Tissue-specific methylomes reveal epigenetic memory in adult mouse tissue
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ABSTRACT: Cytosine methylation of DNA is an evolutionarily conserved mechanism from plants to animals with crucial roles in gene regulation. However, the variation between methylomes of normal tissues is largely unexplored. To better understand the epigenetic variation of a normal individual, we profiled DNA methylation using whole genome bisulfite sequencing in 17 tissues isolated from an individual mouse. We observed a unique distribution of CpG methylation for each tissue, which cluster based on cell lineage. Global analysis identified only one-eighth of the genome as tissue-specifically methylated. Remarkably, the vast majority of these regions exhibit hallmarks of cis-regulatory activity. Our results also reveal a novel class of dormant enhancers in adult tissues which retain an epigenetic memory of regulatory elements active during development. Together, these results expand the repertoire of regulatory information encoded within the methylome, and suggest mapping it as an alternative method to identify cell-type specific regulatory elements. whole genome bisulfite sequencing of mouse adult tissue
Project description:During mammalian development DNA methylation patterns need to be reset in primordial germ cells (PGC) and preimplantation embryos. However, many retro-transposons and imprinted genes are resistant to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that some of these sequences are immune to widespread erasure of DNA methylation in the mouse embryonic stem cells (mESCs) lacking de novo DNA methyltransferases. Persistence of DNA methylation at these loci in mESCs depends on the histone H3K9 methyltransferase Setdb1, as deletion of Setdb1 results in reduction of H3K9me3 and DNA methylation levels concomitant with an increase in 5-hydroxymethylation (5hmC). In addition, depletion of H3K9 methyltransferase G9a leads to genome-wide reduction of DNA methylation but to a lesser extent at the above sequences. Taken together, these data reveal that Setdb1 ensures the fidelity of DNA methylation at specific loci in mESCs, which may reflect mechanisms functioning in vivo during key developmental stages. Examination of genome-wide DNA methylation status in 2 cell types.
Project description:During mammalian development DNA methylation patterns need to be reset in primordial germ cells (PGC) and preimplantation embryos. However, many retro-transposons and imprinted genes are resistant to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that some of these sequences are immune to widespread erasure of DNA methylation in the mouse embryonic stem cells (mESCs) lacking de novo DNA methyltransferases. Persistence of DNA methylation at these loci in mESCs depends on the histone H3K9 methyltransferase Setdb1, as deletion of Setdb1 results in reduction of H3K9me3 and DNA methylation levels concomitant with an increase in 5-hydroxymethylation (5hmC). In addition, depletion of H3K9 methyltransferase G9a leads to genome-wide reduction of DNA methylation but to a lesser extent at the above sequences. Taken together, these data reveal that Setdb1 ensures the fidelity of DNA methylation at specific loci in mESCs, which may reflect mechanisms functioning in vivo during key developmental stages. Examination of genome-wide DNA methylation status in 3 cell types.
Project description:There are 64 replication-dependent histone genes with different isotypes associated with each class of the five histone types, H1, H2a, H2b, H3 and H4 in the human genome. In a previous report, we have showed that HIST1H2ac serves as a master regulator of ERalpha-dependent gene activation via ERalpha recruitment, and mediates chromatin looping of regulatory elements of estrogen receptor-targeted genes. Here, we performed whole genome bisulfite sequencing of MCF-7 upon suppressing H2ac expression through siRNA transfection using two different siRNAs to determine the knockdown effect on DNA methylation. Our analysis showed the DNA methylation of ~99% of the genome were mostly unchanged comparing to normal MCF-7 (methylation levels difference within 0.2).
Project description:The study of 5-hydroxylmethylcytosines (5hmC), the sixth base of the mammalian genome, as an epigenetic mark has been hampered by a lack of method to map it at single-base resolution. Previous affinity purification-based methods could not precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach for mapping 5hmC at base resolution. Application of this new method to the embryonic stem cells not only confirms widespread distribution of 5hmC in mammalian genome, but also reveals a strong sequence bias and strand asymmetry at sites of 5hmC. Additionally, the relative abundance of 5hmC varies significantly depending on the types of functional sequences, suggesting different mechanisms for 5hmC deposition and maintenance. Furthermore, we observe high levels of 5hmC and reciprocally low levels of 5mC at transcription factor binding sites, revealing a dynamic DNA methylation process at cis-regulatory elements. Base resolution sequencing of 5 hydroxymethylcytosine in human and mouse embryonic stem cells
Project description:Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation, an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, which presents a major roadblock for dissecting the precise roles of PRC2 activity during lineage commitment. While recent studies suggest that loss of H3K27me3 leads to changes in DNA methylation in ESCs, how these two pathways coordinate to regulate gene expression programs during lineage commitment is poorly understood. Here, we analyzed gene expression and DNA methylation levels in several PRC2 mutant ESC lines that maintain varying levels of H3K27me3. We found that maintenance of intermediate levels of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). However, genes that function to specify other lineages failed to be repressed, suggesting that PRC2 activity is necessary for lineage fidelity. We also found that H3K27me3 is antagonistic to DNA methylation in cis. Furthermore, loss of H3K27me3 leads to a gain in promoter DNA methylation in developmental genes in ESCs and in lineage genes during differentiation. Thus, our data suggest a role for PRC2 in coordinating dynamic gene repression while protecting against inappropriate promoter DNA methylation during differentiation. Embryonic Stem Cell (ESC) lines mutant for PRC2 core components Suz12 (Suz12GT and Suz12delta) and Eed (Eednull) were subjected to in vitro directed differentiation down the spinal motor neuron lineage. ESCs and day 5 differentiated cells from the three mutant lines and wild-type were used for Reduced Representation Bisulfite Sequencing (RRBS).
