Project description:Transgene line containing the histone gene fused to GFP was established and characterized at genomic, transcriptional and protein level. The GFP tag was then used for ChIP Seq analysis of the DNA sequences bound by H3.2. The analysis revealed interesting ocular specific targets including Ephrin family genes. Altered incorporation of H3.2 was found in the mutant, specifically around the ligand Efna5 and the receptor Ephb2. The effect of this altered incorporation on Ephrin signaling was further analyzed by QPCR and immunohistochemistry. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for GFP (sc-9996)
Project description:The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells. Crosslinking ChIP-seq: Examination of 1 histone variant (H3.3), 2 histone modifications, and Serine-5 phosphorylated RNA polymerase in 2 different cell types (H3.3-HA ES samples 1-4, and H3.3-HA NPC samples 7-10). Examination of 1 histone variant (H3.2), and one histone modification (H3K36me3) in 2 different cell types (H3.2-HA ES samples 5-6, and H3.2-HA NPC samples 11-12). Examination of 1 histone variant (H3.3), input control, and one histone modification (H3K36me3) in one cell type (H3.3-HA hybrid ES, samples 13-15). Examination of 1 histone variant (H3.1S31), input control, and one histone modification (H3K36me3) in one cell type (H3.1S31-HA hybrid ES, samples 16-18). Native ChIP-seq: Examination of 1 histone variant (H3.3), input control, and one histone modification (H3K4me3) in one cell type (H3.3-HA ES, samples 19-21). Examination of 1 histone variant (H3.2), input control, and two histone modifications (H3K4me3 and H3K27me3) in one cell type (H3.2-HA ES, samples 22-25). Examination of 1 histone variant (H3.3), input control, and two histone modifications (H3K4me1 and H3K36me3) in one cell type (H3.3-EYFP ES, samples 26-29). Examination of 1 histone variant (H3.3), input control, and two histone modifications (H3K4me1 and H3K36me3) in one cell type (Hira -/- H3.3-EYFP ES, samples 30-33). Examination of 1 histone variant (H3.3) and input control in one cell type (Atrxflox H3.3-EYFP ES, samples 34-37). Examination of HA antibody background in one cell type (wild-type ES, sample 38).
Project description:The extraembryonic lineage in mammals is unique in its unipotent differentiation ability and gestation-limited development. Here we identified the epigenomic characters of mouse trophoblast stem cells (TSCs) focusing on the genome-wide enrichment of histone H3.1/H3.2 and H3K9me3. Comparative ChIP-sequencing (ChIP-seq) analysis of TSCs and embryonic stem cells (ESCs) revealed that the TSC genome uniquely contained large (over 1Mb) H3.1/H3.2-H3K9me3 domains in the intergenic regions. This feature was common to extraembryonic cells in mice and humans. Depletion of CAF1, a H3.1/H3.2 chaperone, led to downregulation of TSC marker genes such as Cdx2 and Elf5 and ectopic expression of Oct3/4, an ESC marker. Nuclear transfer cloning using TSCs resulted in extremely poor embryonic development, but removal of H3K9me3 from their genome resulted in birth of the first TSC-cloned mice. Thus, the signature of the extraembryonic epigenome is safeguarded by the H3.1/H3.2-H3K9me3 enrichment, which protects the genome from cell fate transition.
Project description:The extraembryonic lineage in mammals is unique in its unipotent differentiation ability and gestation-limited development. Here we identified the epigenomic characters of mouse trophoblast stem cells (TSCs) focusing on the genome-wide enrichment of histone H3.1/H3.2 and H3K9me3. Comparative ChIP-sequencing (ChIP-seq) analysis of TSCs and embryonic stem cells (ESCs) revealed that the TSC genome uniquely contained large (over 1Mb) H3.1/H3.2-H3K9me3 domains in the intergenic regions. This feature was common to extraembryonic cells in mice and humans. Depletion of CAF1, a H3.1/H3.2 chaperone, led to downregulation of TSC marker genes such as Cdx2 and Elf5 and ectopic expression of Oct3/4, an ESC marker. Nuclear transfer cloning using TSCs resulted in extremely poor embryonic development, but removal of H3K9me3 from their genome resulted in birth of the first TSC-cloned mice. Thus, the signature of the extraembryonic epigenome is safeguarded by the H3.1/H3.2-H3K9me3 enrichment, which protects the genome from cell fate transition.
