Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation

Abstract

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand-dependent interactions of the MED1 subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is little information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice. Interestingly, these mice are both viable and fertile and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. Consistent with this phenotype, as well as loss of the strong ligand-dependent estrogen receptor (ER)α-Mediator interaction, expression of a number of known ERα-regulated genes was down-regulated in MED1-mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen-stimulated mammary duct growth in MED1-mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor- and cell-specific in vivo role for MED1 LxxLL motifs, through Mediator-ERα interactions, in mammary gland development.

Nuclear receptors are a family of transcription factors that, in response to small lipophilic ligands, specifically regulate expression of target genes in development, differentiation, metabolism, homeostasis, and reproduction (1, 2). These nuclear receptors are expressed in a developmental stage- and tissue-specific manner and play important roles, not only in normal physiology, but also in pathological conditions (1, 2). Nuclear receptors have characteristic domains that include an N-terminal AF-1 domain that mediates ligand-independent transcription, a central DNA-binding domain that binds to regulatory elements in cognate target genes, and a C-terminal ligand-binding domain with an associated (ligand-induced) AF-2 domain that mediates ligand-dependent activation of target genes (1, 2).

The ultimate action of nuclear receptors following binding to target genes is to enhance the recruitment and/or function of the general transcription machinery, including RNA polymerase II and general transcription factors, on cognate core promoter elements (3). Interactions of the AF-1 and AF-2 domains with various transcription coactivators are required for this process (4–6). Among these diverse coactivators, Mediator, a large (∼1.6 MDa) protein complex composed of at least 25–30 distinct subunits, has emerged as the main bridge for direct communication between transcriptional activators and RNA polymerase II (7–9). In general, individual Mediator subunits interact specifically with their corresponding transcription factors and deletions of these Mediator subunits often affect expression primarily of target genes and pathways controlled by their corresponding transcription factor(s) (9). In the case of nuclear receptors, the Mediator interactions are ligand- and AF-2-dependent and mediated through the LxxLL motifs in the MED1 (a.k.a. TRAP220/PBP/DRIP205) subunit (10–13). Importantly, in relation to the present study, in vitro assays with intact Mediator complexes containing mutated MED1 LxxLL motifs have established direct roles for the LxxLL motifs in strong (ligand-dependent) nuclear receptor-Mediator interactions and in Mediator-dependent transcription by nuclear receptors (14, 15).

LxxLL motifs were initially discovered in SRC1 and CREB-binding protein/p300 (16) and later reported in many other nuclear receptor coactivators including MED1 and SRC/p160 and PGC-1 family members (4, 17). Crystal structures of the agonist-bound nuclear receptor AF-2 domains with LxxLL motifs have provided considerable mechanistic insights into their interactions (17–20). Although the LxxLL motif itself is sufficient to mediate nuclear receptor interactions, sequences flanking the LxxLL motifs play key roles in determining receptor binding specificity (19, 20) and allow division of LxxLL motifs into several classes according to their flanking sequences (reviewed in ref. 17). The fact that diverse coactivators such as SRC/p160, MED1, and PGC-1 have overlapping interaction sites on the AF-2 domain has led to the concept of cofactor exchange during transcriptional activation (4, 21). Interestingly, and related, recent studies have identified AF-2 domain mutations that result either in selective loss of TRβ binding to SRC/p160 relative to PGC-1α (22) or in selective loss of ERα binding to SRC/p160 family members relative to MED1 (23). These findings have led to the idea that these motifs could be targets for the development of reagents that selectively disrupt nuclear receptor interactions with certain coactivator complexes to achieve tissue- and gene-specific therapeutic interventions (6).

Although it has been shown that the LxxLL motifs of MED1 are required for its interactions with nuclear receptors and for nuclear receptor-mediated transcription in vitro, there is no available information regarding the functional significance of MED1 LxxLL motifs in vivo. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice and, surprisingly, found that these mice are apparently fertile and grossly normal. However, they do exhibit profound defects in virgin mammary gland development. Consistent with this observation, we observed significant impairments both in ERα-dependent gene expression in mammary epithelial cells and in estrogen-stimulated mammary ductal growth. Moreover, we found that MED1 is differentially expressed in mammary epithelial cells and plays a role in luminal lineage determination.

