Postmitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal chromatin, transcriptome, and identity
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ABSTRACT: Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide long-term histone replacement in postmitotic cells, including neurons. Beyond replenishment, histone variants also play active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover. Here, we uncover crucial roles for the histone H3 variant H3.3 in neuronal development. We find that newborn cortical excitatory neurons, which have only just completed replication-coupled deposition of canonical H3.1 and H3.2, substantially accumulate H3.3 immediately post mitosis. Co-deletion of H3.3-encoding genes H3f3a and H3f3b from newly postmitotic neurons abrogates H3.3 accumulation, markedly alters the histone posttranslational modification (PTM) landscape, and causes widespread disruptions to the establishment of the neuronal transcriptome. These changes coincide with developmental phenotypes in neuronal identities and axon projections. Thus, preexisting, replication-dependent histones are insufficient for establishing neuronal chromatin and transcriptome; de novo H3.3 is required. Stage-dependent deletion of H3f3a and H3f3b from (1) cycling neural progenitor cells, (2) neurons immediately post mitosis, or (3) several days later, reveals the first postmitotic days to be a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, co-deletion of H3f3a and H3f3b does not lead to immediate H3.3 loss, but causes progressive H3.3 depletion over several months without widespread transcriptional disruptions or cellular phenotypes. Our study thus uncovers key developmental roles for de novo H3.3 in establishing neuronal chromatin, transcriptome, identity, and connectivity immediately post mitosis that are distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.
Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. Gene expression profiles were measured by mRNA-Sequencing in undifferentiated ESCs (D0), neurodevelopment (D8) and differentiated neurons (D12) to assess the impact of the mutation on gene expression and development.
Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. To assess the effect of the K4A mutation on Pol II activity, nascent RNA levels were mesaured by PRO-seq.
Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. Genomic localization of H3.3 protein was determined by ChIP-Sequencing in ESCs (D0). Histone modifications patterns of H3K4me1, H3K4me3 and H3K27ac were measured by ChIP-Sequencing in ESCs (D0) to assess the impact of the H3.3K4A mutation on the epigenetic landscape. Levels of H3K36me3 were measured by ChIP-Sequencing in WT and H3.3K36A mutant ESCs (D0), NPCs (D8) and neurons (D12) to assess the impact of the H3.3K36A mutation on H3K36me3 levels in development.
Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. Genomic localization of H3.3 protein was determined by ChIP-Sequencing in ESCs (D0). Distribution patterns of RNA Polymerase II Phosphorylated on Serine 5 (RNA Pol II Ser5P), of histone modification H3K27me3 and chromatin remodeler components Brg1/Smarca4 (Swi/Snf) and Chd4 (NuRD) were measured by ChIP-Sequencing in ESCs (D0) to assess the impact of the H3.3K4A mutation on the epigenetic landscape. Distribution patterns of H3.3 were assessed by ChIP-Sequencing in HEK293T cells after depletion of Brg1/Smarca4 (Swi/Snf) and Chd4 (NuRD).
Project description:Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout-first approach to target the H3f3a gene for inactivation in C57BL6/J mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 embryos had some potential morphological differences from WT littermates including smaller size and reduced head size, while an E18.5 null embryo was smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post-translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho-Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell-type-specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes.
Project description:Minor histone variants replace canonical histones in replication-independent manner, altering chromatin structure and thereby affecting gene expression. This constitutes a distinct mechanism of genome regulation, extending the function of nucleosomes beyond ‘simple’ DNA packaging. In an unusual genomic arrangement, two unique genes (H3F3A and H3F3B), encode the same protein – a developmentally essential histone H3.3. Mutations in each of these genes occur in different cancers, including pediatric brain tumors. To investigate this phenomenon we performed an integrative analysis of the expression, regulatory sequences, and mutability of these genes. We report that H3F3A and H3F3B have distinct expression patterns in human cell types. This difference is maximal between differentiated and stem-like cells, whose expression profile resembles that of cancers. The transcription factors, including Oct4/Sox2 and N-Myc, can differentially regulate these genes, and we demonstrate that Oct4 and Sox2 upregulate H3f3a but not H3f3b in mouse ESCs. Notably, the increased H3F3A contribution to the total H3.3 pool correlates with tumor malignancy. We infer that a similar increase in the H3F3A ‘transcriptional dosage’ in stem-like cells enables the mutations in this gene to impact cell fate determination. Collectively, our findings provide new insights into the interplay between gene expression and DNA mutations in chromatin-associated factors.
Project description:We used H3f3b-/- testes to analyze the impact of H3f3b derived H3.3 on spermatogenesis. Analysis of Histone H3 (Lys 4) tri-methylation in H3f3b WT/WT and H3f3b -/- testes tissue.
Project description:Background. The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. Results. H3f3b KO mice exhibit a semi-lethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in MEFs with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. Conclusions. Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development. Examination of H3K9Ac and H3K4me3 in wild-type and H3.3 null MEFs
Project description:Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9, K27 or K79. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to profile the changes in histone modifications, in the different H3.3 mutant mESC lines. The following histone modifications were profiled: H3K27ac (39685, Active Motif), H3K9ac (C5B11-9649, Cell Signalling Technology), H3K27me3 (C36B11, Cell Signalling Technology), H3K9me2 (D85B4, Cell Signalling Technology), H3K9me3 (D4W1U, Cell Signalling Technology). H3.3 (09-838, Merck-Millipore, purified polyclonal) ChIP-seq was performed in order to profile the deposition of this histone variant in the H3.3 K9A and K27A mESC mutant lines. SUZ12 (D39F6-3737, Cell Signalling Technology) ChIP-seq was performed to profile the genomic distribution of the Polycomb repressive complex 2 (i.e., PRC2). Two to three independent replicates per condition were grown and harvested for each ChIP experiment. Matched input controls were also generated in every experiment. Sequencing libraries were prepared using the NEBNext Ultra II library preparation kit (New England Biolabs) and sequenced on NextSeq500 or NextSeq2000 platforms in single-end mode.
Project description:Background. The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. Results. H3f3b KO mice exhibit a semi-lethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in MEFs with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. Conclusions. Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.