The rat mammary carcinoma susceptibility locus <i>1a</i> (<i>Mcs1a</i>) is located in a gene desert and confers resistance to three distinctly acting carcinogenic treatments.

<p>A) Genetic map of the congenic lines contributing to the positional identification of the <i>Mcs1a</i> locus on rat chromosome <i>2</i>. Each congenic line, as defined by genotyping the genetic markers indicated along the vertical scale bar (also listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s005" target="_blank">Table S1</a>), represents a segment from the resistant Copenhagen (Cop) inbred strain introgressed into the susceptible Wistar-Furth (WF) genetic background. The critical interval for the <i>Mcs1a</i> resistance allele is defined by resistant congenic lines (filled bars) showing a <i>7,12</i>-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma multiplicity that is lower than that of the susceptible congenic control line (WF.Cop), and susceptible congenic lines (open bars) showing a DMBA-induced mammary carcinoma multiplicity not different than that of the susceptible congenic control line. The grey boxes illustrate the areas of recombination. The coordinates (in bp) along the vertical axis are from the 2004 version of the rat genome (UCSC Genome Browser, rn4). B) DMBA-induced mammary carcinoma multiplicity phenotype for <i>Mcs1a</i> resistant congenic lines Q (n = 83), R3 (n = 24), V4 (n = 24), W4 (n = 28), Y4 (n = 45), and W5 (n = 41) and susceptible congenic lines P5 (n = 16), V5 (n = 56), R5 (n = 30), A4 (n = 24), Y3 (n = 38) and WF.Cop (n = 44). Congenic line Q originally defined the <i>Mcs1a</i> interval in our previous publication and is used as a reference for resistance <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549-Haag1" target="_blank">[33]</a>. C) <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-induced mammary carcinoma multiplicity phenotype of resistant congenic lines W4 (n = 20) and W5 (n = 23), susceptible congenic line R5 (n = 14) and the susceptible congenic control line WF.Cop (n = 28). D) Mammary carcinoma multiplicity phenotype induced by mammary ductal infusion of retrovirus expressing the activated <i>HER2/neu</i> oncogene (<i>HER2/neu</i>) for resistant congenic line R3 (n = 15) and susceptible congenic line A4 (n = 14). In all graphs, resistant congenic lines are displayed as filled bars, susceptible congenic lines are displayed as open bars. Significant difference (P<0.05) from the susceptible congenic control line (panels B and C) or from susceptible congenic line A4 (panel D) is indicated by an asterisk.</p

The non-protein coding <i>Mcs1a</i> resistance locus regulates transcript levels of <i>Nr2f1</i> in the mammary gland, mammary epithelium and mammary carcinomas.

<p>A–C) Q-PCR analysis of <i>Nr2f1</i> transcript levels in mammary gland (MG; panel A), rat mammary epithelial cell (RMEC, panel B) and DMBA- or MNU-induced mammary carcinoma (carc.; panel C) samples from resistant congenic (res.; n = 54 panel A, n = 18 panel B, n = 12 each panel C) and WF.Cop susceptible congenic control (susc.; n = 19 panel A, n = 11 panel B, n = 6 each panel C) rat lines. Data derived from both the W4 and W5 congenic lines are used in the <i>Mcs1a</i> resistant congenic data points. <i>Nr2f1</i> transcript levels are shown relative to the transcript level of the <i>ActB</i> endogenous control gene. D) Chromosome conformation capture (3C) assay for the <i>Nr2f1</i> promoter and the <i>Mcs1a</i> critical interval. The region is shown as a UCSC Genome Browser view (version rn4 of rat genome) and the location of the <i>Mcs1a</i> critical interval in indicated as a horizontal black line. The evolutionary sequence conservation track is also shown. The locations of the 3C assay primers are shown as vertical purple lines. The fixed primer in the <i>Nr2f1</i> promoter is shown with respect to <i>Bgl</i>II restriction sites in the <i>Nr2f1</i> gene span. Graphed is the average relative interaction frequency (+/− sem) of the <i>Bgl</i>II fragment in the <i>Nr2f1</i> promoter containing the fixed primer with each of the <i>Bgl</i>II fragments in <i>Mcs1a</i> containing the 3C assay primers (n = 4 or more templates). Significantly increased relative interaction frequency is indicated with 1 asterisk for a background cut-off interaction frequency of 0.05 and 2 asterisks for a background cut-off interaction frequency of 0.1. The horizontal axis indicates the genomic distance (in Kb) from the 3C assay primers in <i>Mcs1a</i> to the fixed primer in the <i>Nr2f1</i> promoter. The main peak in the interaction profile coincides with blocks of strong evolutionary sequence conservation (to zebrafish and frog, <i>X. tropicalis</i>). Sequence variation within the interacting <i>Mcs1a</i> region is outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s002" target="_blank">Figure S2</a>. E) Schematic drawing of the higher-order chromatin interaction of <i>Mcs1a</i> with the <i>Nr2f1</i> promoter. The <i>Mcs1a</i> critical interval is indicated as a thick area in the black line that represents the DNA. The green, orange and red shapes represent the putative DNA-binding proteins involved in the structure.</p

Reduced <i>NR2F1</i> transcript levels in human breast cancer correlate with histological grade 3 tumors.

