Specific Gene Networks Research Articles

The nuclear receptor co-repressor RIP140 controls the expression of metabolic gene networks

NRs (nuclear receptors) regulate the expression of specific gene networks in target cells by recruiting cofactor complexes involved in chromatin remodelling and in the assembly of transcription complexes. The importance of activating gene expression, in metabolic tissues, is well established, but the contribution of transcriptional inhibition is less well defined. In this review, we highlight a crucial role for RIP140 (receptor-interacting protein 140), a transcriptional co-repressor for NR, in the regulation of metabolic gene expression. Many genes involved in lipid and carbohydrate metabolism are repressed by RIP140 in adipose and muscle. The repressive function of RIP140 results from its ability to bridge NRs to repressive enzyme complexes that modify DNA and histones. In the absence of RIP140, expression from many metabolic genes is increased so that mice exhibit a lean phenotype and resistance to high-fat-diet-induced obesity and display increased glucose tolerance and insulin sensitivity. We propose that a functional interplay between transcriptional activators and the co-repressor RIP140 is an essential process in metabolic regulation.

Progress in Using Mouse Inbred Strains, Consomics, and Mutants to Identify Genes Related to Stress, Anxiety, and Alcohol Phenotypes

This article summarizes the proceedings of a symposium that took place at the 2005 meeting of the Research Society on Alcoholism. The organizers/chairs were Daniel Goldowitz and Katheen A. Grant. The presentations were as follows: (1) High-Throughput Screening for Ethanol Phenotypes, by Douglas B. Matthews and Kristin M. Hamre; (2) Genetic Basis of Schedule-Induced Polydipsia in Mice, by Guy Mittleman and Elissa J. Chesler; (3) Effects of Stress and Ethanol Dependence on Ethanol Self-administration in Inbred and Mutant Mice, by Howard C. Becker and Marcelo F. Lopez; (4) Changes in Dopaminergic Mechanisms Associated With Ethanol Dependence, by Sara R. Jones and Tiffany A. Mathews; and (5) Defining Brain Region-Specific Gene Networks Relevant to Ethanol Behaviors, by Michael F. Miles and Robnet Kerns.

Use of the Progesterone Receptor Antagonist RU 486 to Identify Novel Progesterone Receptor-Regulated Pathways in Implantation

The steroid hormone progesterone (P) is a critical regulator of embryo implantation and maintenance of pregnancy. P acting through the nuclear progesterone receptors (PRs) regulates the expression of specific gene networks that in turn control the extensive cell proliferation, differentiation, and remodeling that occur in various uterine cell types during the progressive phases of implantation. To identify the P-regulated pathways that underlie the implantation process in the mouse, we employed RU 486, a well-characterized PR antagonist that binds to the receptor and blocks its gene regulatory function. We performed messenger RNA (mRNA) profiling in the peri-implantation uterus using oligonucleotide microarrays to analyze changes in mRNA levels in response to RU 486. This analysis provided, for the first time, a comprehensive profile of PR-regulated gene networks with potential roles during implantation. Our study identified a variety of novel PR-regulated molecules, such as growth factors, protease inhibitors, metabolic enzymes, peptide hormones, transcription factors, immune response molecules, cytoskeletal proteins, and cell adhesion molecules, that are potential mediators of P action in the peri-implantation mouse uterus. This article provides a brief description of the expression and function of these newly identified molecular pathways.

Nuclear retinoid receptors and the transcription of retinoid-target genes.

The pleiotropic effects of retinoids are mediated by nuclear retinoid receptors (RARs and RXRs) which are ligand-activated transcription factors. In response to retinoid binding, RAR/RXR heterodimers undergo major conformational changes and orchestrate the transcription of specific gene networks, through binding to specific DNA response elements and recruiting cofactor complexes that act to modify local chromatin structure and/or engage the basal transcription machinery. Then the degradation of RARs and RXRs by the ubiquitin-proteasome controls the magnitude and the duration of the retinoid response. RARs and RXRs also integrate a variety of signaling pathways through phosphorylation events which cooperate with the ligand for the control of retinoid-target genes transcription. These different modes of regulation reveal unexpected levels of complexity in the dynamics of retinoid-dependent transcription.

Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase

BackgroundIn the endometrium the steroid hormone progesterone (P), acting through its nuclear receptors, regulates the expression of specific target genes and gene networks required for endometrial maturation. Proper endometrial maturation is considered a requirement for embryo implantation. Endometrial receptivity is a complex process that is spatially and temporally restricted and the identity of genes that regulate receptivity has been pursued by a number of investigators.MethodsIn this study we have used high density oligonucleotide microarrays to screen for changes in mRNA transcript levels between normal proliferative and adequate secretory phases in Rhesus monkey artificial menstrual cycles. Biotinylated cRNA was prepared from day 13 and days 21–23 of the reproductive cycle and transcript levels were compared by hybridization to Affymetrix HG-U95A arrays.ResultsOf ~12,000 genes profiled, we identified 108 genes that were significantly regulated during the shift from a proliferative to an adequate secretory endometrium. Of these genes, 39 were up-regulated at days 21–23 versus day 13, and 69 were down-regulated. Genes up-regulated in P-dominant tissue included: secretoglobin (uteroglobin), histone 2A, polo-like kinase (PLK), spermidine/spermine acetyltransferase 2 (SAT2), secretory leukocyte protease inhibitor (SLPI) and metallothionein 1G (MT1G), all of which have been previously documented as elevated in the Rhesus monkey or human endometrium during the secretory phase. Genes down-regulated included: transforming growth factor beta-induced (TGFBI or BIGH3), matrix metalloproteinase 11 (stromelysin 3), proenkephalin (PENK), cysteine/glycine-rich protein 2 (CSRP2), collagen type VII alpha 1 (COL7A1), secreted frizzled-related protein 4 (SFRP4), progesterone receptor membrane component 1 (PGRMC1), chemokine (C-X-C) ligand 12 (CXCL12) and biglycan (BGN). In addition, many novel/unknown genes were also identified. Validation of array data was performed by semi-quantitative RT-PCR of two selected up-regulated genes using temporal (cycle day specific) endometrial cDNA populations. This approach confirmed up-regulation of WAP four-disulfide core domain 2 (WFDC2) and SLPI during the expected window of receptivity.ConclusionThe identification of P-regulated genes and gene pathways in the primate endometrium is expected to be an important first step in elucidating the cellular processes necessary for the development of a receptive environment for implantation.

The ERE-luc reporter mouse.

Estrogens, by signaling through two nuclear receptors (ERs), ERα and ERβ), can control specific gene networks and modulate target cell activities. In the last decade, cell-based approaches have provided major insights into the transcriptional regulation of ERs at the promoter of target genes where the interplay between ERs and coactivators and corepressors provides the receptors with tissue specificity of action. This is particularly highlighted by the analysis of transcription efficiency of synthetic ligands for ERs such as selective estrogen receptor modulators (SERMs) that may act as agonist in certain organs and antagonists in others. Furthermore, these molecular studies revealed a multiplicity of mechanisms controlling ER transcriptional activation (Maggi et al. 2004). Indeed, the cognate ligand, estrogen, appears not to be an absolute requirement for the activation of ERs (Bunone et al. 1996; Kato et al. 1995; Ma et al. 1994; Power et al. 1991). Several authors reported that growth factors through phosphorylation might stimulate unliganded ER to induce the transcription of selected target genes (Bunone et al. 1996; Kato et al. 1995; Patrone et al. 1996).

Immune-responsive gene 1 is a novel target of progesterone receptor and plays a critical role during implantation in the mouse.

The steroid hormone progesterone (P) is a critical regulator of uterine receptivity during blastocyst implantation. The hormone acts through nuclear P receptors (PRs) to modulate the expression of specific gene networks in various uterine cell types. To identify the P-regulated pathways underlying uterine receptivity, we previously used oligonucleotide microarrays to analyze uterine mRNA profiles at the time of implantation in response to RU486, a PR antagonist. We reported that the mRNA corresponding to the immune-responsive gene 1 (Irg1), a previously described lipopolysaccharide-inducible gene, is one of the several mRNAs that are markedly down-regulated by RU486 in the preimplantation uterus. In the present study, we performed in situ hybridization to show that P stimulates Irg1 mRNA synthesis in the luminal epithelial cells of uteri of ovariectomized wild-type but not PR knockout mice. We also report that Irg1 mRNA was induced in the luminal epithelium of pregnant uterus between d 3 and 5, overlapping the window of implantation. To investigate the function of Irg1 during implantation, we administered sense or antisense oligodeoxynucleotides into preimplantation mouse uteri. Treatment with antisense oligodeoxynucleotides led to suppression in Irg1 mRNA expression without affecting unrelated mRNAs in the pregnant uterus. This intervention was also accompanied by impairment in embryo implantation, indicating that the phenotype is linked to the suppression of Irg1 mRNA. Collectively, our studies identified Irg1 as a novel target of PR in the pregnant uterus and also revealed that it is a critical regulator of the early events leading to implantation.

Progesterone receptor-regulated gene networks in implantation.