Project description:While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe the first complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive and complementary relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells and suggesting a possible new approach toward the development of cancer therapeutics. ChIP-methylC-Seq on H3K9me3, H3K27me3, and H3K36me3 in breast cancer HCC1954. 36 cycles of sequencing on Illumina platform.
Project description:We develop a single cell methylome analysis technique based on RRBS, which works robustly for mouse embryonic stem cells (mESCs), sperm, metaphase II oocytes, and zygotes. In total, 36 samples were analyzed, including 8 single mouse embryonic stem cells (mESCs), pooled-5, 10, 20 mESCs, bulk mESCs, 7 single sperms, 10 single pronuclei from 5 individual zygotes, 2 metaphase II oocytes, 2 the first polar bodies and 3 negative controls.
Project description:Intrauterine growth restriction (IUGR) is one of the most common adverse pregnancy outcomes with high risk of perinatal morbidity and mortality, and affects up to 7% of pregnancies. Here, seven pairs of placentas were employed for whole genomic promoter DNA methylation profiling and some of the candidate differentially methylated promoters were further validated in additional twelve pairs of samples. Consistent with previous report, our results further indicated that IUGR associated placentas harbored a distinct promoter DNA hypomethylation pattern and the result was further confirmed byultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) In this study, seven pairs of MC twins that were diagnosed as severely growth-discordant (one twin with IUGR but another one was normal) were enrolled for DNA methylation identification.Promoters are defined as the regions crossing the upstream 2200bp and downstream 500bp of the transcriptional start site. A complete set of 34163 genes located in the 22 autosomes and the XY sex chromosomes were prepared based on the RefSeq gene files (http://genome.ucsc.edu/, hg19). The promoters overlapped with each other were merged to form a large region, resulting in 20882 merged candidate regions ranging from 2700bp to 8864bp. The capture probes were designed and synthesized by Roche Nimblegen Incorporation, consisted of 150,407 oligonucleotides.
Project description:Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide and has a poor prognosis. Promoters represent an essential regulatory element of gene transcription in the human genome. In order to understand the promoter methylation in relation with gene transcription in HCCs, we applied a liquid hybridization capture-based bisulfite sequencing (LHC-BS) approach to examine the promoter methylome of HCCs, for which we customized 150,407 capture probes and enabled coverage of 91.8% of the RefSeq gene promoters within the human genome. We found the differential promoter DNA methylation between HCCs and peripheral normal tissues. Then we integrated promoter methylomic and transcriptomic profiling and described gene expression and regulation in HCCs. Lastly, we validated the key genes in a larger number of samples and screened candidate genes aberrantly regulated by DNA methylation in human HCCs. Capture-based whole genome promoter bisulfite-seq for 8 pairs of HCC tumor and non-tumor liver (NTL) samples.
Project description:Significant interest has been devoted to the isolation of human pluripotent stem cells displaying the naive state of mouse embryonic stem cells. However, to what extent naive human cells isolated in culture resemble pluripotent cells in vivo remains unclear. Here we present three lines of evidence indicating that naive pluripotent stem cells generated in the absence of transgenes share defining molecular signatures with the human pre-implantation embryo. First, a comprehensive analysis of the transposcriptome reveals that naive human cells have a retroelement expression profile of human cleavage stage embryos. Second, base-resolution mapping of the DNA methylome in naive human cells reveals a genome-wide reduction in CpG and non-CpG methylation levels. Third, female naive cells exhibit an X chromosome status that is similar to that of the human blastocyst. Our work demonstrates that pluripotent stem cells with epigenomic hallmarks of the early human embryo can be directly captured in vitro. Examine the methylomes of 6 naïve, 2 primed and 2 re-primed human embryonic stem cells