Results

Med1^KI/KI Mice Exhibit Profound Defects in Pubertal Mammary Gland Development.

To study the role of the MED1 LxxLL motifs in a physiological context, we generated MED1 LxxLL motif-mutant knockin mice (Med1^KI/KI). The point mutations that were introduced result in conversion of both MED1 LxxLL motifs to LxxAA motifs (Fig. 1A and Fig. S1), which previously was shown to disrupt strong Mediator-nuclear receptor interactions in vitro (14, 15). These mutations had no effect on the expression level of the MED1 protein (Fig. S1). Surprisingly, in contrast to the embryonic lethality of a total MED1 knockout (24), Med1^KI/KI mice were viable, fertile, and grossly normal. However, they did exhibit profound defects in mammary gland development. In these studies, mammary glands of 8-week-old Med1^KI/KI and wild-type virgin mice were isolated, fixed in Carnoy’s solution, and then stained with Carmine. As shown morphologically in Fig. 1B and quantitated in Fig. 1C, mammary gland ductal growth and branch morphogenesis were significantly impaired in Med1^KI/KI mice relative to wild-type mice. Similar defects were observed throughout pubertal mammary gland development at different ages (Fig. S2).

Fig. 1.

MED1 LxxLL mutations impair mammary gland development. (A) Diagram indicating the four L to A point mutations introduced into the two MED1 LxxLL motifs in the mutant MED1 knockin mice. (B) Mammary glands from 8-week-old wild-type and MED1 mutant mice. Glands were fixed and subjected to whole-mount staining. (C) Mammary duct lengths. The distances of mammary ducts from the lymph node were measured and quantified in five individual mice. (D) BrdU immunohistochemial staining of mammary gland sections from 7- to 8-week-old wild-type and MED1 mutant virgin mice. (E) Percentage of BrdU-positive staining mammary epithelial cells from wild-type and MED1 mutant mice are shown (N = 5). Scale bar, 1 mm (B) and 50 μm (D).

To determine if the mammary gland defects observed in Med1^KI/KI mice resulted from disrupted cell proliferation, we performed BrdU incorporation assays (25). Seven- to eight-week-old wild-type and Med1^KI/KI age-matched female mice were injected intraperitoneally with BrdU 2 h prior to sacrifice. Mammary glands were harvested, fixed, and then subjected to BrdU staining. Ten random areas of 100 cells in each sample were selected and counted to estimate the percentage of total epithelial cells that were BrdU-positive (Fig. 1 D and E). As expected, about 20% of mammary epithelial cells stained positive for BrdU in wild-type mice. In contrast, there were about 4 times fewer BrdU-positive mammary epithelial cells in the Med1^KI/KI mice. These data support the idea that the observed mammary gland defects in Med1^KI/KI mice are caused, at least in part, by decreased cell proliferation.

Med1^KI/KI Mice Show Impaired ERα Target Gene Expression in Mammary Epithelial Cells.

Estrogen is the dominant hormone promoting mammary epithelial cell proliferation at the stage of mammary gland development that we studied. Thus, we reasoned that the mutations in the MED1 LxxLL motifs exerted their effects by disrupting the estrogen signaling pathway, either by influencing the production of estrogen or by directly affecting ERα-mediated transcription. To discriminate between these possibilities, we first examined the blood estrogen levels of 8-week-old adult mice by ELISA (Cayman). We found that the MED1 LxxLL motif mutations did not affect the production of estrogen (Fig. 2A). To determine whether mutations disrupted ERα interactions with Mediator, immobilized GST and GST-ERα (ligand-binding domain) fusion proteins were independently incubated with nuclear extracts from either wild-type or Med1^KI/KI mouse embryo fibroblast cells. As expected (26), GST-ERα (ligand-binding domain), but not GST alone, bound Mediator from wild-type nuclear extract in a ligand (estrogen)-dependent manner (Fig. 2B). However, it failed to bind Mediator from Med1^KI/KI nuclear extract, even in the presence of estrogen. As a control, a GST-VP16 activation domain fusion protein, which interacts with the MED17 subunit of Mediator, interacted equally well with Mediator in extracts from wild-type or mutant mice. These data confirm that the strong ligand-dependent ERα-Mediator interaction is effectively and selectively disrupted by the LxxLL to LxxAA mutations.

