Epigenetics & Chromatin最新文献

Background: Epigenetic reprogramming is involved in luteinizing hormone (LH)-induced ovulation; however, the underlying mechanisms are largely unknown.

Results: We here observed a rapid histone deacetylation process between two waves of active transcription mediated by the follicle-stimulating hormone (FSH) and the LH congener human chorionic gonadotropin (hCG), respectively. Analysis of the genome-wide H3K27Ac distribution in hCG-treated granulosa cells revealed that a rapid wave of genome-wide histone deacetylation remodels the chromatin, followed by the establishment of specific histone acetylation for ovulation. HDAC2 phosphorylation activation coincides with histone deacetylation in mouse preovulatory follicles. When HDAC2 was silenced or inhibited, histone acetylation was retained, leading to reduced gene transcription, retarded cumulus expansion, and ovulation defect. HDAC2 phosphorylation was associated with CK2α nuclear translocation, and inhibition of CK2α attenuated HDAC2 phosphorylation, retarded H3K27 deacetylation, and inactivated the ERK1/2 signaling cascade.

Conclusions: This study demonstrates that the ovulatory signal erases histone acetylation through activation of CK2α-mediated HDAC2 phosphorylation in granulosa cells, which is an essential prerequisite for subsequent successful ovulation.

Background: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts.

Results: We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes.

Conclusion: Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

Polycomb group RING finger protein 6 (PCGF6) plays an important role as a regulator of transcription in a variety of cellular processes, including tumorigenesis. However, the function and expression of PCGF6 in papillary RCC (pRCC) remain unclear. In the present study, we found that PCGF6 expression was significantly elevated in pRCC tissues, and high expression of PCGF6 was associated with poor survival of patients with pRCC. The overexpression of PCGF6 promoted while depletion of PCGF6 depressed the proliferation of pRCC cells in vitro. Interestingly, myc-related zinc finger protein (MAZ), a downstream molecular of PCGF6, was upregulated in pRCC with hypomethylation promoter. Mechanically, PCGF6 promoted MAZ expression by interacting with MAX and KDM5D to form a complex, and MAX recruited PCGF6 and KDM5D to the CpG island of the MAZ promoter and facilitated H3K4 histone demethylation. Furthermore, CDK4 was a downstream molecule of MAZ that participated in PCGF6/MAZ-regulated progression of pRCC. These results indicated that the upregulation of PCGF6 facilitated MAZ/CDK4 axis expression and pRCC progression by hypomethylation of the MAZ promoter. The PCGF6/MAZ/CDK4 regulatory axis may be a potential target for the treatment of ccRCC.

Background: KDM5 family proteins are multi-domain regulators of transcription that when dysregulated contribute to cancer and intellectual disability. KDM5 proteins can regulate transcription through their histone demethylase activity in addition to demethylase-independent gene regulatory functions that remain less characterized. To expand our understanding of the mechanisms that contribute to KDM5-mediated transcription regulation, we used TurboID proximity labeling to identify KDM5-interacting proteins.

Results: Using Drosophila melanogaster, we enriched for biotinylated proteins from KDM5-TurboID-expressing adult heads using a newly generated control for DNA-adjacent background in the form of dCas9:TurboID. Mass spectrometry analyses of biotinylated proteins identified both known and novel candidate KDM5 interactors, including members of the SWI/SNF and NURF chromatin remodeling complexes, the NSL complex, Mediator, and several insulator proteins.

Conclusions: Combined, our data shed new light on potential demethylase-independent activities of KDM5. In the context of KDM5 dysregulation, these interactions may play key roles in the alteration of evolutionarily conserved transcriptional programs implicated in human disorders.

Background: Allele-specific methylation of the imprinting control region (ICR) is the molecular basis for the genomic imprinting phenomenon that is unique to placental mammals. We previously showed that the ICR at the mouse H19 gene locus (H19 ICR) was unexpectedly established after fertilization and not during spermatogenesis in transgenic mice (TgM), and that the same activity was essential for the maintenance of paternal methylation of the H19 ICR at the endogenous locus in pre-implantation embryos. To examine the universality of post-fertilization imprinted methylation across animal species or imprinted loci, we generated TgM with two additional sequences.

Results: The rat H19 ICR, which is very similar in structure to the mouse H19 ICR, unexpectedly did not acquire imprinted methylation even after fertilization, suggesting a lack of essential sequences in the transgene fragment. In contrast, the mouse IG-DMR, the methylation of which is acquired during spermatogenesis at the endogenous locus, did not acquire methylation in the sperm of TgM, yet became highly methylated in blastocysts after fertilization, but only when the transgene was paternally inherited. Since these two sequences were evaluated at the same genomic site by employing the transgene co-placement strategy, it is likely that the phenotype reflects the intrinsic activity of these fragments rather than position-effect variegation.

