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

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

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 to active CpG rich promoters independent of cell type.

<p>A. The Venn diagram depicts the overlap of transcripts with an MBD3 peak within 3-7 and 9,310 in MDA-231 were included in the overlap analysis. A total of 4292 transcripts had MBD3 peaks at TSS in both cell lines. B. The heatmap displays MBD3-DamID signal, H3K4me3 signal (MCF-7 ChIP-seq data is from UW, GEO accession number GSM945269, ENCODE Project Consortium 2011; MDA-231 ChIP-seq, this study) and gene expression in MCF-7 and MDA-231 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Khaitan1" target="_blank">[54]</a>. 8,207 genes were selected for display as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. Genomic intervals from −7 to +3 kb relative to TSS were binned into 20 equal bins and ranked by MBD3-DamID signal. H3K4me3 and gene expression were displayed in the same order. The color scale for interpretation of signal intensity is located at the bottom of the heatmap. C. Refseq transcript 5′ ends were selected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. The plot depicts average DamID signal for all transcripts across the interval from −3 to +3 kb relative to TSS. MCF-7 and MDA-231 plots are in green and tan, respectively. D. For each cell type, promoters were subdivided as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Weber1" target="_blank">[16]</a>. The plot displays the average DamID signal for each promoter class by cell type across the indicated genomic interval. Promoter class is indicated by color coding as indicated in the figure. E. A composite gene model for genes selected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. DamID signal intensity was averaged for genes in the two cell lines and displayed in the plot. The location of TSS and TES is indicated. Cell lines are distinguished by color coding as indicated. F. The column graph depicts the enrichment percentage derived from Functional Analysis. Genes were selected as MBD3 targets if there was an MBD3 peak within 3 kb of TSS. The luminal and basal discriminatory genes were as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-CharafeJauffret1" target="_blank">[47]</a>.</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

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

MBD3 localizes to active promoters and to other genomic regions.

<p>A. The heatmap was prepared in a manner similar to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen-1004028-g002" target="_blank">Figure 2B</a>. Genes were binned (500 bp per bin) from −7 to +3 kb relative to TSS, and rank ordered by MBD3 density. H3K4me3 and gene expression were displayed in the same order. Intensity signal scale is indicated in the figure. B. Refseq transcript 5′ ends were selected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. The plot depicts average MBD3 ChIP-seq signal for all transcripts across the interval from −3 to +3 kb relative to TSS. Color coding is as depicted in the figure. C. For each cell type, promoters were subdivided as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Weber1" target="_blank">[16]</a>. The plot displays the average MBD3 ChIP-seq signal for each promoter class by cell type across the indicated genomic interval. Promoter class is indicated by color coding as indicated in the figure. D. The column graph depicts the overlap of MBD3 peaks with the indicated genomic features. Peaks that overlap promoters (−3 to +3 kb relative to TSS) or the indicated features by at least one base are included in the columns. Note that a single peak could overlap with more than one type of feature in this analysis. E. A composite gene model for genes selected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. MBD3 ChIP-seq signal intensity was averaged for genes in the two cell lines and displayed in the plot. The location of TSS and TES is indicated. Cell lines are distinguished by color coding as indicated. F. The column graph depicts the enrichment score derived from Functional Analysis. Genes were selected as MBD3 targets if there was an MBD3 peak within 3 kb of TSS. The luminal and basal discriminatory genes were as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-CharafeJauffret1" target="_blank">[47]</a>.</p

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

<div><p>Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01087307" target="_blank">NCT01087307</a></p></div

Structural changes detected in skin fibroblast clones D1-L-F1 and D2-L-F.

<p>(A) All genome changes detected in D1-L-F1 and D2-L-F clones. The tracks numbered from innermost are as follows: 1—structural changes. Green = deletions, black = duplications, blue = inversions and red = translocations. 2—deletions as detected by read-depth analyses. 3—amplifications as detected by read-depth analyses. 4—LOH events. 5—somatic SNVs, black dots are heterozygous SNVs and red dots are homozygous SNVs. (B) Schematic describing the chr19, chr20 translocation in D1-L-F1. Black rectangles depict region wherein translocation event was detected with a concomitant change in copy number.</p