Fig. 2.

MED1 mutations abolish the ligand-dependent ERα-Mediator interaction and ERα target gene expression. (A) Estrogen levels in sera from 8-week-old wild-type (black bar) and Med1^KI/KI (gray bar) mice (n = 5). (B) ERα-Mediator interactions. GST-pulldown assays employed equivalent amounts of purified GST, GST-ERα(LBD) and GST-VP16 proteins and nuclear extracts from wild-type and mutant mouse embryonic fibroblasts as sources of Mediator. (C) ERα target gene expression. Real-time PCR was carried out after reverse transcription of total RNA from mammary epithelial cells of Med1^KI/KI and control wild-type mice. Relative expression levels of the indicated ERα target genes (normalized to 18S rRNA content) are shown (Value = Mean ± STD; ^∗P < 0.05).

To determine whether expression of ERα target genes was affected in the mammary glands of Med1^KI/KI mice, mammary epithelial cells were first isolated from mammary glands of 7- to 8-week-old Med1^KI/KI mice and control wild-type mice. Total RNA was isolated and subjected to semiquantitative real-time PCR analyses following reverse transcription. We examined expression of a number of known ERα target genes, including WNT1-inducible signaling pathway protein 2 (WISP2), diamine oxidase (Dio), cyclin D1, pS2, and progesterone receptor (PR) (Fig. 2C). We found that expression of most of these ERα target genes (pS2, cyclin D, Dio, WISP2) was significantly reduced in Med1^KI/KI mammary epithelial cells. Interestingly, the expression of another ERα target gene, PR, was not significantly altered, which is consistent with our finding that these mammary epithelial cells were still able to differentiate during pregnancy (Fig. S3). Furthermore, we found that the expression levels of both ERα and PGC-1α, another ERα coactivator (27), also were not changed. These data are consistent with the idea that MED1 LxxLL motifs play an important in vivo role in ERα-mediated transcription in the mammary gland.

Estrogen-Induced Expression of ERα Target Genes Is Blocked in Med1^KI/KI Mammary Epithelial Cells.

We next carried out experiments to determine whether the impaired expression of the ERα target genes in Med1^KI/KI mice was caused by a disruption in the response to estrogen stimulation. Mammary epithelial cells again were isolated from both wild-type and Med1^KI/KI mice and then grown in DMEM/F12 phenol red-free medium with charcoal-stripped serum for 2 d. The cells then were treated with vehicle or estrogen for different durations (0, 5, 20, 120 min), after which they were collected. Total RNAs were isolated and reverse transcribed for real-time PCR analyses to assess expression of representative ERα target genes (pS2, cyclin D, and Dio). Estrogen-dependent expression of these genes was observed in wild-type mammary epithelial cells, as expected, but abrogated in Med1^KI/KI mammary epithelial cells (Fig. 3A). These data are consistent with the notion that impaired expression of these genes in Med1^KI/KI mammary epithelial cells is caused by disrupted ERα-mediated transcription.

Fig. 3.

MED1 LxxLL motif mutations block estrogen-dependent gene expression and estrogen-stimulated mammary duct growth. (A) Gene expression levels. Wild-type mammary epithelial cells were isolated from wild-type and Med1^KI/KI mice and treated with vehicle or 10 nM estrogen for the indicated time. Total RNAs were isolated and expression of the indicated ERα target genes was measured by real-time PCR as above (Value = Mean ± STD; ^∗P < 0.05). (B) Mammary duct growth. 3-week-old wild-type and Med1^KI/KI mice were ovariectomized and implanted with slow-release E2 pellets for 21 d. Whole-mount Carmine stainings of inguinal glands from these mice are shown.

