Epigenetics & Chromatin最新文献

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.

Background: Many human disease phenotypes manifest differently by sex, making the development of methods for incorporating X and Y-chromosome data into analyses vital. Unfortunately, X and Y chromosome data are frequently excluded from large-scale analyses of the human genome and epigenome due to analytical complexity associated with sex chromosome dosage differences between XX and XY individuals, and the impact of X-chromosome inactivation (XCI) on the epigenome. As such, little attention has been given to considering the methods by which sex chromosome data may be included in analyses of DNA methylation (DNAme) array data.

Results: With Illumina Infinium HumanMethylation450 DNAme array data from 634 placental samples, we investigated the effects of probe filtering, normalization, and batch correction on DNAme data from the X and Y chromosomes. Processing steps were evaluated in both mixed-sex and sex-stratified subsets of the analysis cohort to identify whether including both sexes impacted processing results. We found that identification of probes that have a high detection p-value, or that are non-variable, should be performed in sex-stratified data subsets to avoid over- and under-estimation of the quantity of probes eligible for removal, respectively. All normalization techniques investigated returned X and Y DNAme data that were highly correlated with the raw data from the same samples. We found no difference in batch correction results after application to mixed-sex or sex-stratified cohorts. Additionally, we identify two analytical methods suitable for XY chromosome data, the choice between which should be guided by the research question of interest, and we performed a proof-of-concept analysis studying differential DNAme on the X and Y chromosome in the context of placental acute chorioamnionitis. Finally, we provide an annotation of probe types that may be desirable to filter in X and Y chromosome analyses, including probes in repetitive elements, the X-transposed region, and cancer-testis gene promoters.

Conclusion: While there may be no single "best" approach for analyzing DNAme array data from the X and Y chromosome, analysts must consider key factors during processing and analysis of sex chromosome data to accommodate the underlying biology of these chromosomes, and the technical limitations of DNA methylation arrays.

Background: Regulatory elements such as promoters, enhancers, and insulators interact each other to mediate molecular processes. To capture chromatin interactions of regulatory elements, 3C-derived methods such as Hi-C and Micro-C are developed. Here, we generated and analyzed Hi-C, Micro-C, and promoter capture Micro-C datasets with different sequencing depths to study chromatin interactions of regulatory elements and nucleosome positions in human prostate cancer cells.

Results: Compared to Hi-C, Micro-C identifies more high-resolution loops, including ones around structural variants. By evaluating the effect of sequencing depth, we revealed that more than 2 billion reads of Micro-C are needed to detect chromatin interactions at 1 kb resolution. Moreover, we found that deep-sequencing identifies additional long-range loops that are longer than 1 Mb in distance. Furthermore, we found that more than 50% of the loops are involved in insulators while less than 10% of the loops are promoter-enhancer loops. To comprehensively capture chromatin interactions that promoters are involved in, we performed promoter capture Micro-C. Promoter capture Micro-C identifies loops near promoters with a lower amount of sequencing reads. Sequencing of 160 million reads of promoter capture Micro-C resulted in reaching a plateau of identifying loops. However, there was still a subset of promoters that are not involved in loops even after deep-sequencing. By integrating Micro-C with NOMe-seq and ChIP-seq, we found that active promoters involved in loops have a more accessible region with lower levels of DNA methylation and more highly phased nucleosomes, compared to active promoters that are not involved in loops.

Conclusion: We determined the required sequencing depth for Micro-C and promoter capture Micro-C to generate high-resolution chromatin interaction maps and loops. We also investigated the effect of sequencing coverage of Hi-C, Micro-C, and promoter capture Micro-C on detecting chromatin loops. Our analyses suggest the presence of distinct regulatory element groups, which are differently involved in nucleosome positions and chromatin interactions. This study does not only provide valuable insights on understanding chromatin interactions of regulatory elements, but also present guidelines for designing research projects on chromatin interactions among regulatory elements.

Background: Epigenome-edited animal models enable direct demonstration of disease causing epigenetic mutations. Transgenic (TG) mice stably expressing epigenome-editing factors exhibit dramatic and stable changes in target epigenome modifications. Successful germline transmission of a transgene from founder mice to offspring will yield a sufficient number of epigenome-edited mice for phenotypic analysis; however, if the epigenetic mutation has a detrimental phenotypic effect, it can become difficult to obtain the next generation of animals. In this case, the phenotype of founder mice must be analyzed directly. Unfortunately, current TG mouse production efficiency (TG founders per pups born) is relatively low, and improvements would increase the versatility of this technology.