Project description:The extraembryonic lineage in mammals is unique in its unipotent differentiation ability and gestation-limited development. Here we identified the epigenomic characters of mouse trophoblast stem cells (TSCs) focusing on the genome-wide enrichment of histone H3.1/H3.2 and H3K9me3. Comparative ChIP-sequencing (ChIP-seq) analysis of TSCs and embryonic stem cells (ESCs) revealed that the TSC genome uniquely contained large (over 1Mb) H3.1/H3.2-H3K9me3 domains in the intergenic regions. This feature was common to extraembryonic cells in mice and humans. Depletion of CAF1, a H3.1/H3.2 chaperone, led to downregulation of TSC marker genes such as Cdx2 and Elf5 and ectopic expression of Oct3/4, an ESC marker. Nuclear transfer cloning using TSCs resulted in extremely poor embryonic development, but removal of H3K9me3 from their genome resulted in birth of the first TSC-cloned mice. Thus, the signature of the extraembryonic epigenome is safeguarded by the H3.1/H3.2-H3K9me3 enrichment, which protects the genome from cell fate transition.
Project description:The extraembryonic lineage in mammals is unique in its unipotent differentiation ability and gestation-limited development. Here we identified the epigenomic characters of mouse trophoblast stem cells (TSCs) focusing on the genome-wide enrichment of histone H3.1/H3.2 and H3K9me3. Comparative ChIP-sequencing (ChIP-seq) analysis of TSCs and embryonic stem cells (ESCs) revealed that the TSC genome uniquely contained large (over 1Mb) H3.1/H3.2-H3K9me3 domains in the intergenic regions. This feature was common to extraembryonic cells in mice and humans. Depletion of CAF1, a H3.1/H3.2 chaperone, led to downregulation of TSC marker genes such as Cdx2 and Elf5 and ectopic expression of Oct3/4, an ESC marker. Nuclear transfer cloning using TSCs resulted in extremely poor embryonic development, but removal of H3K9me3 from their genome resulted in birth of the first TSC-cloned mice. Thus, the signature of the extraembryonic epigenome is safeguarded by the H3.1/H3.2-H3K9me3 enrichment, which protects the genome from cell fate transition.
Project description:The extraembryonic lineage in mammals is unique in its unipotent differentiation ability and gestation-limited development. Here we identified the epigenomic characters of mouse trophoblast stem cells (TSCs) focusing on the genome-wide enrichment of histone H3.1/H3.2 and H3K9me3. Comparative ChIP-sequencing (ChIP-seq) analysis of TSCs and embryonic stem cells (ESCs) revealed that the TSC genome uniquely contained large (over 1Mb) H3.1/H3.2-H3K9me3 domains in the intergenic regions. This feature was common to extraembryonic cells in mice and humans. Depletion of CAF1, a H3.1/H3.2 chaperone, led to downregulation of TSC marker genes such as Cdx2 and Elf5 and ectopic expression of Oct3/4, an ESC marker. Nuclear transfer cloning using TSCs resulted in extremely poor embryonic development, but removal of H3K9me3 from their genome resulted in birth of the first TSC-cloned mice. Thus, the signature of the extraembryonic epigenome is safeguarded by the H3.1/H3.2-H3K9me3 enrichment, which protects the genome from cell fate transition.
Project description:Nucleosomes are the principal packaging units of chromatin and critical for gene regulation and genome stability. In mammals, a subset of nucleosomes fail to be replaced by protamines during spermatogenesis and are retained in mature spermatozoa providing opportunities for paternal epigenetic transmission. In humans, the remaining 10% localize at regulatory elements of genes. To assess evolutionary conservation and to dissect the molecular logic underlying nucleosome retention, we determined the genome wide nucleosome occupancy in mouse spermatozoa that only contain 1% residual histones. In striking contrast to mammalian somatic cells and haploid round spermatids, we observe high enrichment of nucleosomes at CpG-rich sequences throughout the genome, at conserved regulatory sequences as well as at intra- and intergenic regions and repetitive DNA. This preferred occupancy occurs mutually exclusive with DNA methylation both in mouse and human sperm. At unmethylated CpG-rich sequences, residing nucleosomes are largely composed of the H3.3 histone variant, and trimethylated at lysine 4 (H3K4me3). Both canonical H3.1/H3.2 and H3.3 variant histones are present at promoters marked by Polycomb-mediated H3K27me3, which is strongly predictive for gene repression in pre-implantation embryos. Our data indicate important roles of DNA sequence composition, DNA methylation, variant H3.3 and canonical H3.1/H3.2 histones and associated modifications in nucleosome retention versus eviction during the histone-to-protamine remodeling process in elongating spermatids and potentially in epigenetic inheritance by nucleosomes between generations. Identification of histone, histone variant and histone modification states in round spermatids and sperm
Project description:Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming both for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA into oocytes in somatic cell nuclear transfer (SCNT) embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity. Transcriptome sequencing of 4-cell NT embryos, Luciferase 4-cell SCNT embryos, 4-cell NT embryos_H3.3KD, 4-cell NT embryos_H3.3KD+H3.3mRNA, H3.3 KD + H3.2 mRNA SCNT embryos