Med1^KI/KI Mammary Ducts Fail to Respond to Estrogen-Stimulated Growth in Vivo.

To determine, in vivo, whether the observed defects in mammary gland ductal growth and branch morphogenesis in Med1^KI/KI mice are due to an impaired response to estrogen, 21-day-old wild-type and Med1^KI/KI littermates were first ovariectomized and then implanted with slow-release pellets containing estradiol (E2) or placebo (Innovative Research of America). After 21 d, inguinal glands were excised and processed for whole-mount staining, while uteri were also collected as positive controls for the efficacy of estrogen treatment. We found that uterus weights in both control and Med1^KI/KI mice were increased by more than 10-fold, indicating successful E2 treatments (Fig. S4A). We also found that placebo treatment did not stimulate mammary ductal growth (Fig. S4B). Importantly, whereas a significant induction of mammary ductal growth by E2 treatment was observed in wild-type mice, the growth of mammary ducts was greatly inhibited in the Med1^KI/KI mice despite E2 treatment (Fig. 3B). These data further support the idea that the mammary gland developmental defects in Med1^KI/KI mice are due to a failed estrogen response and that MED1 LxxLL motifs play critical in vivo roles in mediating estrogen functions in the mammary gland.

MED1, Like ERα, Is Highly Expressed in Luminal Epithelial Cells.

Of the two major types of mammary epithelial cells, luminal and basal cells, ERα is expressed mainly in the luminal epithelial cells. To better understand the role of MED1 in mammary gland development, we determined the pattern of MED1 expression in these cells. Mammary gland tissue sections from 8-week-old mice were prepared as above and subjected to coimmunofluorescence staining using anti-ERα and anti-MED1 antibodies. Alexa 555- and 488-conjugated secondary goat-anti-mouse and goat-anti-rabbit antibodies were used to detect ERα and MED1, respectively. As expected, we observed a high level of ERα staining in the nuclei of mammary luminal epithelial cells but not in the basal cells (Fig. 4A, Top). Interestingly, the staining pattern for MED1 was very similar to that of ERα—almost undetectable in basal epithelial cells and high in luminal epithelial cells, with the strong ERα overlap clearly evident in the merged images. To further confirm this observation, we carried out coimmunofluorescence staining experiments with antibodies against MED1 and the basal epithelial cell marker CK14. The results clearly show that MED1 and CK14 have a mutually exclusive expression pattern (Fig. 4A, Bottom). Coimmunostaining with antibodies against MED1 and the luminal epithelial marker CK18 further confirmed preferential expression of MED1 in luminal epithelial cells (Fig. S5). Interestingly, and in contrast to the differential expression of MED1, the core Mediator subunit MED30 (indicative of the Mediator complex) is expressed in both luminal and basal mammary epithelial cells at very comparable levels (Fig. S6). These data indicate that MED1 is differentially expressed in the different types of mammary epithelial cells, with high expression in the luminal mammary epithelial cells in the normal mammary gland.

Fig. 4.

MED1 is differentially expressed in mammary epithelial cells and plays a role in luminal epithelial cell differentiation. (A) Mammary glands from wild-type and MED1 mutant mice were isolated, fixed, and subjected to coimmunofluorescent staining with antibodies against MED1 and ERα (Top) or MED1 and basal epithelial marker CK14 (Bottom). (B) Mammary gland sections of wild-type and Med1^KI/KI mice were subjected to coimmunofluorescent staining using antibodies against the cytokeratin markers CK14 and CK18 (luminal epithelial marker). Scale bar, 50 μm. (C) Mammary epithelial cells from wild-type and Med1^KI/KI mice were stained with the indicated PE- or FITC-conjugated antibodies and analyzed by FACS.

