Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation Pingping Jianga,b,1, Qiuping Hua,1, Mitsuhiro Itoc,1,3, Sara Meyera, Susan Waltza, Sohaib Khana, Robert G. Roederc,2, and Xiaoting Zhanga,2 aDepartment of Cancer and Cell Biology, College of Medicine, University of Cincinnati, 3125 Eden Avenue, OH 45267; bCollege of Life Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China; and cLaboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 Contributed by Robert G. Roeder, February 16, 2010 (sent for review January 18, 2010) Mediator recently has emerged as a central player in the direct (7–9). In general, individual Mediator subunits interact transduction of signals from transcription factors to the general specifically with their corresponding transcription factors and de- transcriptional machinery. In the case of nuclear receptors, in vitro letions of these Mediator subunits often affect expression primar- studies have shown that the transcriptional coactivator function of ily of target genes and pathways controlled by their corresponding the Mediator involves direct ligand-dependent interactions of the transcription factor(s) (9). In the case of nuclear receptors, the MED1 subunit, through its two classical LxxLL motifs, with the re- Mediator interactions are ligand- and AF-2-dependent and ceptor AF2 domain. However, despite the strong in vitro evidence, mediated through the LxxLL motifs in the MED1 (a.k.a. there currently is little information regarding in vivo functions of TRAP220/PBP/DRIP205) subunit (10–13). Importantly, in rela- the LxxLL motifs either in MED1 or in other coactivators. Toward tion to the present study, in vitro assays with intact Mediator com- this end, we have generated MED1 LxxLL motif-mutant knockin plexes containing mutated MED1 LxxLL motifs have established mice. Interestingly, these mice are both viable and fertile and do direct roles for the LxxLL motifs in strong (ligand-dependent) not exhibit any apparent gross abnormalities. However, they do nuclear receptor-Mediator interactions and in Mediator-depen- BIOCHEMISTRY exhibit severe defects in pubertal mammary gland development. dent transcription by nuclear receptors (14, 15). Consistent with this phenotype, as well as loss of the strong li- LxxLL motifs were initially discovered in SRC1 and CREB- gand-dependent estrogen receptor (ER)α-Mediator interaction, ex- binding protein/p300 (16) and later reported in many other pression of a number of known ERα-regulated genes was down- nuclear receptor coactivators including MED1 and SRC/p160 regulated in MED1-mutant mammary epithelial cells and could and PGC-1 family members (4, 17). Crystal structures of the no longer respond to estrogen stimulation. Related, estrogen-sti- agonist-bound nuclear receptor AF-2 domains with LxxLL motifs mulated mammary duct growth in MED1-mutant mice was also have provided considerable mechanistic insights into their inter- greatly diminished. Finally, additional studies show that MED1 is actions (17–20). Although the LxxLL motif itself is sufficient to differentially expressed in different types of mammary epithelial mediate nuclear receptor interactions, sequences flanking the cells and that its LxxLL motifs play a role in mammary luminal LxxLL motifs play key roles in determining receptor binding epithelial cell differentiation and progenitor/stem cell determina- specificity (19, 20) and allow division of LxxLL motifs into several tion. Our results establish a key nuclear receptor- and cell-specific classes according to their flanking sequences (reviewed in ref. 17). in vivo role for MED1 LxxLL motifs, through Mediator-ERα interac- The fact that diverse coactivators such as SRC/p160, MED1, and tions, in mammary gland development. PGC-1 have overlapping interaction sites on the AF-2 domain has led to the concept of cofactor exchange during transcriptional MED1/TRAP220 ∣ estrogen receptor ∣ progenitor/stem cell activation (4, 21). Interestingly, and related, recent studies have identified AF-2 domain mutations that result either in selective uclear receptors are a family of transcription factors that, in loss of TRβ binding to SRC/p160 relative to PGC-1α (22) or in Nresponse to small lipophilic ligands, specifically regulate selective loss of ERα binding to SRC/p160 family members rela- expression of target genes in development, differentiation, meta- tive to MED1 (23). These findings have led to the idea that these bolism, homeostasis, and reproduction (1, 2). These nuclear motifs could be targets for the development of reagents that se- receptors are expressed in a developmental stage- and tissue- lectively disrupt nuclear receptor interactions with certain coac- specific manner and play important roles, not only in normal phy- tivator complexes to achieve tissue- and gene-specific therapeutic siology, but also in pathological conditions (1, 2). Nuclear recep- interventions (6). tors have characteristic domains that include an N-terminal AF-1 Although it has been shown that the LxxLL