The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation (original) (raw)
Figure 3
The non-protein coding Mcs1a resistance locus regulates transcript levels of Nr2f1 in the mammary gland, mammary epithelium and mammary carcinomas.
A–C) Q-PCR analysis of Nr2f1 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 Mcs1a resistant congenic data points. Nr2f1 transcript levels are shown relative to the transcript level of the ActB endogenous control gene. D) Chromosome conformation capture (3C) assay for the Nr2f1 promoter and the Mcs1a critical interval. The region is shown as a UCSC Genome Browser view (version rn4 of rat genome) and the location of the Mcs1a 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 Nr2f1 promoter is shown with respect to _Bgl_II restriction sites in the Nr2f1 gene span. Graphed is the average relative interaction frequency (+/− sem) of the _Bgl_II fragment in the Nr2f1 promoter containing the fixed primer with each of the _Bgl_II fragments in Mcs1a 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 Mcs1a to the fixed primer in the Nr2f1 promoter. The main peak in the interaction profile coincides with blocks of strong evolutionary sequence conservation (to zebrafish and frog, X. tropicalis). Sequence variation within the interacting Mcs1a region is outlined in Figure S2. E) Schematic drawing of the higher-order chromatin interaction of Mcs1a with the Nr2f1 promoter. The Mcs1a 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.