The BRG1 Chromatin Remodeler Protects Against Ovarian Cysts, Uterine Tumors, and Mammary Tumors in a Lineage-Specific Manner

<div><p>The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of <em>Brg1</em> null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (<em>Brg1^null/+</em>) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized <em>Wap-Cre</em> to mutate both <em>Brg1</em> floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that <em>Brg1^Wap-Cre</em> conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a <em>Brm</em>-deficient background. However, <em>Brg1^Wap-Cre</em> mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express <em>Brg1</em> and <em>Wap</em>. Thus, tumor formation in <em>Brg1</em> mutant mice appears to be confined to particular cell types that require BRG1 and also express <em>Wap</em>. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent <em>ARID1A/BAF250a</em> mutations in human ovarian and endometrial tumors.</p> </div

Expression of <i>Brg1</i>, <i>Brm</i>, and <i>Wap</i> in CD45⁻Ter119⁻CD31⁻ mammary gland subpopulations.

<p>A. Representative FACS plot of the subpopulations remaining after removing the hematopoietic and endothelial (CD45⁺Ter119⁺CD31⁺) cells from a suspension prepared from dissociated mammary tissue from a normal adult virgin mouse. The CD24⁺CD49f^low/− subset is enriched in luminal (Lum) cells; the CD24⁺CD49f⁺ subset is enriched in mature myoepithelial (Myo) cells; and the CD24⁺CD49^high subset is enriched in mammary stem cells (referred to as mammary repopulating units or MRU). B, C. RT-qPCR analysis of <i>Brg1</i> (B) and <i>Brm</i> (C) mRNA levels normalized to <i>Gapdh</i> levels in wild-type, flow-sorted mammary cell populations from adult virgin female mice. The Lum, Myo, and MRU subsets are as defined in panel A. Each histogram represents the mean ± SE from 3 independent experiments. D. RT-qPCR analysis of <i>Wap</i> mRNA levels normalized to <i>Gapdh</i> mRNA levels measured in the same 3 subsets. Lum, Myo, and MRU cells were isolated from pregnant females (E17.5 and E13.5) and virgin/nulliparous females (for representative FACS plots, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031346#pone-0031346-g001" target="_blank">Figure 1</a>). Asterisks indicate that the <i>Wap</i> signal was below the limit of detection. Each histogram represents the mean ± SE from 2 (E17.5) or 3 (E13.5, virgin/nulliparous) independent experiments.</p

Phenotype of <i>Brg1^Wap-Cre</i> mutant mice.

<p>*First pregnancy was at 2–3 months of age, and all mice were analyzed at 15–19 months of age.</p>1<p>Tg was hemizygous in each case (<i>Wap-Cre^+/0</i>).</p>2<p>Mutant genotypically but considered a control because Cre transgene expression is induced by pregnancy, which does not occur in males.</p>3<p>Did not score for ovarian or uterine phenotype.</p

Lineage-specific deletion of <i>Brg1</i> in the mammary gland.

<p>A. Whole-mount preparation of control (top) and mutant (bottom) mammary glands from multiparous females. Asterisks, lymph nodes. B. Ethidium bromide-stained gels showing <i>Brg1</i> Δfl (top) and fl (bottom) PCR products. MW, molecular-weight standard (500-, 400-, 300-, 200-, and 75-bp fragments are visible); MG, mammary gland; NP, nulliparous; MP, multiparous. C. Mammary gland section from multiparous mouse carrying the <i>Wap-Cre</i> transgene on a <i>R26R</i> background. Cre activity, visualized as blue X-Gal staining, is restricted to luminal cells (arrowhead). Basal/myoepithelial cells (arrow) are negative and appear pink because of nuclear fast red counterstain. D, E. IHC staining of BRG1 showing strong staining in nuclei throughout the mammary gland in controls (D) but absent in luminal cells of mutant mice (E). Arrows, luminal cells; asterisks, adipocyte nuclei. 400× magnification.</p

Mutant luminal cells do not undergo aberrant apoptosis.

<p>A, B. TUNEL assay of control (A) and mutant (B) adult mammary gland sections counterstained with methyl green. C. Lymphocytes induced to undergo apoptosis have brown nuclei (arrow) and serve as a positive control. D. qPCR analysis of <i>Brg1</i> Δfl genomic DNA levels normalized to <i>Gapdh</i> genomic DNA levels in mammary glands. Each histogram represents the mean ± SE from 3 independent experiments.</p