The bHLH/PAS transcription factor singleminded 2s promotes mammary gland lactogenic differentiation

Abstract

We have previously demonstrated that the bHLH/PAS transcription factor, singleminded 2s (Sim2s), is required for proper mammary ductal morphogenesis and luminal epithelial differentiation. Furthermore, loss of Sim2s in breast cancer cells resulted in downregulation of epithelial markers and acquisition of a basal-like phenotype. The objective of this study was to further define the role of Sim2s in mammary differentiation. We found that Sim2s is developmentally regulated throughout mammary gland development with highest expression during lactation. Mammary glands from nulliparous mice expressing Sim2s driven by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter were morphologically indistinguishable from wild-type mice but displayed hallmarks of precocious lactogenic differentiation. These included elevated expression of the milk protein genes Wap and Csn2, and apical localization of the lactation marker Npt2b. Consistent with the in vivo results, Sim2s enhanced prolactin-mediated Csn2 expression in HC11 and CIT3 mouse mammary epithelial cells, and downregulation of Sim2s by shRNA in HC11 cells inhibited Csn2 expression. Chromatin immunoprecipitation (ChIP) analyses of the Csn2 gene found that Sim2s associates with the Csn2 promoter and re-ChIP experiments showed that Sim2s interacted with the RNA II polymerase (RNAPII) complex. Together, these data demonstrate, for the first time, that Sim2s is required for establishing and maintaining mammary gland differentiation.

Fig. 1.

Sim2s expression is developmentally regulated in mouse mammary tissues. (A) Total RNA isolated from C57 mouse mammary glands (n=3 animals per group) at weeks 4 and 10 of virgin development (V4 and V10), days 6 and 16 of pregnancy (P6 and P16), days 1 and 7 of lactation (L1 and L7) and day 3 of involution (I3) was used for qRT-PCR analysis of Sim2s expression. (B) In situ hybridization analysis of Sim2s mRNA from 5-week-old virgin (top) and L7 (middle) mouse mammary glands. The sense control is shown in the bottom panel. Left images are Hematoxylin and Eosin (H&E)-stained adjacent sections. (C) Immunohistochemical (ICH) staining of L7 FVB mouse mammary glands for Sim2s (left panel) showing nuclear localization in the mammary epithelium. Normal rabbit IgG (right panel) was used as a negative control.

Fig. 2.

Sim2s expression patterns correlate with a subset of milk protein genes during lactation. (A-E) Total RNA isolated from V8, L1, L4 and L7 FVB mammary glands (n=5 animals per group) was used for qRT-PCR analysis of (A) Sim2s, (B) Wap, (C) Csn2, (D) Lalba and (E) Expi expression. Cldn7 was used to normalize all mammary qRT-PCR data.

Fig. 3.

Generation of MMTV-Sim2s-HA transgenic mice. (A) Schematic diagram of the MMTV-Sim2s-HA targeting construct. The MMTV-LTR construct was provided by Dr Jeffery Rosen at Baylor College of Medicine. Exons 2 and 3 (E2, E3) and Intron 2 (I2) are derived from the rabbit β-globin gene (RβG). The poly-A signal is derived from the bovine growth hormone gene (bGHpA). (B)RT-PCR analysis of pooled samples from three transgenic females showing expression of the Sim2s-HA transgene exclusively in the mammary gland. Hrt, heart; Hyp, hypothalamus; Kid, kidney; Lng, lung; Lvr, liver; Neg, negative control (water); MG, mammary gland; Pit, pituitary; Ovr, ovary; SG, salivary gland; Spl, spleen; Utr, uterus; WAT, white adipose tissue. Gapdh was used as a loading control. (C) IHC analysis of HA localization in mammary glands from wild-type (WT) and transgenic (Sim2s-HA) V8 females.

Fig. 4.

Assessment of mammary morphology in Sim2s-HA transgenic and non-transgenic female mice. (A-F) Mammary glands were isolated from (A) V5, (B) V8, (C) P6, (D) P16, (E) L2 and (F) I14 FVB females, fixed in paraformaldehyde, sectioned and stained with H&E. Scale bars: 100 μm.

Fig. 5.

Sim2s expression results in precocious expression of a subset of milk proteins and alveolar epithelial cell surface markers. (A-D) qRT-PCR was used to evaluate gene expression in samples from nulliparous wild type (WT) and transgenic (Sim2s) females (n=5 animals per group). (A) Csn2 expression. (B) Wap expression. (C) Lalba expression. (D) Expi expression. *, P<0.05. Cldn7 expression was used to normalize qRT-PCR data. (E) Immunohistochemical analysis of aquaporin 5 (Aqp5, top), the sodium-inorganic phosphate co-transport protein Npt2b (middle) and β-casein (bottom) in mammary glands from V8 WT and transgenic females.

Fig. 6.

Sim2s expression is upregulated in differentiated HC11 and CIT3 cells. (A) qRT-PCR was used to detect Sim2s in undifferentiated HC11 cells (UN) and cells treated with HC + Prl for 1 or 4 days (PRL1 and PRL4). (B) Western blot analysis of Sim2s and β-casein protein levels in undifferentiated (UN) HC11 cells and PRL4 cells. (C) Western blot analysis of Sim2s levels in UN CIT3 cells and PRL3 cells. β-actin was used as a loading control for western analyses.

Fig. 7.

Sim2s is necessary and sufficient for robust prolactin-mediated Csn2 expression. (A) qRT-PCR analysis of Csn2 expression in control and Sim2s-overexpressing HC11 cells. Total RNA was isolated from UN cells or cells treated for 4, 8 or 24 hours with HC + Prl. Csn2 expression in control cells is shown on a smaller scale in the inset panel. (B) Western blot analysis of β-casein production in UN and HC + Prl treated control and Sim2s-overexpressing HC11 cells. (C) qRT-PCR analysis of Csn2 expression in control and Sim2s-overexpressing CIT3 cells. Total RNA was isolated from UN cells or PRL3 cells. (D)qRT-PCR analysis of Csn2 expression in HC11 cells stably expressing a scrambled shRNA sequence (control) or expressing shRNA targeting Sim2s (siSim2s). (E) Western blot analysis of Sim2s and β-casein in control and stable shSim2s UN cells or PRL4 cells. *, P<0.05.

Fig. 8.

Sim2s alters the binding of regulatory factors to the Csn2 promoter region. (A) Schematic diagram of the Csn2 gene. White boxes represent non-coding exons and black boxes represent coding exons (II-VIII). Arrows indicate the location of proximal promoter primers. (B) ChIP analyses of the proximal promoter were performed on chromatin isolated from UN HC11 cells or cells treated for 4, 8 or 24 hours with HC + Prl using the antibodies indicated to the left. (C) Re-ChIP analysis of Sim2s and RNAPII at the proximal promoter of Csn2 in control cells at PRL4. Following an initial pull down with Sim2s antibody, chromatin was re-immunoprecipitated with RNAPII antibody and the Csn2 promoter sequence was amplified using PCR. (D) ChIP analysis of Sim2s and AcH3 occupancy of Actnb coding region in control and Sim2s-overexpressing HC11 cells is included as a control.