Regarding the issue of cell-specific MED1 expression and function, the differentiation of mammary epithelial cells during pregnancy involves, in addition to ERα (28), other nuclear receptors (e.g., ERβ and PR) that also have been shown to interact with MED1 in vitro (29, 30). Since we found that MED1 is expressed in mammary epithelial cells during pregnancy (Fig. S7A), our observation that Med1^KI/KI mice exhibit a severe phenotype in mammary gland pubertal development, but not during pregnancy, reflects differential (cell- and receptor-specific) MED1 LxxLL motif requirements for ERα, ERβ, and PR function in vivo. This is also consistent with the lack of any apparent phenotype in Med1^KI/KI mice in other steroid responsive tissues (e.g. uterus) that also express MED1 (Fig. S7B).

MED1 LxxLL Motif Mutations Affect Mammary Epithelial Cell Lineage Determination.

In view of our finding that MED1 is not expressed universally in all mammary epithelial cells but, rather, is expressed mainly in luminal epithelial cells, we carried out experiments to determine whether MED1 LxxLL motif mutations have an impact on the lineage determination of mammary epithelial cells. Immunofluorescent staining was carried out using antibodies against luminal (CK18) and basal (CK14) epithelial markers, as described above, on paraffin-embedded 8-week-old adult mammary gland sections. As expected, mammary epithelial cells in wild-type mice were well differentiated into distinct luminal (CK18+) and basal (CK14+) epithelial cells. In contrast, Med1^KI/KI mice contained a significant population of CK14 and CK18 double-positive progenitor cells (Fig. 4B, merged images). These data suggest an important role for MED1 in mammary luminal epithelial cell lineage determination.

It has been reported that the cell-surface markers CD24 and CD29 can be used to distinguish mammary progenitors with luminal-cell fate (CD29^lowCD24⁺) (31). We therefore examined CD24 and CD29 expression profiles to determine the specific cell populations that are affected by the MED1 LxxLL-motif mutations. Wild-type and Med1^KI/KI mammary epithelial cells were isolated and subjected to FACS analyses after staining with antibodies against these markers. As shown in Fig. 4C, we found a significant increase in CD29^lowCD24⁺ mammary epithelial cell populations from Med1^KI/KI mice relative to wild-type mice. These data suggest that MED1 LxxLL motifs play a role in controlling the luminal progenitor cell population in pubertal mice. To confirm these findings, we examined the expression of another well-known mammary stem/progenitor cell marker, stem cell antigen-1 (Sca1) (32), in the mammary epithelial cells of these mice, as it has been shown previously that the CD24⁺Sca1^- population is enriched in luminal progenitor cells. Consistent with the above observations, we found a significant increase in the CD24⁺Sca1^- mammary epithelial cell population from the mammary glands of Med1^KI/KI mice (Fig. 4C). Taken together, these results indicate that MED1 LxxLL motifs play critical roles in luminal progenitor cell determination.

Discussion

It is well known that mammary gland development is under precise temporal control by specific hormonal and signaling pathways during each developmental stage (reviewed in refs. 33, 34). The estrogen signaling pathway has been shown to be the key pathway involved in ductal growth and branch morphogenesis in the stages from prepuberty to puberty (33, 34). We previously have shown that MED1 is required for optimal ERα-dependent transcription in vitro (26, 35). In this study, we provide in vivo evidence that the MED1 LxxLL motifs play important roles in ERα-mediated functions in early mammary gland development. We found that mammary ductal growth and branch morphogenesis are significantly impaired in virgin Med1^KI/KI mice relative to wild-type mice. Consistent with this observation, Med1^KI/KI mammary epithelial cells show a significant reduction in the expression of a number of known ERα target genes. Furthermore, the expression of these genes is no longer responsive to estrogen stimulation in isolated mammary epithelial cells. Importantly, we provide further evidence showing that exogenous estrogen-stimulated mammary duct growth in ovariectomized MED1 mutant mice is also blocked in vivo.

