Search

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1β complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1β coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1β complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes., Competing Interests: Competing interests statement:C.K.G. is a cofounder, equity holder and member of the Scientific Advisory Board of Asteroid Therapeutics.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1β with the NCoR/HDAC3 complex, resulting in the activation of PGC1β and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts., Competing Interests: Declaration of interests C.K.G. is a co-founder, equity holder, and member of the Scientific Advisory Board of Asteroid Therapeutics. J.T.K. is on the Molecular Cell advisory board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer's disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the FosB proto-oncogene (FOSB) and ERCC excision repair 1 (ERCC1) genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Stolzenburg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The purpose of this study was to identify critical pathways promoting survival of tamoxifen-tolerant, estrogen receptor α positive (ER + ) breast cancer cells, which contribute to therapy resistance and disease recurrence. Gene expression profiling and pathway analysis were performed in ER + breast tumors of patients before and after neoadjuvant tamoxifen treatment and demonstrated activation of the NF-κB pathway and an enrichment of epithelial-to mesenchymal transition (EMT)/stemness features. Exposure of ER + breast cancer cell lines to tamoxifen, in vitro and in vivo , gives rise to a tamoxifen-tolerant population with similar NF-κB activity and EMT/stemness characteristics. Small-molecule inhibitors and CRISPR/Cas9 knockout were used to assess the role of the NF-κB pathway and demonstrated that survival of tamoxifen-tolerant cells requires NF-κB activity. Moreover, this pathway was essential for tumor recurrence following tamoxifen withdrawal. These findings establish that elevated NF-κB activity is observed in breast cancer cell lines under selective pressure with tamoxifen in vitro and in vivo , as well as in patient tumors treated with neoadjuvant tamoxifen therapy. This pathway is essential for survival and regrowth of tamoxifen-tolerant cells, and, as such, NF-κB inhibition offers a promising approach to prevent recurrence of ER + tumors following tamoxifen exposure. IMPLICATIONS: Understanding initial changes that enable survival of tamoxifen-tolerant cells, as mediated by NF-κB pathway, may translate into therapeutic interventions to prevent resistance and relapse, which remain major causes of breast cancer lethality., (©2020 American Association for Cancer Research.)

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages., Competing Interests: KC, JS, AK, GL, SU, VN, CG, AN No competing interests declared, (© 2019, Cohen Katsenelson et al.)

Mechanisms by which members of the AP-1 family of transcription factors play non-redundant biological roles despite recognizing the same DNA sequence remain poorly understood. To address this question, here we investigate the molecular functions and genome-wide DNA binding patterns of AP-1 family members in primary and immortalized mouse macrophages. ChIP-sequencing shows overlapping and distinct binding profiles for each factor that were remodeled following TLR4 ligation. Development of a machine learning approach that jointly weighs hundreds of DNA recognition elements yields dozens of motifs predicted to drive factor-specific binding profiles. Machine learning-based predictions are confirmed by analysis of the effects of mutations in genetically diverse mice and by loss of function experiments. These findings provide evidence that non-redundant genomic locations of different AP-1 family members in macrophages largely result from collaborative interactions with diverse, locus-specific ensembles of transcription factors and suggest a general mechanism for encoding functional specificities of their common recognition motif.

