Expression of BPAG1 in normal human melanocytes and human melanoma cell lines.

<p>(A) The expression of BPAG1 and BPAG2 mRNA was quantified by RT-PCR in normal human melanocytes (NHM) and human melanoma cell lines A375 and G361. Normal human keratinocyte (NHK) mRNA was used as a positive control. β-actin was amplified as a loading control for cDNA. NTC; no template control. (B) The expression of BPAG1 protein was detected by IP-western blotting in human melanoma cell lines A375 and G361. A431 was used as positive control for BPAG1. The arrow indicates BPAG1.</p

Overview of the rapid method for isolating auto-antibody against tumor-associated antigen (TAA) using a scFv library.

<p>The tumor-homing scFv-presenting phages were collected from tumors that were injected with a scFv library. The collected phages were infected to HB2151 for scFv secretion. The secreted scFvs from HB2151 were transferred to nitrocellulose membranes by colony lift. The membranes were incubated with tumor lysate followed by serum from a tumor-bearing mouse. The scFv-tumor protein complex was detected by auto-antibodies. The complex was digested into peptide by trypsin and analyzed using MALDI-TOF mass spectrometry for identification.</p

Quantification of anti-BPAG1 auto-antibodies in melanoma patients.

<p>(A) The levels of anti-BPAG1 auto-antibodies in sera collected from healthy volunteers and melanoma patients were quantified using a MESACUP BP230 ELISA Kit. The INDEX values were plotted and the average INDEX values as shown (±S.E.M.) for control subjects (1.64±0.27) and melanoma patients (3.47±0.40). (B) The melanoma patients were classified using the American Joint Committee on Cancer (AJCC) 2002 staging criteria. <i>In situ</i>, stage I or stage II patients were categorized as “early”, while stage III or stage IV patients were categorized as “advanced”. The average INDEX values (±S.E.M.) of early and advanced melanoma patients were 4.14±0.83 and 3.15±0.43, respectively; the bars indicate the average INDEX value.</p

Identification of bpag1 as a tumor antigen recognized by auto-antibodies.

<p>(A) An example of the screening output. ScFv-tumor antigen complex was detected with auto-antibodies in tumor-bearing mouse serum. (B) Eight candidates were identified by MALDI-TOF mass spectrometry with statistical significance (p<0.05); expect  =  expectation value. (C) Comparison of bpag1, tbc1d13 and c7orf30 expression in NIH-3T3 cells (white bar), F10 melanoma cells (black bar) and F10 melanoma tumors (grey bar) by SYBR Green real-time PCR.</p

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

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

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