Interestingly, whereas Mediator is generally thought to be essential for all RNA polymerase II-transcribed genes, Med1^KI/KI mice did not exhibit some defects (e.g., in uterine development, fertility, or estrogen production) that are observed in ERα null mice (25). This lack of detectable effects on other tissues could reflect a previously unrecognized tissue-specific requirement for MED1 and its LxxLL motifs in mediating ERα function, similar to what has been previously shown for other nuclear receptor cofactors such as SRC/p160 family members (reviewed in ref. 21). This tissue-specific role of MED1 LxxLL motifs could be caused by the differential expression of other compensatory coactivators. Thus, it has been reported that ERα shows distinct responses to antiestrogens in different (mammary vs. uterine) tissues because of variations in coactivator expression levels (36). Furthermore, it recently was found that Mediator can be recruited to ERα target genes by other coactivators such as CCAR1 (37) and to other nuclear receptor-promoter complexes by PGC-1α (38), which also interacts directly with ERα (27). We also have found that the MED1 LxxLL motifs, but not MED1 itself, is dispensable for PPARγ-mediated adipogenesis (15). Therefore, the presence or high level expression of certain other ERα-interacting cofactors in these estrogen-responsive tissues could bypass the requirement for the MED1 LxxLL motifs. We also note that the mammary gland developmental defects in Med1^KI/KI mice, although profound, are not as severe as those in ERα null mice. Consistent with this observation, we have already observed that not all ERα target genes are affected in the mammary epithelial cells of these mice. Therefore, even if the same sets of genes are affected in tissues that do not exhibit severe defects, this could be because those genes do not play the same important roles in those tissues.

Our current studies have focused on the role of the MED1 LxxLL motifs in mammary gland development because defects in this tissue were the first and most dramatic that were observed in Med1^KI/KI mice. Although linked to ERα in this tissue, MED1 also has been reported to interact with a number of other nuclear receptors [e.g. ERβ, PR, VDR (vitamin D receptor), PPARγ (peroxisome proliferator-activated receptor γ), TR (thyroid receptor), AhR (aryl hydrocarbon receptor), and GR (glucocorticoid receptor)] in vitro through its LxxLL motifs. However, these nuclear receptors are not likely to contribute in a significant way to the specific mammary gland developmental defects observed in Med1^KI/KI mice, as corresponding receptor deletions do not result in comparably severe phenotypes in pubertal mammary gland development. It is notable, however, that whereas Med1^KI/KI mice exhibit a deficiency in early (pubertal) mammary gland development, they show normal mammary epithelial differentiation during pregnancy—a process that involves ERβ and PR, as well as ERα (28–30). Our data, altogether, are thus indicative of both nuclear receptor- and tissue-specific functions of the MED1 LxxLL motifs—similar conceptually to what has been demonstrated in vivo for one of the two LxxLL motifs in coactivator NCoA6 (39). However, we cannot exclude the possibility that the functions of these steroid receptors are affected by MED1 LxxLL motif mutations in other tissues where they might normally function, especially since some phenotypic abnormalities might be exhibited only during certain stress conditions that elicit demands for higher levels of nuclear receptor function. Indeed, we have found that mice containing muscle-specific deletions of MED1 show normal glucose and insulin levels but significant alterations in both glucose tolerance and insulin sensitivity. Therefore, it will be particularly interesting to determine whether MED1 LxxLL motifs play a role in eliciting these responses under these stress conditions. These observations further emphasize the importance of studying the role of these cofactors in vivo, in specific tissues, and under the appropriate conditions.