We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor α, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor α and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen-receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKβ establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKβ signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tamoxifen has shown great success in the treatment of breast cancer; however, long-term treatment can lead to acquired tamoxifen (TOT) resistance and relapse. TOT classically antagonizes estradiol (E2) -dependent breast cancer cell growth, but exerts partial agonist/antagonist behavior on gene expression. Although both E2 and TOT treatment of breast cancer cells results in recruitment of the estrogen receptor (ER) to common and distinct genomic sites, the mechanisms and proteins underlying TOT preferential recruitment of the ER remains poorly defined. To this end, we performed in silico motif-enrichment analyses within the ER-binding peaks in response to E2 or TOT, to identify factors that would specifically recruit ER to genomic binding sites in the presence of TOT as compared to E2. Intriguingly, we found Nkx3-1 and Oct-transcription factor homodimer motifs to be enriched in TOT preferential binding sites and confirmed the critical role of Oct-3/4 (aka Oct-4) in directing ER recruitment to TOT preferential genomic binding sites, by chromatin immunoprecipitation (ChIP) analyses. Further investigation revealed Oct-4 expression to be basally repressed by Nkx3-1 in MCF-7 cells and TOT treatment appeared to elevate Nkx3-1 degradation through a p38MAPK-dependent phosphorylation of the E3 ligase, Skp2 at serine-64 residue, as observed by quantitative mass-spectrometry analyses. Consistently, Oct-4 upon induction by phospho-Ser64-Skp2-mediated proteasomal degradation of Nkx3-1, participated in ER transcriptional complexes along with p38MAPK and Skp2 in a tamoxifen-dependent manner leading to TOT-dependent gene activation and cell proliferation of the TOT-resistant MCF-7-tam r breast cancer cells. Notably, Oct-4 levels were highly elevated in MCF-7-tam r cells, and appeared critical for their TOT sensitivity in cell proliferation assays. Furthermore, overexpression of Oct-4 enhanced tumor growth in the presence of tamoxifen in mice in vivo. Collectively, our work presents a novel mechanism for tamoxifen-specific gene activation by ER, secondary to its TOT preferential recruitment to genomic sites by specific activation of Oct-4, a phenomenon that appears to underlie tamoxifen resistance in breast cancer cells and in xenograft tumor models, and could be useful in designing therapeutic interventions to improve treatment outcome.

In normal human cells, centrosome loss induced by centrinone-a specific centrosome duplication inhibitor-leads to irreversible, p53-dependent G1 arrest by an unknown mechanism. A genome-wide CRISPR/Cas9 screen for centrinone resistance identified genes encoding the p53-binding protein 53BP1, the deubiquitinase USP28, and the ubiquitin ligase TRIM37. Deletion of TP53BP1, USP28, or TRIM37 prevented p53 elevation in response to centrosome loss but did not affect cytokinesis failure-induced arrest or p53 elevation after doxorubicin-induced DNA damage. Deletion of TP53BP1 and USP28, but not TRIM37, prevented growth arrest in response to prolonged mitotic duration. TRIM37 knockout cells formed ectopic centrosomal-component foci that suppressed mitotic defects associated with centrosome loss. TP53BP1 and USP28 knockouts exhibited compromised proliferation after centrosome removal, suggesting that centrosome-independent proliferation is not conferred solely by the inability to sense centrosome loss. Thus, analysis of centrinone resistance identified a 53BP1-USP28 module as critical for communicating mitotic challenges to the p53 circuit and TRIM37 as an enforcer of the singularity of centrosome assembly., (© 2016 Meitinger et al.)

Estrogen receptor (ER) and NFκB are two widely expressed, pleiotropic transcription factors that have been shown to interact and affect one another's activity. While the ability of ER to repress NFκB activity has been extensively studied and is thought to underlie the anti-inflammatory activity of estrogens, how NFκB signaling affects ER activity is less clear. This is a particularly important question in breast cancer since activation of NFκB in ER positive tumors is associated with failure of endocrine and chemotherapies. In this review, we provide an update on the multiple mechanisms by which NFκB can influence ER activity, including down-regulation of ER expression, enhanced ER recruitment to DNA, and increased transcriptional activity of both liganded and unliganded ER. Additionally, a novel example of NFκB potentiation of ER-dependent gene repression is reviewed. Together, these mechanisms can alter response to endocrine therapies and may underlie the poor outcome for women with ER positive tumors that have active NFκB signaling., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Macrophages reside in essentially all tissues of the body and play key roles in innate and adaptive immune responses. Distinct populations of tissue macrophages also acquire context-specific functions that are important for normal tissue homeostasis. To investigate mechanisms responsible for tissue-specific functions, we analyzed the transcriptomes and enhancer landscapes of brain microglia and resident macrophages of the peritoneal cavity. In addition, we exploited natural genetic variation as a genome-wide "mutagenesis" strategy to identify DNA recognition motifs for transcription factors that promote common or subset-specific binding of the macrophage lineage-determining factor PU.1. We find that distinct tissue environments drive divergent programs of gene expression by differentially activating a common enhancer repertoire and by inducing the expression of divergent secondary transcription factors that collaborate with PU.1 to establish tissue-specific enhancers. These findings provide insights into molecular mechanisms by which tissue environment influences macrophage phenotypes that are likely to be broadly applicable to other cell types., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs.