Conclusions: Our results suggested that post-fertilization imprinted methylation is a versatile mechanism for protecting paternal imprinted methylation from reprogramming during the pre-implantation period.

Background: Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin.

Results: We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant.

Conclusion: Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading.

Background: With the advance of bisulfite sequencing technologies, massive amount of methylation data have been generated, which provide unprecedented opportunities to study the epigenetic mechanism and its relationship to other biological processes. A commonly seen feature of the methylation data is the correlation between nearby CpG sites. Although such a spatial correlation was utilized in several epigenetic studies, its interaction to other characteristics of the methylation data has not been fully investigated.

Results: We filled this research gap from an information theoretic perspective, by exploring the impact of the spatial correlation on the methylation entropy (ME). With the spatial correlation taken into account, we derived the analytical relation between the ME and another key parameter, the methylation probability. By comparing it to the empirical relation between the two corresponding statistics, the observed ME and the mean methylation level, genomic loci under strong epigenetic control can be identified, which may serve as potential markers for cell-type specific methylation. The proposed method was validated by simulation studies, and applied to analyze a published dataset of mouse brain methylome.

Conclusions: Compared to other sophisticated methods developed in literature, the proposed method provides a simple but effective way to detect CpG segments under strong epigenetic control (e.g., with bipolar methylation pattern). Findings from this study shed light on the identification of cell-type specific genes/pathways based on methylation data from a mixed cell population.

Background: Cellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium.

Results: Using whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6's ability to interact with sites containing one or two methylated CpG dinucleotides.

Conclusions: Our study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.

Background: Preconception exposure to phthalates such as the anti-androgenic dibutyl-phthalate (DBP) impacts both male and female reproduction, yet how this occurs largely remains unknown. Previously we defined a series of RNAs expressly provided by sperm at fertilization and separately, and in parallel, those that responded to high DBP exposure. Utilizing both populations of RNAs, we now begin to unravel the impact of high-DBP exposure on those RNAs specifically delivered by the father.

Results: Enrichment of RNAs altered by DBP exposure within the Molecular Signature Database highlighted cellular stress, cell cycle, apoptosis, DNA damage response, and gene regulation pathways. Overlap within each of these five pathways identified those RNAs that were specifically (≥ fivefold enriched) or primarily (≥ twofold enriched) provided as part of the paternal contribution compared to the oocyte at fertilization. Key RNAs consistently altered by DBP, including CAMTA2 and PSME4, were delivered by sperm reflective of these pathways. The majority (64/103) of overlapping enriched gene sets were related to gene regulation. Many of these RNAs (45 RNAs) corresponded to key interconnected CRREWs (Chromatin remodeler cofactors, RNA interactors, Readers, Erasers, and Writers). Modeling suggests that CUL2, PHF10, and SMARCC1 may coordinate and mechanistically modulate the phthalate response.

Conclusions: Mediated through a CRREW regulatory network, the cell responded to exposure presenting stressed-induced changes in the cell cycle-DNA damage-apoptosis. Interestingly, the majority of these DBP-responsive epigenetic mediators' direct acetylation or deacetylation, impacting the sperm's cargo delivered at fertilization and that of the embryo.

Background: Sex determination and differentiation are complex and delicate processes. In female chickens, the process of sex differentiation is sensitive and prone to be affected by the administration of aromatase inhibitors, which result in chicken sex reversal and infertility. However, the molecular mechanisms underlying sex differentiation and infertility in chicken sex reversal remain unclear. Therefore, we established a sex-reversed chicken flock by injecting an aromatase inhibitor, fadrozole, and constructed relatively high-resolution profiles of the gene expression and chromatin accessibility of embryonic gonads.

Results: We revealed that fadrozole affected the transcriptional activities of several genes, such as DMRT1, SOX9, FOXL2, and CYP19A1, related to sex determination and differentiation, and the expression of a set of gonadal development-related genes, such as FGFR3 and TOX3, by regulating nearby open chromatin regions in sex-reversed chicken embryos. After sexual maturity, the sex-reversed chickens were confirmed to be infertile, and the possible causes of this infertility were further investigated. We found that the structure of the gonads and sperm were greatly deformed, and we identified several promising genes related to spermatogenesis and infertility, such as SPEF2, DNAI1, and TACR3, through RNA-seq.

Conclusions: This study provides clear insights into the exploration of potential molecular basis underlying sex differentiation and infertility in sex-reversed chickens and lays a foundation for further research into the sex development of birds.