Project description:Whole-Genome Bisulfite Sequencing of HCHC mutants To extend our understanding of the role of the HCHC complex in Neurospora, we carried out whole-genome bisulfite sequencing (WGBS) of cdp-2, chap, and hda-1 mutants. Consistent with prior analyses, the WGBS revealed both hypomethylated and hypermethylated regions in the three HCHC mutants while the ; sequences with a lower Combined RIP Index (CRI) tend to show reduced methylation in the mutants, while sequences with higher CRI scores show increased methylation. Analysis of the WGBS data also demonstrated that shorter methylated regions, regardless of their degree of methylation in wild type, commonly have reduced methylation in the HCHC mutants, while longer methylated sequences tend to have elevated methylation. In addition, the borders of normally methylated regions typically lose methylation and show a contraction of boundary methylation in the HCHC mutants. Sequences near telomeres that are normally methylated were found to lose methylation in the mutants, while most of the increased DNA methylation of the three HCHC mutants is found at centromeres. CHAP in vitro DNA-affinity purification HT-seq and CHAP DamID-Seq To test whether the CHAP AT-hook motifs bind AT-rich RIP’d DNA, we performed in vitro DNA-affinity purification with the recombinant CHAP-N-terminus (1-274 residues) containing the two AT-hook motifs and analyzed the purified DNA with high throughput sequencing (HT-Seq). To complement this approach, we also assessed binding of CHAP in vivo with DamID-seq using the CHAP-Dam strain. Together, these techniques gave us a detailed genomic view of the specific localization and binding of CHAP to AT-rich RIP’d DNA, which is nearly coincident with methylated DNA regions.
Project description:The role of the lineage-determining transcription factor, Interferon regulatory factor (IRF8), in microglia remains elucidated. We report the genome-wide methyl-CpG status in adult wild-type (WT) and IRF8-knockout (IRF8KO) microglia by whole-genome bisulfite-Seq (WGBS). Deep-sequencing and subsequent differential analysis revealed that IRF8KO microglia exhibited a cell-intrinsic methylation profile. Furthermore, the differentially methylated regions showed a significant correlation with IRF8-dependent chromatin accessibility in microglia. This study provides a novel insight into understanding epigenetic regulation in microglia.
Project description:While DNA methylation in other tissues can be approximated through model species, the dynamic distribution and regulatory significance of DNA methylation in the rumen, a unique organ in ruminant, remain largely unknown. Here, we employed whole-genome bisulfite sequencing (WGBS), transcriptomics, and histone modification data to compare fetal and adult stages of bovine rumen with other tissues, including pluripotent stem cells (PSCs) approximating pre-implantation embryos. We found extensive methylation differences, including CG methylation (mCG) and non-CG methylation (mCH; H represents A, C and T) between the rumen at fetal and adult stages and other tissues and PSCs. These differentially methylated regions (DMRs) are closely associated with other epigenetic regulatory components, such as transcription factors (TFs) and histone modifications. These DMRs can also combine to form large hypo CG-DMRs to regulate a cluster of functionally related genes. We elucidated the reasons for morphological and functional differences between fetal and adult rumen at the epigenetic level and the interactions between epigenetic modifications and gene expression. This study highlights the differences in methylation patterns between the rumen and other tissues during development and the role of DNA methylation in controlling gene expression and establishing tissue-specific functions.
Project description:Using MethylC-Seq to provide single-base resolution of DNA methylation status in WT, nrpd1-3, nrpe1-11, ros1-4 single mutants and ros1-4nrpd1-3 double mutant
Project description:Using MethylC-Seq to provide single-base resolution of DNA methylation status in 35S-SUC2 WT and anti-silencing mutants( arp6-5, pie1-7, h2a.z-2, idm1-9 and ros1-14）
Project description:DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well understood in Arabidopsis, mutations in RdDM genes cause no visible developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 in rice. Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH methylation levels. Moreover, male and female reproductive development was disturbed, which led to the sterility of osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation likely regulates the expression of multiple key genes involved in stamen development and meiosis. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH DNA methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity including OsRDR2 activity. Our results, which revealed a global increase in DNA methylation through enhancement of RdDM activity in reproductive organs, demonstrate the crucial role of OsRDR2 in the sexual reproduction of rice.
Project description:Using MethylC-Seq to provide single-base resolution of DNA methylation status in Ws background wild-type (WT), ape1l-1 (Ws background), arp-1 (Col-0 background) and zdp-1 (Col-0 background) mutants
Project description:Using WGBS we assessed global DNA methylation changes in Dnmt3aKO or Dnmt3bKO mouse embryonic stem cells. Compared with WT cells, Dnmt3aKO cells but not Dnmt3bKO cells showed genome-wide hypomethylation.