Recent studies suggest a hierarchical model in which lineage-determining factors act in a collaborative manner to select and prime cell-specific enhancers, thereby enabling signal-dependent transcription factors to bind and function in a cell-type-specific manner. Consistent with this model, TLR4 signaling primarily regulates macrophage gene expression through a pre-existing enhancer landscape. However, TLR4 signaling also induces priming of ∼3,000 enhancer-like regions de novo, enabling visualization of intermediates in enhancer selection and activation. Unexpectedly, we find that enhancer transcription precedes local mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). H3K4 methylation at de novo enhancers is primarily dependent on the histone methyltransferases Mll1, Mll2/4, and Mll3 and is significantly reduced by inhibition of RNA polymerase II elongation. Collectively, these findings suggest an essential role of enhancer transcription in H3K4me1/2 deposition at de novo enhancers that is independent of potential functions of the resulting eRNA transcripts., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Toll-like receptors (TLRs) play important roles in initiation of innate immune responses and promotion of pathological forms of inflammation. Recent technological advances have enabled the visualization of transcription factor binding and histone modifications in response to TLR signaling at genome-wide levels. Findings emerging from these studies are beginning to provide a picture of how signal-dependent transcription factors regulate the inflammatory response in a cell-specific manner by controlling the recruitment of nucleosome remodeling factors and histone modifying enzymes. Of particular interest, new small molecule inhibitors have been developed that influence inflammatory responses by altering the reading or erasure of histone modifications required for inflammatory gene activation. These findings suggest new approaches for treatment of inflammatory diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of coregulator complexes that function to read, write, and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll-like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 trimethylation/demethylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The estrogen receptor α (ERα) is a master regulator of gene expression and works along with cooperating transcription factors in mediating the actions of the hormone estradiol (E2) in ER-positive tissues and breast tumors. Here, we report that expression of paired-like homeodomain transcription factor (PITX1), a tumor suppressor and member of the homeobox family of transcription factors, is robustly up-regulated by E2 in several ERα-positive breast cancer cell lines via ERα-dependent interaction between the proximal promoter and an enhancer region 5' upstream of the PITX1 gene. Overexpression of PITX1 selectively inhibited the transcriptional activity of ERα and ERβ, while enhancing the activities of the glucocorticoid receptor and progesterone receptor. Reduction of PITX1 by small interfering RNA enhanced ERα-dependent transcriptional regulation of a subset of ERα target genes. The consensus PITX1 binding motif was found to be present in 28% of genome-wide ERα binding sites and was in close proximity to estrogen response elements in a subset of ERα binding sites, and E2 treatment enhanced PITX1 as well as ERα recruitment to these binding sites. These studies identify PITX1 as a new ERα transcriptional target that acts as a repressor to coordinate and fine tune target-specific, ERα-mediated transcriptional activity in human breast cancer cells.

Nuclear receptor estrogen receptor alpha (ER alpha) controls the expression of hundreds of genes responsible for target cell phenotypic properties, but the relative importance of direct versus tethering mechanisms of DNA binding has not been established. In this first report, we examine the genome-wide chromatin localization of an altered-specificity mutant ER with a DNA binding domain deficient in binding to estrogen response element (ERE)-containing DNA (DBDmut ER) versus wild-type ER alpha. Using high-throughput sequencing of ER chromatin immunoprecipitations (ChIP-Seq) and mRNA transcriptional profiling, we show that direct ERE binding is required for most of (75%) estrogen-dependent gene regulation and 90% of hormone-dependent recruitment of ER to genomic binding sites. De novo motif analysis of the chromatin binding regions in MDA-MB-231 human breast cancer cells defined unique transcription factor profiles responsible for genes regulated through tethering versus direct ERE binding, with Runx motifs enriched in ER-tethered sites. We confirmed a role for Runx1 in mediating ER alpha genomic recruitment and regulation of tethering genes. Our findings delineate the contributions of direct receptor ERE binding versus binding through response elements for other transcription factors in chromatin localization and ER-dependent gene regulation, paradigms likely to underlie the gene regulatory actions of other nuclear receptors as well.

