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. 2014 Nov 15;395(2):245-54.
doi: 10.1016/j.ydbio.2014.09.012. Epub 2014 Sep 16.

The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation

Kyung Hyun Yoo  1 , Keunsoo Kang  2 , Yonatan Feuermann  1 , Seung Jin Jang  1 , Gertraud W Robinson  1 , Lothar Hennighausen  3
Affiliations

The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation

Kyung Hyun Yoo et al. Dev Biol. .

Abstract

The transcription factor STAT5 mediates prolactin signaling and controls functional development of mammary tissue during pregnancy. This study has identified the miR-193b locus, also encoding miRNAs 365-1 and 6365, as a STAT5 target in mammary epithelium. While the locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells. Inactivation of the miR-193b locus in mice resulted in elevated mammary stem/progenitor cell activity as judged by limiting dilution transplantation experiments of primary mammary epithelial cells. Colonies formed by mutant cells were larger and contained more Ki-67 positive cells. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Cav3 and elevated levels of Elf5. Normal colony development was partially obtained upon ectopically expressing Cav3 or upon siRNA-mediated reduction of Elf5 in miR-193b-null primary mammary epithelial cells. This study reveals a previously unknown link between the mammary-defining transcription factor STAT5 and a microRNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells.

Keywords: Alveoli, Differentiation; Development; Mammary; Micro RNAs; STAT5; Stem cells; miR-193b.

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Figures

Figure 1
Figure 1. Features of the miR-193b locus
(A) Integrative analysis of available ChIP-seq data (Chang et al., 2013; Kang et al., 2014; Lain et al., 2013; Lemay et al., 2013; Rijnkels et al., 2013; Yamaji et al., 2013) revealed regulatory regions (gray bars) at the miR-146b, miR-148a and miR-193b loci. HFSC, hair follicle stem cells; PR, prolactin receptor; MG, mammary gland; NFIB motif, TGGCC and STAT5 motif, TTCnnnGAA. (B) Analysis of RNA-seq and available histone ChIP-seq data reveals an active miR-193b locus in mammary tissue while it is silent in liver. Grey bar indicates putative transcription start site of the miR-193b transcript as described previously (Feuermann et al., 2013). MiR-193b was the first STAT5-regulated miRNA identified in this lab and the respective locus was inactivated in the mouse prior to the identification of the STAT5-controlled miR-146 and miR-148a.
Figure 2
Figure 2. Increased stem cell activity in the absence of the miR-193b locus
(A) FACS analysis of mammary epithelium from 8–12 week-old nulliparous mice (wild type on left). (B) Graph showing MEC population from three wild type and three mutant mice. (C) Representative images of wholemounts of repopulated fat pads injected with the indicated numbers of MECs. (D) Ratio of the number of repopulated fat pads per number of fat pads injected with 1000, 200 and 100 MECs, respectively.
Figure 3
Figure 3. Enhanced progenitor cell activity in the absence of miR-193b
(A) Pictures were taken at days 0 and 7 after placing primary MECs in a colony formation culture system (Scale bar: 50 μm). (B) Representative pictures of colonies stained with crystal violet at day 10. (C) Graphs with the number and size of colonies. Three biological replicates were analyzed. (D) Confocal images of colonies stained for E-cadherin (Green), α-SMA (Green) and ZO-1 (Red) at day 7 and Ki-67 (Red), pH3 (Red) and K14 (Red) at day 4 with DAPI (Blue) as a counterstain (Scale bar: 20 μm). (E) Graphs with the number of positive cells stained with Ki-67 and pH3.
Figure 4
Figure 4. Reduced NKCC1 in ductal epithelium in the absence of the miR-193b locus
H&E staining of histological sections from mammary tissue from control (A) and miR-193b mutant (B) nulliparous mice. Immunofluorescence for NKCC1 (red) and a-SMA (green) in control (C, E) and mutant (D, F) tissues. Arrows point to luminal cells that express NKCC1.
Figure 5
Figure 5. Precocious differentiation of mammary epithelial activity in the absence of the miR-193b locus
Low (A and B) and high (C and D) magnification images of hematoxylin and eosin stained sections of p13 mammary tissues show increased alveolar development and accelerated differentiation in mutant (A and C) compared to controls (B and D). Arrows point to alveoli.
Figure 6
Figure 6. Identification of differentially expressed genes in the absence of miR-193b
(A) Clustering analysis of RNA-seq result reveals a marked difference of gene expression between wild-type and mutant mammary tissues obtained at p13. Three biological replicates were used. (B) Percentage of STAT5-dependent genes among 456 induced genes is shown. (C) Expression levels of Stat5a, Stat5b, Wap and Csn1s2b are shown. *FDR-adjusted p value < 0.05.
Figure 7
Figure 7. Identification of functional roles of ELF5 and caveolin3 in miR-193b−/− cultures
(A) Colonies generated by miR-193b−/− MECs treated with Elf5-siRNAs. (B) Colonies generated by miR-193b−/− MECs with restored Cav3 expression. Whole mount assay (top, original magnification; x5) and representative pictures of colonies (lower, scale bar: 50 μm) at day 6. Quantitative real-time qPCR analysis_ of Elf5 and Cav3 (left). Graph with the size of colonies (right).
Figure 8
Figure 8
Proposed model linking the miR-193b locus to the biology of mammary stem cells and differentiating epithelium. The miR-193b locus is under STAT5 control and negatively controls the activity of mammary stem and progenitor cells. This conclusion is based on mouse genetics, where loss of miR-193b resulted in enhanced stem and progenitor cell activity. Loss of miR-193b also resulted in precocious differentiation of mammary epithelium during pregnancy and a concomitant induction of Elf5 expression and reduced Cav3 expression. The functional contribution of ELF5 and caveolin3 in mammary alveolar differentiation was determined in in vivo loss-of-function and gain-of-function studies using colony formation assays in this research.
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