Another significant finding of this study is that MED1 is not only differentially expressed in different types of mammary epithelial cells but also plays a role in mammary luminal epithelial cell lineage determination. Having been considered a more general transcriptional cofactor, MED1 was thought to exist in all Mediator complexes and to be uniformly expressed. However, our previous biochemical analyses provided evidence both for an otherwise intact Mediator complex in Med1^-/- cells (14) and for compositional heterogeneity within Mediator complexes, as evidenced by the existence of MED1 in only a subpopulation of Mediator complexes with unique subunit compositions (35). Importantly, our finding that Mediator core subunit MED30 is comparably expressed in both basal and luminal mammary epithelial cells indicates that the absence of MED1 in basal epithelial cells is not likely due to an overall decrease in the Mediator levels in these cells. With regard to the issue of MED1-related Mediator heterogeneity and estrogen signaling, it is relevant to note that breast cancer has currently been categorized into different subtypes [e.g. luminal type (A and B), basal type, and triple negative] according to their distinct signature gene expression profiles and altered signaling pathways (40). Importantly, these different subtypes of breast cancer show different prognoses and require different treatment strategies (41). Interestingly, MED1 has been found to be overexpressed and amplified in more than 40–50% of human breast cancers (42), which was further confirmed by a number of genome-wide microarray analyses of human breast cancer patient samples (Oncomine). Therefore, future determination of the role of MED1 in breast tumorigenesis and its expression in these different subtypes of human breast cancer may have important prognostic or therapeutic implications.

Materials and Methods

Generation and Maintenance of Med1^KI/KI Mice.

To generate Med1^KI/KI mice, the linearized targeting vector was electroporated into E14 embryonic stem cells. Positive clones were selected, confirmed by Southern blot analyses and microinjected into blastocysts of C57BL6/J mice. These mice were extensively backcrossed (10 times) to a C57BL6/J background. All animal were housed in AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-approved facilities at the University of Cincinnati and at Rockefeller University. All procedures were approved by Institutional Animal Care and Use Committees, and in accordance with the National Institutes of Health guidelines outlined in the Guide for Care and Use of Laboratory Animals.

Isolation of Mouse Mammary Epithelial Cells.

Mammary glands (thoracic and inguinal glands) were excised, minced, and digested for 2 h at 37 °C in DMEM/F12 medium containing 0.1% collagenase. The digest was centrifuged at 1,000 rpm for 5 min and the pellet was resuspended and digested with 2 U/ml DNase I. Mammary epithelial cells were further purified by repeated washes with phosphate buffered saline followed by brief centrifugation (10 s) at low speed (900 rpm). Pooled cell pellets containing epithelial organoids were filtered through a 400-mm polyester mesh (PE400; Clarcon, Warrington, UK).

Estrogen Measurement and E2-Stimulated Responses.

Blood was drawn from the heart of wild-type and MED1 mutant mice immediately after sacrifice. Serum E2 (17-βestradiol) levels were measured by an Estradiol EIA kit according to the manufacturer’s protocol (Caymen). To examine E2-stimulated mammary gland growth, 21-day-old virgin females were ovariectomized and implanted with 21-day slow-release pellets (0.025 mg, Innovative Research of America). After 21 d, inguinal glands were excised for whole-mount staining with carmine (Sigma).

Fluorescence Immunohistochemistry.

Inguinal glands were fixed and embedded in paraffin blocks as described above. Following heat-induced antigen retrieval in citrate buffer, tissue sections were incubated with primary antibodies against MED1, ERα (clone 6F11, Novocastra), CK14 (clone LL002, Thermo Scientific), or CK18 (Fitzgerald) overnight at 4 °C. Alexa Fluor 488- or 555-conjugated goat anti-rabbit or anti-mouse secondary antibodies were purchased from Invitrogen. The images were visualized and captured using an Axioplan Imaging 2e fluorescence microscope (Zeiss).

FACS Analysis.

Mammary epithelial cells were isolated from the thoracic and inguinal mammary glands, as above, followed by a disassociation step with trypsin. Cell suspensions were incubated at room temperature for 1 hr with the following antibodies: PE-CD24 (1∶200, BD Biosciences), CD29 (1∶200, AbD Serotec), and FITC-Sca-1 (1∶200, BD Biosciences). Cells were sorted using a FACSCanto II system (BD Biosciences) and the data were analyzed with FACSDiva 6.1.1 software.