Results: In the current study, we describe an approach to generate epigenome-edited TG mice using a combination of both the dCas9-SunTag and piggyBac (PB) transposon systems. Using this system, we successfully generated mice with demethylation of the differential methylated region of the H19 gene (H19-DMR), as a model for Silver-Russell syndrome (SRS). SRS is a disorder leading to growth retardation, resulting from low insulin-like growth factor 2 (IGF2) gene expression, often caused by epimutations at the H19-IGF2 locus. Under optimized conditions, the efficiency of TG mice production using the PB system was approximately threefold higher than that using the conventional method. TG mice generated by this system showed demethylation of the targeted DNA region and associated changes in gene expression. In addition, these mice exhibited some features of SRS, including intrauterine and postnatal growth retardation, due to demethylation of H19-DMR.

Conclusions: The dCas9-SunTag and PB systems serve as a simple and reliable platform for conducting direct experiments using epigenome-edited founder mice.

Intellectual disability is a well-known hallmark of Down Syndrome (DS) that results from the triplication of the critical region of human chromosome 21 (HSA21). Major studies were conducted in recent years to gain an understanding about the contribution of individual triplicated genes to DS-related brain pathology. Global transcriptomic alterations and widespread changes in the establishment of neural lineages, as well as their differentiation and functional maturity, suggest genome-wide chromatin organization alterations in trisomy. High Mobility Group Nucleosome Binding Domain 1 (HMGN1), expressed from HSA21, is a chromatin remodeling protein that facilitates chromatin decompaction and is associated with acetylated lysine 27 on histone H3 (H3K27ac), a mark correlated with active transcription. Recent studies causatively linked overexpression of HMGN1 in trisomy and the development of DS-associated B cell acute lymphoblastic leukemia (B-ALL). HMGN1 has been shown to antagonize the activity of the Polycomb Repressive Complex 2 (PRC2) and prevent the deposition of histone H3 lysine 27 trimethylation mark (H3K27me3), which is associated with transcriptional repression and gene silencing. However, the possible ramifications of the increased levels of HMGN1 through the derepression of PRC2 target genes on brain cell pathology have not gained attention. In this review, we discuss the functional significance of HMGN1 in brain development and summarize accumulating reports about the essential role of PRC2 in the development of the neural system. Mechanistic understanding of how overexpression of HMGN1 may contribute to aberrant brain cell phenotypes in DS, such as altered proliferation of neural progenitors, abnormal cortical architecture, diminished myelination, neurodegeneration, and Alzheimer's disease-related pathology in trisomy 21, will facilitate the development of DS therapeutic approaches targeting chromatin.

Background: Nucleosome-mediated chromatin compaction has a direct effect on the accessibility of trans-acting activators and repressors to DNA targets and serves as a primary regulatory agent of genetic expression. Understanding the nature and dynamics of chromatin is fundamental to elucidating the mechanisms and factors that epigenetically regulate gene expression. Previous work has shown that there are three types of canonical sequences that strongly regulate nucleosome positioning and thus chromatin accessibility: putative nucleosome-positioning elements, putative nucleosome-repelling sequences, and homopolymeric runs of A/T. It is postulated that these elements can be used to remodel chromatin in C. elegans. Here we show the utility of such elements in vivo, and the extreme efficacy of a newly discovered repelling sequence, PRS-322.

Results: In this work, we show that it is possible to manipulate nucleosome positioning in C. elegans solely using canonical and putative positioning sequences. We have not only tested previously described sequences such as the Widom 601, but also have tested additional nucleosome-positioning sequences: the Trifonov sequence, putative repelling sequence-322 (PRS-322), and various homopolymeric runs of A and T nucleotides.

Conclusions: Using each of these types of putative nucleosome-positioning sequences, we demonstrate their ability to alter the nucleosome profile in C. elegans as evidenced by altered nucleosome occupancy and positioning in vivo. Additionally, we show the effect that PRS-322 has on nucleosome-repelling and chromatin remodeling.

Background: The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood.

Results: The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast.

Conclusion: Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.