Gene expression in unfertilized eggs and the MBT stage of zebrafish embryos
ABSTRACT: Very little is known on the nature of epigenetic states in developing zebrafish despite its growing importance as a model organism in developmental biology. We report histone modifications on promoters of pluripotency genes in zebrafish embryos at the mid-late blastula transition (MBT+) stage. We identify three classes of expressed genes based on these profiles: (1) those with a promoter occupied by marks of active genes without any repressive marks; (2) those co-occupied by both activating and repressive modifications; of these genes, klf4 was notably found to be mosaically expressed in the embryo, possibly accounting for this epigenetic pattern; (3) those occupied by repressive marks with, surprisingly, little not acetylated H3K9 or H4. Culture of embryo-derived cells results in a switch from histone acetylation to K9 and K27 trimethylation on genes transcriptionally inactivated, resulting in a profile similar to that of fibroblasts. All promoters retain H3K4me3, indicating no correlation between H3K4me3 occupancy and gene expression. Our results illustrate a complex chromatin state on the promoter of pluripotency-associated genes in the zebrafish embryo, shortly after the embryonic genome is turned on. Overall design: We assessed gene expression in unfertilized eggs and the MBT stage of zebrafish embryos (~ 3.5 hpf) using a 44K custom designed chip from Agilent (3 and 4 replicates, respectively). Genes of interest were extracted and evaluated according to expression dynamics and levels.
Project description:Very little is known on the nature of epigenetic states in developing zebrafish despite its growing importance as a model organism in developmental biology. We report histone modifications on promoters of pluripotency genes in zebrafish embryos at the mid-late blastula transition (MBT+) stage. We identify three classes of expressed genes based on these profiles: (1) those with a promoter occupied by marks of active genes without any repressive marks; (2) those co-occupied by both activating and repressive modifications; of these genes, klf4 was notably found to be mosaically expressed in the embryo, possibly accounting for this epigenetic pattern; (3) those occupied by repressive marks with, surprisingly, little not acetylated H3K9 or H4. Culture of embryo-derived cells results in a switch from histone acetylation to K9 and K27 trimethylation on genes transcriptionally inactivated, resulting in a profile similar to that of fibroblasts. All promoters retain H3K4me3, indicating no correlation between H3K4me3 occupancy and gene expression. Our results illustrate a complex chromatin state on the promoter of pluripotency-associated genes in the zebrafish embryo, shortly after the embryonic genome is turned on. We assessed gene expression in unfertilized eggs and the MBT stage of zebrafish embryos (~ 3.5 hpf) using a 44K custom designed chip from Agilent (3 and 4 replicates, respectively). Genes of interest were extracted and evaluated according to expression dynamics and levels.
Project description:After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal-zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal-zygotic transition.
Project description:In mature human sperm, genes of importance for embryo development (i.e., transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent, key factors that may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, whereas H3K36me3 is located in the 3' coding region of heavily transcribed genes in somatic cells, H3K36me3 is present in the promoters of "silent" developmental regulators in sperm, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tDNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm.
Project description:We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb repressive complex 1 (PRC1)-like 4 (PRC1L4), given the copresence of PcG proteins RING1, RING2, and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/HP1?, known to function in transcriptional repression. PRC1L4-mediated repression necessitated L3MBTL2 that compacted chromatin in a histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT domain-containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genes that exhibited a significant reduction in H2A lysine 119 monoubiquitination. Our findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks.
Project description:Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) requires a resetting of the epigenome in order to facilitate a cell fate transition. Previous studies have shown that epigenetic modifying enzymes play a central role in controlling induced pluripotency and the generation of iPSC. Here we show that RNF40, a histone H2B lysine 120 E3 ubiquitin-protein ligase, is specifically required for early reprogramming during induced pluripotency. Loss of RNF40-mediated H2B monoubiquitination (H2Bub1) impaired early gene activation in reprogramming. We further show that RNF40 contributes to tissue-specific gene suppression via indirect effects by controlling the expression of the polycomb repressive complex-2 histone methyltransferase component EZH2, as well as through more direct effects by promoting the resolution of H3K4me3/H3K27me3 bivalency on H2Bub1-occupied pluripotency genes. Thus, we identify RNF40 as a central epigenetic mediator of cell state transition with distinct functions in resetting somatic cell state to pluripotency.
Project description:Epigenetic regulation of gene expression is fundamental for cell type-specific gene expression. However, integrated comparative transcriptomic and epigenomic analyses in various adult primary differentiated cells remain underrepresented. We generated promoter landscapes of DNA methylation and three important histone methylation marks (H3K4me3, H3K9me2, and H3K27me3) in two primary cell types (B lymphocytes and liver) from adult mice. In line with previous studies, we also observed distinct H3K4me3 patterns at promoters dictated by CpG content in differentiated primary cells. We further explored the distribution of initiating RNA polymerase II and elongating RNA polymerase II across genes within different promoter classes, suggesting different rate-limiting steps at CpG-rich vs. CpG-poor genes. Examination of differentially expressed genes revealed that regulation of tissue-specific genes is closely related to gene function regardless of promoter type. Although repressive chromatin marks displayed differential preference to promoters based on CpG content, we observed fine-tuning of the pattern of association of these marks with specific promoter types in a cell type-specific manner. The distribution of H3K9me2 and H3K27me3, relative to CpG content, differed substantially between the two cell types. Cell-type specific accumulation of repressive chromatin marks was also observed at silent genes in both cell types, suggesting that differentiated primary cells may exhibit cell-type specificity in the distribution of repressive chromatin marks. Epigenetic regulation of gene expression and the association of specific histone marks with promoter sequence classes are fine-tuned in a cell type-specific manner. This unexpected finding underscores the value of extensive study of epigenetic marks across cell and tissue types.
Project description:Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.
Project description:Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.
Project description:Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types, and that this variation is likely determined by a combination of the chromatin landscape, cell-specific DNA-binding transcription factors, and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells, there is presently little understanding of this aspect of the Pol III system in human ES cells. Here, we determine the occupancy profiles of Pol III components in human H1 ES cells, and also induced pluripotent cells, and compare to known profiles of chromatin, transcription factors, and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly, we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells, especially NANOG. Notably, in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases, a peak of H3K4me3 and/or RNA Pol II, between the H3K27me3 and Pol III binding peaks, suggesting that H3K4me3 and Pol II activity may "insulate" Pol III from neighboring repressive H3K27me3. Further, we find iPSCs have a larger Pol III repertoire than their precursors. Finally, the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together, our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity.
Project description:Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using k-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.