SWI/SNF complexes are ATP-dependent chromatin remodelling enzymes that have been implicated in the regulation of gene expression in yeast and higher eukaryotes. BRG1, a catalytic subunit in the mammalian SWI/SNF complex, is required for transcriptional activation by the estrogen receptor, but the mechanisms by which the complex is recruited to estrogen target genes are unknown. Here, we have identi®ed an interaction between the estrogen receptor and BAF57, a subunit present only in mammalian SWI/SNF complexes, which is stimulated by estrogen and requires both a functional hormone-binding domain and the DNA-binding region of the receptor. We also found an additional interaction between the p160 family of coactivators and BAF57 and demonstrate that the ability of p160 coactivators to potentiate transcription by the estrogen receptor is dependent on BAF57 in transfected cells. Moreover, chromatin immunoprecipitation assays demonstrated that BAF57 is recruited to the estrogen-responsive promoter, pS2, in a liganddependent manner. These results suggest that one of the mechanisms for recruiting SWI/SNF complexes to estrogen target genes is by means of BAF57.

Hey1 is a member of the basic helix-loop-helix-Orange family of transcriptional repressors that mediate Notch signaling. Here we show that transcription from androgen-dependent target genes is inhibited by Hey1 and that expression of a constitutively active form of Notch is capable of repressing transactivation by the endogenous androgen receptor (AR). Our results indicate that Hey1 functions as a corepressor for AF1 in the AR, providing a mechanism for cross talk between Notch and androgen-signaling pathways. Hey1 colocalizes with AR in the epithelia of patients with benign prostatic hyperplasia, where it is found in both the cytoplasm and the nucleus. In marked contrast, we demonstrate that Hey1 is excluded from the nucleus in most human prostate cancers, raising the possibility that an abnormal Hey1 subcellular distribution may have a role in the aberrant hormonal responses observed in prostate cancer.

SRC1, initially identified as a nuclear receptor coactivator, was found to interact with a member of the transcriptional enhancer factor (TEF) family of transcription factors, TEF-4. The interaction, which occurs in both intact cells and in a cell-free system, is mediated by the highly conserved basic helix-loop-helix/Per-ArntSim (bHLH-PAS) domain present in the N-terminal region of SRC1. Moreover, all three members of the p160 family of nuclear receptor coactivators, SRC1, TIF2, and RAC3, are able to potentiate transcription from a TEF response element in transient transfection experiments, and this activation requires the presence of the bHLH-PAS domain. These results suggest that the p160 proteins could be bona fide coactivators of the TEF family of transcription factors.Transcriptional coactivators, recruited by sequence-specific transcription factors, enhance transcriptional activation of target genes via interactions with chromatin remodeling complexes and components of the basal transcriptional apparatus (1, 2). Three related 160-kDa proteins, SRC1, TIF2, and RAC3, encoded by separate genes, form the steroid receptor coactivator (SRC) 1 or p160 family of coactivators (for a review, see Refs. 3 and 4). These proteins are highly homologous and were initially identified as factors that interacted with nuclear receptors (NRs) in the presence of ligand and were able to enhance receptor-dependent transcriptional activation (5-7). The p160 proteins have been reported to potentiate the activity not only of NRs but also a number of other transcription factors (8 -14), although the mechanisms by which the p160s enhance the activity of other signaling pathways are less well characterized.The p160 proteins contain conserved domains responsible for the interaction with NRs (15,16), and protein interaction domains responsible for the recruitment of downstream effectors, such as histone acetyltransferases like CBP/p300 (17, 18) and protein methyltransferases (19). In addition, the p160 coactivators have a highly conserved N-terminal basic helix-loophelix/Per-Arnt-Sim (bHLH-PAS) domain. The bHLH domain is a DNA binding and protein dimerization motif shared by many transcription factors (20), and in the bHLH-PAS subfamily an additional dimerization motif, called PAS domain, extends from the C-terminal end of the HLH domain (21). The bHLH-PAS domain present in the p160 proteins has a striking homology with those from the bHLH-PAS family of transcription factors, and it is also the most conserved region between the three members of the family. Nevertheless, its function remains unclear and it seems to be dispensable for the enhancing of the NR transcriptional activity in cotransfection studies (5). Therefore, the role of this putative protein dimerization motif in the stabilization of competent coactivator complexes, mediating accessory protein-protein interactions and/or the recruitment of p160 coactivators by other transcription factors remains to be established.To understand the molecular mechanisms of SRC1 functions a...

The expression of the ␤-amyloid precursor protein (APP), which plays a key role in the development of Alzheimer's disease, is regulated by a variety of cellular mediators in a cell-dependent manner. In the present study, we present evidence that thyroid hormones negatively regulate the expression of the APP gene in neuroblastoma cells. Transient transfection studies using plasmids that contain progressive deletions of the 5 region of the gene demonstrate that triiodothyronine (T3), the more active form of the thyroid hormones, represses APP promoter activity by a mechanism that requires binding of the nuclear T3 receptor (TR) to a specific sequence located in the first exon. The unliganded receptor increases promoter activity, and T3 reverses that activity to basal levels. The repressive effect of T3 does not exhibit TR isoform specificity, and it is equally mediated by TR␣ and TR␤. Gel mobility shift assays using in vitro synthesized nuclear receptors and nuclear extracts led to the identification of a negative thyroid hormone response element, at nucleotide position ؉80/ ؉96, that preferentially binds heterodimers of TR with the retinoid X receptor. Insertion of sequences containing this element confers negative regulation by T3 to a heterologous TK promoter, thus indicating the functionality of the element.