The steroid hormone progesterone (P) plays a pivotal role in the establishment and maintenance of pregnancy. The well-known function of P during early pregnancy is to regulate (i) uterine receptivity for blastocyst attachment, (ii) progressive phases of embryo-uterine interactions, and (iii) differentiation of the endometrial stroma that maintains an environment conducive for the growth and development of the implanting embryo. The cellular actions of P are mediated through intracellular progesterone receptors (PRs), which are well-studied gene regulators. It is postulated that hormone-occupied PRs trigger the expression of specific gene networks in different cell types within the uterus and the products of these genes mediate the hormonal effects during early pregnancy. In the present article, we provide a brief description of the molecules that have emerged as candidate markers of progesterone action in rodents and humans during implantation.

The program of androgen-responsive genes in neoplastic prostate epithelium.

The human prostate gland is an important target organ of androgenic hormones. Testosterone and dihydrotestosterone interact with the androgen receptor to regulate vital aspects of prostate growth and function including cellular proliferation, differentiation, apoptosis, metabolism, and secretory activity. Our objective in this study was to characterize the temporal program of transcription that reflects the cellular response to androgens and to identify specific androgen-regulated genes (ARGs) or gene networks that participate in these responses. We used cDNA microarrays representing about 20,000 distinct human genes to profile androgen-responsive transcripts in the LNCaP adenocarcinoma cell line and identified 146 genes with transcript alterations more than 3-fold. Of these, 103 encode proteins with described functional roles, and 43 represent transcripts that have yet to be characterized. Temporal gene expression profiles grouped the ARGs into four distinct cohorts. Five uncharacterized ARGs demonstrated exclusive or high expression levels in the prostate relative to other tissues studied. A search of available DNA sequence upstream of 28 ARGs identified 25 with homology to the androgen response-element consensus-binding motif. These results identify previously uncharacterized and unsuspected genes whose expression levels are directly or indirectly regulated by androgens; further, they provide a comprehensive temporal view of the transcriptional program of human androgen-responsive cells.

Isolation and Characterization of a Novel Coactivator Protein, NCoA-62, Involved in Vitamin D-mediated Transcription

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

Steroid receptor coactivator-1 is a histone acetyltransferase.

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors. Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

Glucocorticoid resistant syndromes--molecular basis and clinical presentations.

The mechanisms of action of glucocorticoid hormones are mediated via specific intracellular receptor proteins. The glucocorticoid receptor (GR) regulates expression of specific target genes or gene networks by ligand-dependent transcriptional activation, i.e. ligand-dependent activation of the receptor with subsequent dimer formation and DNA binding. There are a number of factors, such as the receptor concentration, receptor associated proteins, receptor alterations and the effects on the gene network including hormonal regulation of transcription, mRNA splicing and translation, that might influence glucocorticoid responsiveness in a normal and healthy population as well as in different diseases. Several categories of glucocorticoid resistance have been described including inherited GR resistance which has been explained in terms of specific mutations and offers an important model for genetic and clinical studies of steroid sensitivity, and relative glucocorticoid resistance, which occurs naturally in the course of cellular differentiation, cell to cell or tissue to tissue, since all cells possess receptors for glucocorticoids but do not show the same response to them. From a clinical point of view, it is also interesting to consider preexisting genetic susceptibility to glucocorticoids, acquired changes in the GR gene structure and organization, including alterations of noncoding sequences, and the importance of mutations, deletions and other changes in the GR gene affecting receptor function. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance, both generalized inherited glucocorticoid resistance (GIGR) and directed mutagenesis, has identified two regions of clustered mutations in the proximity of previously identified affinity labeled residues directly affecting the steroid binding function. Finally, studies of New Words primates and cell lines derived from hematologic malignancies constitute animal and human models for the molecular basis of glucocorticoid resistance where a number of inherited and acquired mutations in the GR gene have been demonstrated.

Ordered binding of retinoic acid and retinoid-X receptors to asymmetric response elements involves determinants adjacent to the DNA-binding domain.

Retinoic acid, a pleiotropic regulator of development and homeostasis, controls the expression of specific gene networks via direct interactions with nuclear receptors. The retinoic acid receptor (RAR), as a heterodimer with the retinoid-x receptor (RXR), binds to DNA recognition sites, referred to as retinoic acid response elements (RAREs), that are generally composed of a direct repeat of the half-site core motif PuGGTCA spaced by 2 (DR-2) or 5 (DR-5) basepairs. The asymmetric nature of direct repeat RAREs suggests that RAR and RXR bind preferentially to one of the two half-site core motifs. Here we show that RXR occupies the 5'-up-stream half-site, and RAR the 3'-down-stream half-site of the direct repeat in both DR-2 and DR-5 RAREs. We also demonstrate that a region adjacent to the zinc finger region of RAR and RXR is essential for specific and cooperative binding of DNA-binding domain peptides to RAREs. However, differential utilization of these determinants mediate RAR-RXR heterodimer binding to DR-2 and DR-5 RAREs. The demonstration of ordered but nonequivalent binding of RAR-RXR complexes to DR-2 and DR-5 RAREs sets a precedent for the generation of sequence specificities in heterodimeric DNA-binding proteins.

