Comparative genomic map of the <i>Mcs5c</i> locus.

<p>Map coordinates for the <i>Mcs5c</i> locus (dark gray bar) and neighboring genes are depicted for the rat (UCSC Genome Browser, Mar. 2012, rn5), mouse (UCSC, Dec. 2011, mm10) and human (UCSC, Feb. 2009, hg19) genomes, and were determined based on sequence homology. The WKy-homozygous genomic region of congenic line 5C-27 is shown in light gray relative to the rat <i>Mcs5c</i> locus. This congenic line carries the smallest defined region of the locus (275kb) and was the resistant line used for all <i>Mcs5c</i> experiments. The remainder of the 5C-27 line is WF-homozygous (chr–chromosome; <i>Tnfsf15/TNFSF15</i> –Tumor Necrosis Factor (Ligand) Superfamily, Member 15; <i>Tnfsf8/TNFSF8</i> –Tumor Necrosis Factor (Ligand) Superfamily, Member 8; <i>Tnc/TNC</i>–Tenascin C; <i>DEC1</i> –Deleted in Esophageal Cancer 1; <i>Pappa/PAPP-A</i>–Pregnancy-Associated Plasma Protein A).</p

The Non-coding Mammary Carcinoma Susceptibility Locus, <i>Mcs5c</i>, Regulates <i>Pappa</i> Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation

<div><p>In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman’s life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, <i>Mcs5c</i>, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that <i>Mcs5c</i> acts within the mammary gland to regulate expression of the neighboring gene <i>Pappa</i> during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. <i>Mcs5c</i>-mediated regulation of <i>Pappa</i> appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage.</p></div

<i>Mcs5c</i> acts in a mammary gland autonomous manner to influence carcinoma multiplicity.

<p>The four mammary gland transplant groups are listed on the x-axis (R = <i>Mcs5c</i> resistant 5C-27 line, S = <i>Mcs5c</i> susceptible control line), with the genotype of the donor listed first and the genotype of the recipient listed second. The number of animals per transplant group were: R->R, n = 76; R->S, n = 49; S->R, n = 69; S->S, n = 39. The y-axis indicates the percentage of animals in each group that had one or more carcinomas at the transplant site 15 weeks after DMBA administration. Logistic regression analysis found a statistically significant donor effect (p-value = 0.0043; recipient effect p-value = 0.825).</p

<i>In vivo</i> methylation analysis of the <i>Pappa</i> CGI and CGI shore.

<p>(A) The first exon of the <i>Pappa</i> gene is shown in relation to a conserved CGI (green box) and the P4-1 looping fragment (gray box). The location of the 12 shore CG dinucleotides investigated in this report are indicated and numbered, as are the regions covered by the two pre-made CGI pyrosequencing assays. The CGI assays each examined 5 CG dinucleotides within the island. (B) A scatterplot demonstrating a statistically significant negative correlation between 6 week MEC <i>Pappa</i> expression (x-axis) and shore methylation (y-axis; Pearson correlation coefficient, R, = -0.67, n = 18, p-value = 0.0023) is shown. Shore methylation values were obtained by averaging the absolute methylation percentages of the 6 significant shore sites (Sites 1, 3, 6–9) for each individual sample. A linear trend line is shown with the dotted line (slope = -7.88). (C) No correlation was observed between 6 week MEC <i>Pappa</i> expression (x-axis) and CGI methylation (y-axis; Pearson correlation coefficient, R, = 0.16, n = 18, p-value = 0.52). CGI methylation values were obtained by averaging the absolute methylation percentages of the 5 sites examined by the CGI-2 assay for each individual sample. A linear trend line is shown with the dotted line (slope = 0.544).</p

Removal of <i>Mcs5c</i> TCE copies results in decreased <i>Pappa</i> expression <i>in vitro</i>.

<p>(A) CRISPR guides targeting the TCE were transfected into LA7 cells, and clones were screened for removal of the target region. Nine positive clones were assessed for remaining TCE copy number via qPCR, standardized to a non-targeted region within the <i>Pappa</i> gene. Copy number in wild-type LA7 cells (n = 4 independent cultures) was also assessed, and results were normalized to diploid MECs. (B) Interaction frequency (IF, y-axis) was calculated in select positive clones (n = 3) and WT LA7 cells between the TCE and <i>Pappa</i> bait regions P4-1 and P3-3. The IF for a positive control region, two nearby <i>BglII</i> fragments, is shown for reference. (C) <i>Pappa</i> expression in positive clones and WT LA7 cells (n = 6) was analyzed via qPCR and standardized to <i>Tbp</i> expression. (D) A scatterplot of <i>Pappa</i> expression and <i>Mcs5c</i> TCE copy number demonstrate a statistically significant positive correlation between the two (Pearson correlation coefficient, R, = 0.6245, n = 13, p-value = 0.0225). A linear trend line is shown (slope = 5.327). (E) WT LA7 cells were treated with 0μM (n = 4) or 1μM (n = 4) 5-aza-dC for 48hrs. <i>Pappa</i> expression was analyzed via qPCR and standardized to <i>Tbp</i>. (F) Methylation levels at <i>Pappa</i> CGI shore site 12 are shown for WT LA7 cells (n = 8) and CRISPR clones (n = 9). The p-value reflects Bonferroni correction. Scatterplots demonstrating negative correlations for shore site 12 methylation levels and <i>Mcs5c</i> TCE copy number (G) and <i>Pappa</i> expression (H) are shown (R = -0.8034/-0.6022, n = 13/17, p-value = 0.0009/0.011, respectively) along with linear tread lines (slope = -0.046/-0.004, respectively). For all bar graphs, p-values were obtained using the non-parametric Mann-Whitney U test, and standard error bars are shown (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).</p

Summary of <i>Mcs5c</i> activity within MECs.