Estrogen receptors (ERs) play critical roles in both normal mammary gland development and in the formation and progression of breast tumors, constituting a major therapeutic target for breast cancer treatment. We have previously described that ER transcriptional activity is potentiated by BAF57, a core subunit of the mammalian SWI/SNF chromatin remodeling complex. Here we provide evidence demonstrating an important role for BAF57 as regulator of ER functions in breast cancer cells. Different experimental manipulations leading to the abrogation of BAF57 expression and/or function severely reduced the expression of various endogenous ER target genes and blocked estrogen-stimulated proliferation in ZR-75-1 breast cancer cells. Moreover, using a structure-function analysis, we have defined the protein domains required for the functional interaction between ER␣ and BAF57, including a key region within the hinge of ER␣ that is essential for BAF57 recruitment and its function on ER-mediated transcription. Interestingly, we found that BAF57 is an ER subtype-selective modulator that specifically regulates ER␣-mediated transcription. Taken together, our results suggest that targeting BAF57 could represent a new way to effectively inhibit the action of ER␣.Breast cancer is a leading cause of cancer mortality among Western women with an estimated annual incidence of 1 million cases worldwide (see Ref. 1 and references therein). Estrogen hormones play a critical role in the development and normal physiology of the mammary gland, and, whereas various factors contribute to the etiology of breast cancer, estrogens within the context of other signaling pathways are an important influence in the initiation and progression of breast tumors (reviewed in Ref. 2). Estrogen effects are mediated by the related but distinct estrogen receptors ␣ (ER␣) 2 and ␤ (ER␤), although accumulating evidence suggests that ER␣ is the main transducer of estrogen signals promoting cell proliferation in both normal and cancerous breast tissues (3-5).Estrogens can act through nongenomic pathways, although most described estrogen actions are mediated by ER␣ and ER␤ acting as ligand-dependent transcription factors. Estrogen receptors belong to the nuclear receptor superfamily (6), and they regulate many physiological processes in response to their natural ligand, 17␤-estradiol (E2). Upon ligand binding, ERs bind to specific DNA sequences (EREs) present in the promoters of their target genes, triggering the recruitment of many cofactors that must overcome the barrier to transcription that represents the tightly packed chromatin fiber and recruit basal transcription factors and RNA polymerase II. The chromatin remodeling factors are usually divided into two distinct categories: first those enzymes responsible for the covalent modification of the histones, and second, ATP-dependent protein complexes that modify the location and association of nucleosomes with the DNA. Both classes of chromatin remodeling factors, acting together in a concerted manner, contrib...

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Thyroid hormones repress expression of APP (beta-amyloid precursor protein) in cultured cells of neuronal origin. The effect involves binding to the nuclear thyroid hormone receptor (TR) and is mediated by DNA sequences located within the first exon of the gene. These sequences contain a thyroid hormone response element that is necessary, but not sufficient, to mediate the inhibitory effect of the thyroid hormone T(3). In this report, we show that repression by T(3) is mediated by a response unit composed by the thyroid hormone response element and 5'-flanking sequences that bind Sp1 and mediate stimulation by this transcription factor. In that unit, binding sites for TR and Sp1 overlap and a complex mechanism appears to account for the TR-mediated regulation of APP. Unliganded TR does not bind to DNA and allows Sp1 to bind to DNA and stimulate APP basal expression. Binding of ligand T(3), which increases affinity of TR by DNA, precludes binding of Sp1 to DNA and decreases the Sp1-dependent expression of APP.

The hairy/enhancer-of-split related with YRPW motif 1 (HEY1) is a member of the basic-helix-loop-helix-Orange (bHLH-O) family of transcriptional repressors that mediate Notch signaling. Several cancer-related pathways also regulate HEY1 expression, and HEY1 itself acts as an indirect positive regulator of the p53 tumor suppressor protein and a negative regulator of androgen receptor activity. In this study we show how a naturally occurring non-synonymous polymorphism at codon 94 of HEY1, which results in a substitution of leucine by methionine (Leu94Met), converts HEY1 from an androgen receptor corepressor to an androgen receptor co-activator without affecting its intrinsic transcriptional repressive domains. The polymorphism Leu94Met also abolishes HEY1-mediated activation of p53 and suppresses the ability of HEY1 to induce p53-dependent cell-cycle arrest and aberrant cell differentiation in human osteosarcoma U2OS cells. Moreover, expression of HEY1, but not of the variant Leu94Met, confers sensitivity to p53-activating chemotherapeutic drugs on U2OS cells. In addition, we have identified motifs in HEY1 that are critical for the regulation of its subcellular localization and analysed how mutations in those motifs affect both HEY1 and HEY1-Leu94Met functions. These findings suggest that the polymorphism Leu94Met in HEY1 radically alters its biological activities and may affect oncogenic processes.