout fetal livers; Starting Material, Knockout Liver - this is a reference sample for the fourth sample. It contains mononucleosomal DNA from bulk chromatin prepared from macroH2A1/2 double knockout fetal livers.
Project description:The distribution of histone variants H2Abbd and macroH2A in 13 regions of the HG18 assembly have been studied using a variant of the ChIP-on-Chip technique. HeLa S3 cell lines expressing tagged histones H2A, H2Abbd or macroHA were obtained using retroviral transfer. DNA fractions associated with tagged histones were isolated using a two-step purification procedure that involved affinity chromatography on a column with anti-FLAG antibodies, followed by affinity chromatography on a Ni-agarose column. The obtained genomic DNA samples were analyzed by hybridization with custom NimbleGene genomic microarrays. Two samples. Test sample 1 is HeLa S3 cells expressing epitope-tagged histone H2Abbd and test sample 2 is HeLa S3 cells expressing epitope-tagged histone macroH2A . The control for both test sample 1 and test sample 2 is HeLa S3 expressing epitope-tagged histone H2A. Two copies of each probe per array were made.
Project description:MacroH2A1 ChIP-chip was performed on custom Nimblegen genomic tiling arrays, to understand the genomic binding patterns of this histone variant and its relationship to gene expression Keywords: ChIP-chip Two macroH2A biological replicates are included. An H3 ChIP-chip sample is included as a control
Project description:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the novel heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1 (HP1)-like protein Pdd1p. Through the identification and mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore suggest that heterochromatin bodies are assembled by the Pdd1p-RNA interaction. Jub1p and Pdd1p dephosphorylation are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body of itself plays an essential role in DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type different mutant cells at 12 hpm, shared chromatin was immunoprecipitated and precipitated DNA was analyzed by high-throughput sequencing.
Project description:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. We previously reported that dephosphorylation of the HP1-like protein Pdd1p is required for the formation of heterochromatin bodies during the process of programmed DNA elimination in the ciliated protozoan Tetrahymena. Here, we show that the heterochromatin body component Jub4p is required for Pdd1p phosphorylation, heterochromatin body formation and DNA elimination. Moreover, our analyses of unphosphorylatable Pdd1p mutants demonstrate that Pdd1p phosphorylation is required for heterochromatin body formation and DNA elimination, while it is dispensable for local heterochromatin assembly. Therefore, both phosphorylation and the following dephosphorylation of Pdd1p are necessary to facilitate the formation of heterochromatin bodies. We suggest that Jub4p-mediated phosphorylation of Pdd1p creates a chromatin environment that is a prerequisite for subsequent heterochromatin body assembly and DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type and various mutant cells at 12 hpm (hours post-mixing), sheared chromatin was immunoprecipitated andprecipitated DNA was analyzed by high-throughput sequencing
Project description:Heterochromatin protein 1 (HP1) proteins are important regulators of heterochromatin mediated gene silencing and chromosome structure and it is well known as the reader of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me). In Drosophila three different histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9; Su(var)3-9, Setdb1 and G9a. To gain insights on the dependence of HP1a on the three different HKMTs, the division of labor between these methyl transferases and the dependence of HP1a on H3K9me we have studied HP1a binding in relation to H3K9me in mutants of these HKMTs. We show that Su(var)3-9 is responsible for the HP1a H3K9me-dependent binding in pericentromeric regions while Setdb1 controls the HP1a H3K9me-dependent binding to cytological region 2L:31 and together with POF chromosome 4. HP1a binds to the promoters and within gene bodies of active genes in these three regions. More importantly, HP1a bound at promoters of active genes are independent of H3K9me and POF and is associated to heterochromatin protein 2 (HP2) and open chromatin. Our results supports a model where HP1a nucleates with high affinity independent of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites. In total 44 samples; 2 replicates for each genotype and for each ChIP (HP1a, H3K9me2 and H3K9me3)