A global view of transcriptional regulation by nuclear receptors: gene expression, factor localization, and DNA sequence analysis

Recent genomic analyses of transcription factor binding, histone modification, and gene expression have provided a global view of transcriptional regulation by nuclear receptors (NRs) that complements an existing large body of literature on gene-specific studies. The picture emerging from these genomic studies indicates that NRs bind at promoter-proximal and promoter-distal enhancers in conjunction with other transcription factors (e.g., activator protein-1, Sp1 and FOXA1). This binding promotes the recruitment of coregulators that mediate the posttranslational modification of histones at promoters and enhancers. Ultimately, signaling through liganded NRs stimulates changes in the occupancy of RNA polymerase II (Pol II) or the activation of preloaded Pol II at target promoters. Chromosomal looping and/or Pol II tracking may underlie promoter-enhancer communication. Interestingly, the direct target genes of NR signaling represent a limited subset of all the genes regulated by NR ligands, with the rest being regulated through secondary effects. As suggested by previous gene-specific analyses, NR-mediated outcomes are highly cell type- and promoter-specific, highlighting the complexity of transcriptional regulation by NRs and the value of genomic analyses for identifying commonly shared patterns. Overall, NRs share common themes in their patterns of localization and transcriptional regulation across mammalian genomes. In this review, we provide an overview of recent advances in the understanding of NR-mediated transcription garnered from genomic analyses of gene expression, factor localization, and target DNA sequences

Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα +) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy

Allosteric Modulators of Steroid Hormone Receptors : Structural Dynamics and Gene Regulation

Peer reviewedPublisher PD

Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development

Estrogen is a key regulator of normal function of female reproductive system and plays a pivotal role in the development and progression of breast cancer. Here, we demonstrate that JMJD2B (also known as KDM4B) constitutes a key component of the estrogen signaling pathway. JMJD2B is expressed in a high proportion of human breast tumors, and that expression levels significantly correlate with estrogen receptor (ER) positivity. In addition, 17-beta-estradiol (E2) induces JMJD2B expression in an ERα dependent manner. JMJD2B interacts with ERα and components of the SWI/SNF-B chromatin remodeling complex. JMJD2B is recruited to ERα target sites, demethylates H3K9me3 and facilitates transcription of ER responsive genes including MYB, MYC and CCND1. As a consequence, knockdown of JMJD2B severely impairs estrogen-induced cell proliferation and the tumor formation capacity of breast cancer cells. Furthermore, Jmjd2b-deletion in mammary epithelial cells exhibits delayed mammary gland development in female mice. Taken together, these findings suggest an essential role for JMJD2B in the estrogen signaling, and identify JMJD2B as a potential therapeutic target in breast cancer

Activation of Estrogen-Responsive Genes Does Not Require Their Nuclear Co-Localization

The spatial organization of the genome in the nucleus plays a role in the regulation of gene expression. Whether co-regulated genes are subject to coordinated repositioning to a shared nuclear space is a matter of considerable interest and debate. We investigated the nuclear organization of estrogen receptor alpha (ERα) target genes in human breast epithelial and cancer cell lines, before and after transcriptional activation induced with estradiol. We find that, contrary to another report, the ERα target genes TFF1 and GREB1 are distributed in the nucleoplasm with no particular relationship to each other. The nuclear separation between these genes, as well as between the ERα target genes PGR and CTSD, was unchanged by hormone addition and transcriptional activation with no evidence for co-localization between alleles. Similarly, while the volume occupied by the chromosomes increased, the relative nuclear position of the respective chromosome territories was unaffected by hormone addition. Our results demonstrate that estradiol-induced ERα target genes are not required to co-localize in the nucleus

Genes targeted by the estrogen and progesterone receptors in the human endometrial cell lines HEC1A and RL95-2

<p>Abstract</p> <p>Background</p> <p>When the steroid hormones estrogen and progesterone bind to nuclear receptors, they have transcriptional impact on target genes in the human endometrium. These transcriptional changes have a critical function in preparing the endometrium for embryo implantation.</p> <p>Methods</p> <p>382 genes were selected, differentially expressed in the receptive endometrium, to study their responsiveness of estrogen and progesterone. The endometrial cell lines HEC1A and RL95-2 were used as experimental models for the non-receptive and receptive endometrium, respectively. Putative targets for activated steroid hormone receptors were investigated by chromatin immunoprecipitation (ChIP) using receptor-specific antibodies. Promoter occupancy of the selected genes by steroid receptors was detected in ChIP-purified DNA by quantitative PCR (qPCR). Expression analysis by reverse transcriptase (RT)-PCR was used to further investigate hormone dependent mRNA expression regulation of a subset of genes.</p> <p>Results</p> <p>ChIP-qPCR analysis demonstrated that each steroid hormone receptor had distinct group of target genes in the endometrial cell lines. After estradiol treatment, expression of estrogen receptor target genes predominated in HEC1A cells (n = 137) compared to RL95-2 cells (n = 35). In contrast, expression of progesterone receptor target genes was higher in RL95-2 cells (n = 83) than in HEC1A cells (n = 7) after progesterone treatment. RT-PCR analysis of 20 genes demonstrated transcriptional changes after estradiol or progesterone treatment of the cell lines.</p> <p>Conclusions</p> <p>Combined results from ChIP-qPCR and RT-PCR analysis showed different patterns of steroid hormone receptor occupancy at target genes, corresponding to activation or suppression of gene expression after hormone treatment of HEC1A and RL95-2 cell lines.</p

Progesterone Receptor induces bcl-x expression through intragenic binding sites favoring RNA Polymerase II elongation

Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of pro- gestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions sur- rounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-dis- tribution of the active Pol II toward the 30-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene ex- pression by facilitating the proper passage of the polymerase along hormone-dependent genes.Fil: Bertucci, Paola Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nacht, Ana Silvina. Universitat Pompeu Fabra; España. Centro de Regulación Genómica; EspañaFil: Alló, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rocha Viegas, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Ballaré, Cecilia. Universitat Pompeu Fabra; España. Centro de Regulación Genómica; EspañaFil: Soronellas, Daniel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Castellano, Giancarlo. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Zaurin, Roser. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Beato, Miguel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Vicent, Guillermo. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

NELF Potentiates Gene Transcription in the Drosophila Embryo

A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. Here we use a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. We propose that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development

Genomics of Signaling Crosstalk of Estrogen Receptor α in Breast Cancer Cells

BACKGROUND: The estrogen receptor alpha (ERalpha) is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERalpha in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERalpha-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERalpha primarily activates and represses genes, respectively. The combined treatments with the anti-estrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant. CONCLUSIONS/SIGNIFICANCE: The activating signal dictates both target gene selection and regulation by ERalpha, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERalpha-dependent carcinomas