Dynamic regulation of cell shape underlies many developmental and immune functions. Cortical remodeling is achieved under the central control of Rho GTPase pathways that modulate an exquisite balance in the dynamic assembly and disassembly of the cytoskeleton and focal adhesions. Macroautophagy (autophagy), associated with bulk cytoplasmic remodeling through lysosomal degradation, has clearly defined roles in cell survival and death. Moreover, it is becoming apparent that proteins, organelles, and pathogens can be targeted for autophagic clearance by selective mechanisms, although the extent and roles of such degradation are unclear. Here we report a conserved role for autophagy specifically in the cortical remodeling of Drosophila blood cells (hemocytes) and mouse macrophages. Continuous autophagy was required for integrin-mediated hemocyte spreading and Rho1-induced cell protrusions. Consequently, hemocytes disrupted for autophagy were impaired in their recruitment to epidermal wounds. Cell spreading required ref(2)P, the Drosophila p62 multiadaptor, implicating selective autophagy as a novel mechanism for modulating cortical dynamics. These results illuminate a specific and conserved role for autophagy as a regulatory mechanism for cortical remodeling, with implications for immune cell function.

Estrogens generally stimulate the proliferation of estrogen receptor (ER)-containing breast cancer cells, but they also suppress proliferation of some ER-positive breast tumors. Using a genome-wide analysis of gene expression in two ER-positive human breast cancer cell lines that differ in their proliferative response to estrogen, we sought to identify genes involved in estrogen-regulated cell proliferation. To this end, we compared the transcriptional profiles of MCF-7 and MDA-MB-231ER+ cells, which have directionally opposite 17beta-estradiol (E2)-dependent proliferation patterns, MCF-7 cells being stimulated and 231ER+ cells suppressed by E2. We identified a set of approximately 70 genes regulated by E2 in both cells, with most being regulated by hormone in an opposite fashion. Using a variety of bioinformatics approaches, we found the E2F binding site to be overrepresented in the potential regulatory regions of many cell cycle-related genes stimulated by estrogen in MCF-7 but inhibited by estrogen in 231ER+ cells. Biochemical analyses confirmed that E2F1 and E2F downstream target genes were increased in MCF-7 and decreased in 231ER+ cells upon estrogen treatment. Furthermore, RNA interference-mediated knockdown of E2F1 blocked estrogen regulation of E2F1 target genes and resulted in loss of estrogen regulation of proliferation. These results demonstrate that regulation by estrogen of E2F1, and subsequently its downstream target genes, is critical for hormone regulation of the proliferative program of these breast cancer cells, and that gene expression profiling combined with bioinformatic analyses of transcription factor binding site enrichment in regulated genes can identify key components associated with nuclear receptor hormonal regulation of important cellular functions.

How nuclear receptors (NRs) coordinate the sequential, ligand-dependent recruitment of multiple coactivator complexes (e.g., SRC complexes and Mediator) that share similar receptor binding determinants is unclear. We show that although the receptor binding subunits of these complexes (i.e., SRCs and Med220, respectively) share overlapping binding sites on estrogen receptor alpha (ERalpha), information contained in the receptor-coactivator interface allows the receptor to distinguish between them. In support of this conclusion, we have identified an ERalpha AF-2 point mutant (L540Q) that selectively binds and recruits Med220, but not SRCs, both in vitro and in vivo. In cells expressing this mutant, the recruitment of Med220 to the pS2 promoter is delayed, and the expression of the vast majority of estrogen target genes is impaired, suggesting a nearly global functional interdependence of these coactivators. Collectively, our results suggest that "facilitated recruitment," rather than competition, drives the sequential recruitment of SRC complexes and Mediator by NRs.