Human Surfactant Protein B Expression in Humanized Transgenic Mice

Surfactant protein B (SP-B, gene name: sftpb) is essential for normal lung function. It reduces alveoli surface tension, thereby preventing the lung from collapse. A single nucleotide polymorphism (SP-B 1580 C/T) is associated with several lung diseases and altered N-linked glycosylation site at Asn129 of SP-B. This change, present in the human population, has been associated with negative effects on SP-B precursor (proSP-B) processing and function. In this study, hSP-B humanized transgenic mice were generated without mouse SP-B background. Four founding lines, showing only the hSP-B gene, were selected via PCR-based DNA analysis. Genomic sequencing of these mice revealed which allele variant (hSP-B-C/T) they carried. Western blot analysis of BALF samples revealed these hTG mice expressed hSP-B protein in levels significant for survival and comparable to that of a healthy human lung. Characterization of the two hSP-B allele variant hTG strains revealed significant differences in their relative alveolar size and total lipid concentration

The gene desert mammary carcinoma susceptibility locus Mcs1a regulates Nr2f1 modifying mammary epithelial cell differentiation and proliferation.

Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in 'triple-negative' (ER-PR-HER2-) breast cancers as compared with 'receptor-positive' breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk

<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

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

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

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