<p>A) Average (+/− sem) of the normalized median <i>NR2F1</i> probe intensities, obtained from a total of 12 breast cancer expression studies (with 120+ samples each study) available through Oncomine. G = Histological grade, ER = Estrogen receptor, PR = Progestrone receptor, HER2 = Human epidermal growth factor receptor 2, TN = Triple-negative. B) Average (+/− sem) of the normalized <i>NR2F1</i> probe intensity in sample groups organized by grade and ER status (from GSE3494; left panel) or by grade and HER2 status (from GSE5460; right panel). Left panel: ER+G1 n = 62, ER+G2 n = 116, ER+G3 n = 33, ER-G2 n = 11, ER-G3 n = 21. Right panel: HER2+G2 n = 7, HER2+G3 n = 23, HER2-G1 n = 27, HER2-G2 n = 24, HER2-G3 n = 46. Significantly different <i>NR2F1</i> transcript level (P<0.05) is indicated by an asterisk.</p

<i>Mcs1a</i> affects rat mammary epithelial cell (RMEC) growth and differentiation.

<p>A) Results of the limiting-dilution RMEC transplantation assay. Graphed is the percentage of transplant sites with an outgrowth versus the number of RMECs transplanted. Vertical bars represent lower and upper limits of a 95%-confidence interval for a proportion. Outgrowth potential of RMECs from the susceptible congenic control (susc.; light grey) and <i>Mcs1a</i> resistant congenic (res.; dark grey) line (lines W4 and W5 combined) is not significantly different. B) Pseudocolor dot plots from a susceptible congenic control sample, representing the gating strategy used to enrich for clonogenic luminal RMECs using cell sorting on the BD FACS Aria. CD31–CD45− RMECs were divided into luminal and basal cells based on CD24 and CD29 expression (left panel). Based on staining with anti-CD61 and peanut lectin (PNL), the clonogenic luminal cell population (CD45–CD31-CD24hiCD29medCD61+PNLhi; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s003" target="_blank">Figure S3</a>) was selected for Matrigel assays (right panel). C) Graphed is the average (+/− sem) number of spherical mammary colonies formed in Matrigel assays plating 10,000 clonogenic RMECs sorted from the susceptible congenic control (n = 12) and <i>Mcs1a</i> resistant congenic line (n = 10; line W4 only). In the lower panel, a representative picture of a spherical mammary colony in a methylene blue-stained Matrigel is shown. Significantly different colony-forming ability (P<0.05) is indicated by an asterisk.</p

Proposed model for the <i>Mcs1a</i> breast cancer risk-affecting mechanism.

<p>The <i>Mcs1a</i> resistance allele displays increased mammary <i>Nr2f1</i> transcript levels as compared with the susceptible allele. Lower <i>Nr2f1</i> transcript levels in the mammary gland are associated with susceptibility, a lower percentage of luminal rat mammary epithelial cells (RMEC), a higher percentage of basal RMECs and increased colony-forming ability of the clonogenic luminal RMEC population, indicative of increased proliferation.</p

FACS analysis of rat and mouse MEC populations.

<p>A) Quantification of luminal and basal RMEC populations derived from susceptible congenic control (susc.; open bars; n = 6; WF.Cop) and <i>Mcs1a</i> resistant congenic (res.; filled bars; n = 7; Line W4) rats. B) Quantification of the CD61hi and PNLhi gates in RMEC populations derived from susc. (open bars; n = 11; WF.Cop) and res. (filled bars; n = 24; Lines W4 and W5). These gates were quantified in all CD31–CD45− RMECs as well as in the luminal RMECs. PNL = peanut lectin. C) Quantification of luminal and basal MMEC populations derived from wild type (WT; filled bars; n = 30) and megadeletion (MD; open bars; n = 27) mice (FVB and B6 pooled). D) Quantification of mature luminal (ML), luminal progenitor (LP) and mammary stem cell (MaSc) populations derived from WT (filled bars; n = 30) and MD (open bars; n = 27) mice (FVB and B6 pooled). FACS pseudocolor dot plot or histograms in each panel's upper figure illustrate the gating strategies used to quantify specific MEC populations. These dot plots were taken from a susc. sample (RMEC), or from a WT (FVB) sample (MMEC). Graphed in panels A–C are the average (+/−sem) percentages of populations among CD45–CD31− MECs. Graphed in panel D is the average (+/− sem) percentage of population among CD45–CD31− MECs, expressed relative to the WT run on the same day. In all graphs, significantly different (P<0.05) percentages of cells between susc. and <i>Mcs1a</i> or between WT and MD are indicated with an asterisk.</p

Global gene expression analysis reveals potential processes associated with <i>Nr2f1/NR2F1</i> transcript levels.

