Hypermethylated LTR retrotransposon exhibits enhancer activity

<p>LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. They are silenced by hypermethylation of cytosines in CpG dinucleotides and are considered parasitic DNA serving no useful function for the host genome. However, hypermethylated LTRs contain enhancer and promoter sequences and can promote tissue-specific transcription of <i>cis</i>-linked genes. To resolve the apparent paradox of hypermethylated LTRs possessing transcriptional activities, we studied the ERV-9 LTR retrotransposon located at the 5′ border of the transcriptionally active β<i>-globin</i> gene locus in human erythroid progenitor and erythroleukemia K562 cells. We found that the ERV-9 LTR, containing 65 CpGs in 1.7 kb DNA, was hypermethylated (with > 90% methylated CpGs). Hypermethylated LTR possessed transcriptional enhancer activity, since <i>in vivo</i> deletion of the LTR by CRISPR-cas9 suppressed transcription of the globin genes by > 50%. ChIP-qPCR and ChIP-seq studies showed that the hypermethylated LTR enhancer spanning recurrent CCAATCG and GATA motifs associated respectively with key transcription factors (TFs) NF-Y and GATA-1 and -2 at reduced levels, compared with the unmethylated LTR in transfected LTR-reporter gene plasmids. Electrophoretic mobility shift assays with methylated LTR enhancer probe showed that the methylated probe bound both NF-Y and GATA-1 and -2 with lower affinities than the unmethylated enhancer probe. Thus, hypermethylation drastically reduced, but did not totally abolish, the binding affinities of the enhancer motifs to the key TFs to assemble the LTR-pol II transcription complex that activated transcription of <i>cis</i>-linked genes at reduced efficiency.</p

Additional file 2: of Hypomethylation coordinates antagonistically with hypermethylation in cancer development: a case study of leukemia

List of C-DMRs. This is an “xls” file providing coordinates for hyper- and hypo-C-DMRs common across all control tests in separate excel sheets, respectively. (XLS 117 kb

Cirsium tanakae Matsum. forma obvallatum Makino

原著和名: クルマアザミ科名: キク科 = Compositae採集地: 千葉県 佐倉市 生谷 (下総 佐倉市 生谷)採集日: 1985/11/12採集者: 萩庭丈壽整理番号: JH030548国立科学博物館整理番号: TNS-VS-98054

Additional file 7: of Hypomethylation coordinates antagonistically with hypermethylation in cancer development: a case study of leukemia

WGCNA. This is a “docx” file that describes correlation modules in details, together with the step-by-step description of WGCNA analysis. WGCNA description includes data cleaning and preprocessing, adjacency topological overlap matrix construction, module detection, calculation of various module measures, and description of preservation statistics methods used. It includes tables and figures for both 3′UTR methylation and non-coding gene data, and their relation with expression of genes analysis. (DOCX 1555 kb

MBD3 Localizes at Promoters, Gene Bodies and Enhancers of Active Genes

<div><p>The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.</p></div

Methylation level of CpG island with an MBD3 peak that fall into within the indicated levels of DNA methylation as determined by RRBS.

<p>CpG islands bound by MBD3 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>) were binned by methylation level (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>). Enrichment or depletion of MBD3 in each bin was determined by two-tailed t-test. Significantly enriched or depleted bins (p<0.001) are highlighted in bold.</p

MBD3 localizes in a cell-type specific manner by ChIP-seq.

<p>A. Exemplar loci are depicted in genome browser format (hg19, <a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Kent1" target="_blank">[53]</a>). Individual sequencing tracks are indicated to the left of the browser view. Genomic intervals and scale bar are indicated above the tracks. B. The column graph depicts the total number of peaks defined from the current study in each cell type. The colors depict peaks that overlap in the two cell types by at least one base as well as those with no overlap (cell-type specific in the figure).</p

Association of MBD3 in MCF-7 with local histone modifications.

<p>A. The Venn diagrams depict overlap of MBD3 associated TSS's with those marked by H3K4me3, H3K9me3, or H3K27me3 (as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>). The total number of TSS's in each category is indicated in the figure. B. The line graphs depict abundance of MBD3, H3K4me3 and H3K27ac (ChIP-seq data from USC/Farnahm, GEO accession number GSM945854, ENCODE Project Consortium 2011) at MBD3 peaks overlapping TSS or MBD3 peaks not overlapping TSS. C. The column graph depicts the percentage of 1 kb genomic bins bound by MBD3 that display the indicated modification/location patterns. (Presence of MBD3 or histone marks is assigned as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>).</p

Non-TSS MBD3 peaks overlapping H3K27ac are in proximity to promoters.

<p>A. The genome browser view displays the genomic region around the human GATA3 locus. MBD3 ChIP-seq data is depicted at the upper portion of the panel along with a scale bar. ChIA-PET read pairs derived from GSM970209 are depicted in the lower portion of the panel. B. The genome browser view displays the same genomic region of the GATA3 locus depicted in panel A. The location of the anchor primer is depicted in the upper panel of tracks. The region indicated is expanded in the lower set of tracks to display primer sets around GATA3. The location of EcoRI sites within the region is indicated below the tracks. The gel displays PCR products using the anchor primer in PCR with the indicated primers tiling across GATA3. C. MBD3 read depth was plotted for all genes where Pol II ChIA-PET pairs exist with one end anchored at TSS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>). ChIA-PET pairs were rank ordered by MBD3 density at the TSS end (end 1) and divided into 10 groups. The upper panel depicts MBD3 density for the end 1. The lower panel depicts MBD3 density at the distal end (end 2) of the ChIA-PET pair. D. Metagene plots (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>) of nucleosome occupancy from cells with and without depletion of MBD3.</p

The DamID platform for location analysis of human MBD3.

<p>A. MCF-7 and MDA-231 cells express endogenous MBD3 at equivalent levels. The immunoblot depicts endogenous MBD3 protein expression in MCF-7 and in MDA-231 cells. Beta actin is included as a control. B. Dam-fused MBD3 can be incorporated into the NuRD complex. An IP-western analysis was performed on lysates from HeLa cells infected with the pLgw-MBD3-V5-EcoDam using V5 antibody or control IgG. The resulting immunoprecipitates were compared to input lysates and probed for MBD3-Dam fusion and endogenous Mi-2 and MTA3. C. Genome browser (hg18, <a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Kent1" target="_blank">[53]</a>) views of exemplar genes analyzed by DamID. The location of genes is indicated at the bottom of the respective panels. Normalized probe signal within the regions displayed is indicated above the genomic coordinates (hg18).</p