motifs of MED1 domain that mediates ligand-independent transcription, a central are required for its interactions with nuclear receptors and for DNA-binding domain that binds to regulatory elements in cog- nuclear receptor-mediated transcription in vitro, there is no avail- nate target genes, and a C-terminal ligand-binding domain with able information regarding the functional significance of MED1 an associated (ligand-induced) AF-2 domain that mediates ligand-dependent activation of target genes (1, 2). Author contributions: P.J., Q.H., M.I., R.G.R., and X.Z. designed research; P.J., Q.H., S.M., The ultimate action of nuclear receptors following binding to M.I., and X.Z. performed research; P.J., Q.H., S.W., S.K., M.I., R.G.R., and X.Z. analyzed data; target genes is to enhance the recruitment and/or function of the and R.G.R. and X.Z. wrote the paper. general transcription machinery, including RNA polymerase II The authors declare no conflict of interest. and general transcription factors, on cognate core promoter 1P.J., Q.H., and M.I. contributed equally to this work. elements (3). Interactions of the AF-1 and AF-2 domains with 2To whom correspondence may be addressed: E-mail: [email protected] or roeder@ various transcription coactivators are required for this process rockefeller.edu. – (4 6). Among these diverse coactivators, Mediator, a large 3Present address: Laboratory of Hematology, Kobe University Graduate school of Health (∼1.6 MDa) protein complex composed of at least 25–30 distinct Sciences, Kobe 654-0142, Japan subunits, has emerged as the main bridge for direct communica- This article contains supporting information online at www.pnas.org/cgi/content/full/ tion between transcriptional activators and RNA polymerase II 1001814107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1001814107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 26, 2021 LxxLL motifs in vivo. Toward this end, we have generated MED1 impaired in Med1KI∕KI mice relative to wild-type mice. Similar LxxLL motif-mutant knockin mice and, surprisingly, found that defects were observed throughout pubertal mammary gland these mice are apparently fertile and grossly normal. However, development at different ages (Fig. S2). they do exhibit profound defects in virgin mammary gland devel- To determine if the mammary gland defects observed in KI∕KI opment. Consistent with this observation, we observed significant Med1 mice resulted from disrupted cell proliferation, we impairments both in ERα-dependent gene expression in mam- performed BrdU incorporation assays (25). Seven- to eight- KI∕KI mary epithelial cells and in estrogen-stimulated mammary ductal week-old wild-type and Med1 age-matched female mice growth. Moreover, we found that MED1 is differentially ex- were injected intraperitoneally with BrdU 2 h prior to sacrifice. pressed in mammary epithelial cells and plays a role in luminal Mammary glands were harvested, fixed, and then subjected to lineage determination. BrdU staining. Ten random areas of 100 cells in each sample were selected and counted to estimate the percentage of total epithe- Results lial cells that were BrdU-positive (Fig. 1 D and E). As expected, Med1KI∕KI Mice Exhibit Profound Defects in Pubertal Mammary Gland about 20% of mammary epithelial cells stained positive for BrdU Development. in wild-type mice. In contrast, there were about 4 times fewer To study the role of the MED1 LxxLL motifs in a KI∕KI physiological context, we generated MED1 LxxLL motif-mutant BrdU-positive mammary epithelial cells in the Med1 mice. 1KI∕KI These data support the idea that the observed mammary gland knockin mice (Med ). The point mutations that were intro- KI∕KI duced result in conversion of both MED1 LxxLL motifs to LxxAA defects in Med1 mice are caused, at least in part, by motifs (Fig. 1A and Fig. S1), which previously was shown to decreased cell proliferation. disrupt strong Mediator-nuclear receptor interactions in vitro KI∕KI α (14, 15). These mutations had no effect on the expression level Med1 Mice Show Impaired ER Target Gene Expression in of the MED1 protein (Fig. S1). Surprisingly, in contrast to Mammary Epithelial Cells. Estrogen is the dominant hormone pro- the embryonic lethality of a total MED1 knockout (24), moting mammary epithelial cell proliferation at the stage of Med1KI∕KI mice were viable, fertile, and grossly normal. How- mammary gland development that we studied. Thus, we reasoned ever, they did exhibit profound defects in mammary gland devel- that the mutations in the MED1 LxxLL motifs exerted their ef- opment. In these studies, mammary glands of 8-week-old fects by disrupting the estrogen signaling pathway, either by in- KI∕KI fluencing the production of estrogen or by directly affecting Med1 and wild-type virgin mice were isolated, fixed in Car- ERα-mediated transcription. To discriminate between these noy’s solution, and then stained with Carmine. As shown morpho- possibilities, we first examined the blood estrogen levels of 8- logically in Fig.