<p>A) Heatmap of expression correlation clustering analysis of 412 genes (which have 1-1-1 mouse-rat-human orthologues) that are differentially expressed between mammary gland samples from megadeletion (MD) and wild type mice (WT), both FVB. B) Heatmap of expression correlation clustering analysis of the same 412 genes in 243 human breast cancers from GSE3494, downloaded from the Gene Expression Omnibus. For both panels, strong correlation is indicated in blue, strong anti-correlation is indicated in yellow. The position of <i>Nr2f1/NR2F1</i> in group 1 is indicated by a dark blue vertical line. Below the panels, a summary of the gene ontology enrichment analysis is given. Smaller print indicates weak enrichment, larger print indicates strong enrichment (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s009" target="_blank">Tables S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s010" target="_blank">S6</a>).</p

A megadeletion (MD) mouse model for the rat <i>Mcs1a</i> locus reveals <i>Nr2f1</i> as the <i>Mcs1a</i> target gene.

<p>A) Schematic representation of the rat <i>Mcs1a</i> locus and mouse orthologous locus that was deleted using mutagenic insertion and chromosome engineering resource (MICER) vector-assisted targeting. The horizontal bar, genetic markers and chromosomal base pair positions in purple represent the location of the <i>Mcs1a</i> critical interval on rat chromosome <i>2</i> (UCSC Genome Browser, rn4). The marker and base pair position in light purple delineate the location of the distal end of the <i>Mcs1a</i> critical interval at the time of design of the MICER project. The MD on mouse chromosome <i>13</i> is depicted as a red horizontal bar. The coordinates (in bp) are from the 2007 version of the mouse genome (UCSC Genome Browser, mm9). At the indicated base pair positions, a compatible pair of MICER vectors was placed, such that upon Cre-recombinase-driven excision in the embryonic stem-cell stage, a functional <i>Hypoxanthine-guanine phosphoribosyltransferase</i> (<i>HPRT</i>) gene was formed, which allowed for selection of a correctly targeted ES cell clone. Construct MHPP256h04 contained <i>Agouti</i> (<i>A</i>), <i>HPRT</i> exons 3–9, <i>loxP</i> (grey triangle) and a Puromycin resistance gene (<i>puro</i>); Construct MHPN5k06 contained a Neomycin resistance gene (<i>neo</i>), <i>loxP</i> (grey triangle), <i>HPRT</i> exons 1 and 2, and <i>Tyrosinase</i> (<i>TYR</i>). B) Graph showing the delayed eye-opening phenotype in MD mice (n = 83; FVB) as compared with WT littermates (n = 88; FVB). Plotted are on the vertical axis the percentage of mice in each genotype group with both eyes completely open and on the horizontal axis age in days. C) RNA-seq analysis of mammary gland gene expression from MD and WT control mice (n = 4 each; FVB). The transcript levels are shown as the average (+/− sem) counts from the RSEM algorithm output (used to estimate transcript levels <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549-Li1" target="_blank">[37]</a>), normalized to the average of the WT group. Shown are transcripts within 2.5 Mb surrounding the gene desert. Only the transcript level of the orphan nuclear receptor gene <i>Nr2f1</i> was significantly different between MD and WT mice (P<0.05, indicated by an asterisk). D) Quantitative real-time PCR (Q-PCR) analysis verifying the differential transcript level of <i>Nr2f1</i> in mammary gland from MD and WT control mice (n = 12 each; FVB). The <i>Nr2f1</i> transcript level is shown relative to the transcript level of the <i>ActB</i> endogenous control gene.</p

Mammary gland transplantation data and logistic regression analysis.

1<p>Transplant groups (donor:recipient): S:S (susceptible:susceptible); S:F1 (susceptible:F1); R:F1 (resistant:F1); R:R (resistant:resistant). Susceptible = WF inbred strain; Resistant = resistant <i>Mcs1a</i> congenic line Y4; F1 = WFxY4 or Y4×WF.</p>2<p>Logistic regression was used to estimate the effect of donor and recipient and the interaction between donor and recipient for the dependent variable of mammary gland transplant carcinoma susceptibility (outcome = carcinoma presence or absence at transplant site) in susceptible WF and <i>Mcs1a</i> resistant congenic transplant groups.</p>*<p>Statistically significant.</p