<p>Our experimental finding are summarized according to the specific time periods examined in this study. The aWOS is the time period during which <i>Mcs5c</i> is most active in MECs, functioning in a genotype-independent manner with regards to looping, and a genotype-dependent manner with regards to <i>Pappa</i> CGI shore methylation and gene expression. <i>In vivo</i> and <i>in vitro</i> analyses have led to the proposed timeline of events, whereby <i>Mcs5c</i> looping results in differential methylation which subsequently affects gene expression. Ultimately, this leads to the previously reported <i>Mcs5c</i> genotype-dependent differences in MG cancer susceptibility [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006261#pgen.1006261.ref011" target="_blank">11</a>] (MG–mammary gland; Susc.–<i>Mcs5c</i> susceptible rats; directionality of changes indicated with small arrows).</p

Percent change in <i>Mcs5c</i> susceptible MEC methylation at sites within the <i>Pappa</i> CGI shore relative to <i>Mcs5c</i> resistant MECs.

<p>Percent change in <i>Mcs5c</i> susceptible MEC methylation at sites within the <i>Pappa</i> CGI shore relative to <i>Mcs5c</i> resistant MECs.</p

Characterization of rat mammary epithelial cells (RMECs) based on cell surface and intracellular markers.

<p>(<b>A</b>) Representative flow cytometric histograms and dot plots showing gating for propidium iodide (PI)-negative (live) cells (left panel); exclusion of endothelial cells and leukocytes based on CD31 and CD45 expression, respectively (middle left panel); CD61 expression in CD45–CD31– RMECs (middle right panel); CD24 and CD29 expression in CD45–CD31– RMECs identifies two major populations indicated with a red or blue circle (right panel). (<b>B</b>) Dot plots of intracellular cytokeratin (CK) 14 and CK19 expression in CD45–CD31– RMECs (upper left panel); intracellular smooth muscle actin (SMA) staining with phalloidin and CD29 expression in CD45–CD31– RMECs (upper right panel); overlay of dot plots showing CD24 and CD29 expression in CK14+CK19- cells and CK19+CK14- cells (lower left panel); overlay of dot plots of phalloidin bright cells on CD24 and CD29 expression in CD45–CD31– RMECs (lower right panel). Based on CK14, CK19, and SMA expression, the luminal (red) and basal (blue) populations in CD45–CD31– RMECs are identified. (<b>C</b>) Contour plot showing binding of Peanut Lectin (PNL) or anti-Thy-1 in CD45–CD31– RMECs (left panel), overlaid histograms showing CD29 expression on PNL+Thy1-, PNL-Thy-1+ cells (middle left panel), contour plots showing anti-Thy-1 (middle right panel) or PNL binding in CD29med or CD29hi cells (right panel). For all panels, rats of 12 weeks of age were used.</p

Differences between the RMECs from mammary glands of untreated control rats and mammary carcinomas from rats exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU).

<p>(<b>A</b>) Representative pseudo-color dot plots showing CD24 and CD29 expression in the RMECs from the mammary gland of an age-matched (22 weeks of age) untreated control rat (upper left panel) and a DMBA- (upper middle panel) or MNU-induced (upper right panel) carcinoma; bar graphs (lower panel) quantifying mean ± sem percentage cells in the CD24hiD29hi gate within the total (CD45–CD31–) RMECs. A significantly different percentage comparing carcinomas to mammary glands is indicated with an asterisk (p<0.05). (<b>B</b>) Bar graphs showing the mean ± sem percentage of RMECs containing >2n cellular DNA (actively dividing cells in S/G2+M phase of cell cycle). Significantly different percentage comparing RMECs from carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>C</b>) Representative overlaid histograms showing upregulation of CD29 expression (upper left panel), upregulation of CD49f expression (upper middle panel) and downregulation of CD61 expression (upper right panel) in RMECs of a DMBA-induced or MNU-induced carcinoma as compared to a control mammary gland; bar graphs quantifying the mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD29 (lower left panel), CD49f (lower middle panel) and CD61 (lower right panel) on RMECs from control mammary glands and carcinomas. Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative pseudo-color dot plot showing gating for CD29 and focal adhesion kinase (FAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hiFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>E</b>) Representative pseudo-color dot plot showing gating for CD29 and Y397-phosphorylated focal adhesion kinase (pFAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hi pFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). In the entire <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">figure 4</a>, age-matched untreated control mammary glands: n = 16, DMBA-induced mammary carcinomas: n = 10 and MNU-induced mammary carcinomas: n = 10.</p

Epithelial cell differentiation in mammary glands and carcinomas from 7,12-dimethylbenz(a)anthracene (DMBA)-or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-exposed rats.

<p>(<b>A</b>) Schematic representation of the modulation of RMEC differentiation 1 week after exposure of rats to the mammary carcinogens DMBA or MNU. DMBA exposure increases CD49f expression and proliferation (not shown here). MNU exposure disrupts the luminal and basal homeostasis. (<b>B</b>) Schematic representation of the changes of RMEC differentiation in carcinomas as compared to mammary gland from untreated age-matched (22 weeks of age) control rats. Note that the RMECs from animals of 22 weeks of age have a higher percentage of luminal cells as compared to younger animals (comparing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g001" target="_blank">Fig. 1A</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">Fig. 4A</a>). In both panels cell surface expression of CD24, CD29, CD49f and CD61 and intracellular expression of focal adhesion kinase (FAK) and Y397-phosphorylated FAK (pFAK) in basal, luminal and CD24hiCD29hi-gated cells are shown.</p