Analysis of the mechanism of steroid hormone receptor-dependent gene activation in cell-free systems.

I. Introduction STEROID hormones regulate the expression of specific gene networks and thereby exert a wide variety of effects on growth, development, and differentiation in higher eukaryotes (for reviews see Refs. 1–6). The hormonal signals are transduced to the target genes through specific, intracellular receptors. The ligand-free receptor is functionally inactive. The hormone response is triggered by the binding of a steroidal ligand to its cognate receptor, followed by the interaction of the hormonereceptor complex with specific enhancer sequences referred to as steroid response elements (SREs) at the target gene. The receptor regulates gene transcription presumably by interacting with the transcription machinery at the target promoter. During the last several years, good progress has been made toward unravelling the molecular mechanism of signal transduction by steroid hormones. The molecular cloning of virtually all known steroid receptor genes has been accomplished, and precise definition of vario...

Analysis of the mechanism of steroid hormone receptor-dependent gene activation in cell-free systems

STEROID hormones regulate the expression of specific gene networks and thereby exert a wide variety of effects on growth, development, and differentiation in higher eukaryotes (for reviews see Refs. 1–6). The hormonal signals are transduced to the target genes through specific, intracellular receptors. The ligand-free receptor is functionally inactive. The hormone response is triggered by the binding of a steroidal ligand to its cognate receptor, followed by the interaction of the hormonereceptor complex with specific enhancer sequences referred to as steroid response elements (SREs) at the target gene. The receptor regulates gene transcription presumably by interacting with the transcription machinery at the target promoter. During the last several years, good progress has been made toward unravelling the molecular mechanism of signal transduction by steroid hormones. The molecular cloning of virtually all known steroid receptor genes has been accomplished, and precise definition of various SREs has been achieved through gene transfection experiments. However, the mechanisms underlying key molecular events in the steroid-induced gene activation pathway, such as ligand-induced activation of receptors and transactivation of target promoters by the activated receptor, have remained unresolved. One approach to analyzing these mechanisms is to reconstitute hormone and receptor-dependent gene activation in vitro. In a cell-free system one can precisely define the minimal combination and appropriate concentration of components necessary to achieve hormone and tissue-specific gene induction by careful manipulation of well-defined DNA templates, purified receptor and other transcription factors, and hormonal ligand. For many years the establishment of a steroid receptor-regulated gene expression system eluded the researchers in this area. Recently a number of such receptor- and ligand-regulated in vitro gene expression systems have been devised in our laboratory as well as in others (7–11). In this review we will describe how these systems have been exploited to gain useful insights into the mechanism of gene activation by steroid receptors.

Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain.

The rodent and primate basal forebrain is a target of a family of endogenous peptide signaling molecules, the neurotrophins--nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3--and of the gonadal steroid hormone estrogen, both of which have been implicated in cholinergic function. To investigate whether or not these ligands may act on the same neurons in the developing and adult rodent basal forebrain, we combined autoradiography with 125I-labeled estrogen and either nonisotopic in situ hybridization histochemistry or immunohistochemistry. We now report colocalization of intranuclear estrogen binding sites with the mRNA and immunoreactive protein for the low-affinity nerve growth factor receptor, which binds all three neurotrophins, and for the cholinergic marker enzyme choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Colocalization of estrogen and low-affinity nerve growth factor receptors implies that their ligands may act on the same neuron, perhaps synergistically, to regulate the expression of specific genes or gene networks that may influence neuronal survival, differentiation, regeneration, and plasticity. That cholinergic neurons in brain regions subserving cognitive functions may be regulated not only by the neurotrophins but also by estrogen may have considerable relevance for the development and maintenance of neural substrates of cognition. If estrogen-neurotrophin interactions are important for survival of target neurons, then clinical conditions associated with estrogen deficiency could contribute to the atrophy or death of these neurons. These findings have implications for the subsequent decline in those differentiated neural functions associated with aging and Alzheimer disease.

Steroid Receptor Regulated Transcription of Specific Genes and Gene Networks

Steroid Receptor Regulated Transcription of Specific Genes and Gene Networks