Milan K. Bagchi

Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor.

Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed.

Identification and Implantation Stage-Specific Expression of an Interferon-α-Regulated Gene in Human and Rat Endometrium1

Implantation of the developing blastocyst is regulated by multiple effectors, such as steroid hormones, growth factors, and cytokines. To understand how these diverse signaling pathways interact to modulate uterine gene expression, we employed a gene expression screen technique to identify the molecules that are induced in the periimplantation rat uterus. Here we report the isolation of a complementary DNA representing a novel gene, interferon-regulated gene 1 (IRG1). This gene exhibits significant homology to interferon (IFN)-α/β−inducible human genes p27 and 6–16, indicating that these genes may belong to the same family. Consistent with this finding, expression of IRG1 messenger RNA (mRNA) in rat uterus increased about 20-fold in response to IFNα. Uterine expression of IRG1 was also stimulated by estrogen and was partially inhibited by an antiestrogen, ICI 182,780. In pregnant rats, IRG1 expression was high on day 1, but declined on days 2 and 3. The level of IRG1 mRNA again rose transiently on day 4 i...

E1A-Mediated Repression of Progesterone Receptor-Dependent Transactivation Involves Inhibition of the Assembly of a Multisubunit Coactivation Complex

ABSTRACT The steroid hormone progesterone acts via high-affinity nuclear receptors that interact with specific DNA sequences located near the promoter of the hormone-responsive gene. Recent studies suggested that the hormone-occupied progesterone receptor (PR) mediates gene activation by recruiting a cellular coregulatory factor, termed coactivator, to the target promoter. The identity and mechanism of action of the coactivator(s) that regulates transcriptional activity of PR are currently under investigation. Here we provide evidence that the hormone-occupied PR forms a multisubunit receptor-coactivator complex containing two previously described coactivators, CREB-binding protein (CBP) and steroid receptor coactivator 1 (SRC-1, a member of the p160 family of coactivators), in nuclear extracts of human breast tumor T47D cells. The association of CBP and SRC-1/p160 with the receptor complex is entirely hormone dependent. Both CBP and SRC-1/p160 possess intrinsic histone acetyltransferase (HAT) activity, and it has been recently proposed that these coactivators function by modulating chromatin structure at the promoter of the target gene. Interestingly, addition of purified CBP to the nuclear extracts of T47D cells markedly stimulated progesterone- and PR-dependent transcription from a nucleosome-free, progesterone response element (PRE)-linked reporter DNA template. Furthermore, depletion of SRC-1/p160 by immunoprecipitation from these transcriptional extracts also significantly impaired PR-mediated RNA synthesis from a naked PRE-linked DNA template. These results strongly implied that CBP and SRC-1/p160 facilitate receptor-mediated transcription in these cell extracts through mechanisms other than chromatin remodeling. We also observed that the adenoviral oncoprotein E1A, which interacts directly with CBP, repressed PR-mediated transactivation when added to the nuclear extracts of T47D cells. Supplementation with purified CBP overcame this inhibition, indicating that the inhibitory effect of E1A is indeed due to a blockade of CBP function. Most importantly, we noted that binding of E1A to CBP prevented the assembly of a coactivation complex containing PR, CBP, and SRC-1/p160, presumably by disrupting the interaction between CBP and SRC-1/p160. These results strongly suggested that E1A repressed receptor-mediated transcription by blocking the formation or recruitment of coactivation complexes. Collectively, our results support the hypothesis that the assembly of a multisubunit coactivation complex containing PR, CBP, and SRC-1/p160 is a critical regulatory step during hormone-dependent gene activation by PR and that the fully assembled complex has the ability to control transcription through mechanisms that are independent of the histone-modifying activities of its component coactivators.

Cloning and Uterus/Oviduct-specific Expression of a Novel Estrogen-regulated Gene (ERG1)

The steroid hormone estrogen profoundly influences growth and differentiation programs in the reproductive tract of cycling and pregnant mamals. It is thought that estrogen exerts its cellular effects by regulating the expression of specific target genes. We utilized a messenger RNA differential display method to identify the genes whose expression is modulated by estrogen in the preimplantation rat uterus. Here we report the cloning of a novel gene (ERG1) that is tightly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that ERG1 mRNA is expressed in a highly stage-specific manner in the uterus and oviduct, and its expression is restricted to the surface epithelium of both of these tissues. Nucleotide sequence analysis of the full-length ERG1 cDNA indicates that it has an open reading frame of 1821 nuceotides encoding a putative protein of 607 amino acids with a single transmembrane domain and a short cytoplasmic tail. The extracellular part of the protein contains several distinct structural motifs. These include a zona pellucida binding domain, which is present in a number of proteins such as the zona pellucida sperm binding proteins, and uromodulin, In addition, there is a repeat of a motif called CUB domain, which exists in a number of genes involved in development and differentiation such as bone morphogenetic protein 1 (BMP1). Although the precise function of ERG1 eludes us presently, its unique pattern of expression in the uterus and oviduct and its regulation by estrogen, a principal reproductive hormone, lead us to speculate that this novel gene plays an important role in events during the reproductive cycle and early pregnancy.

Calcitonin Gene Expression in the Rat Uterus during Pregnancy

Publisher Summary The C cells of the thyroid gland are known as the major site of synthesis of calcitonin. This polypeptide hormone is released in response to hypercalcemia and mediates calcium homeostasis in both bone and kidney. This chapter presents a study that identifies the uterus as a novel site of calcitonin gene expression during preimplantation stages of pregnancy. The calcitonin synthesized in uterus acts in a paracrine or autocrine fashion to regulate calcium levels in certain uterine cells. Calcium controls many cellular processes, including muscle contraction, nerve conduction, and numerous enzymatic reactions. In the uterus, calcitonin regulates processes such as myocontraction and, therefore, plays a crucial role in embryonic implantation and maintenance of pregnancy. The antiprogestin RU486, which effectively blocks implantation and terminates pregnancy, also inhibits calcitonin expression. RU486-induced termination of pregnancy in preimplantation stage animals results from down regulation of certain genes, including calcitonin, which are primarily regulated by progesterone and may be crucial for implantation.

Progesterone Signaling in the Endometrium

Progesterone, acting through the progesterone receptor isoforms, PGR-A and PGR-B, is one of the most critical regulators of endometrial functions. During early pregnancy, PGR-regulated pathways control uterine epithelial receptivity and stromal differentiation, known as decidualization, which are crucial steps toward the establishment of pregnancy. The PGR isoforms control a substantially large cistrome and transcriptome during decidualization in the mouse and the human. Genetically engineered mouse models have established that several PGR-target genes, such as Ihh, Bmp2, Hoxa10, and Hand2 , are essential for implantation and decidualization. This article summarizes literature describing the key PGR-regulated pathways that govern critical uterine functions during the establishment of pregnancy.

Uterus: Growth Factors and Cytokines

Physiology of the mammalian uterus is maintained by a variety of cytokines and growth factors. Many of these factors are expressed in response to ovarian steroid hormones in uterine epithelial and stromal cells, and act in a paracrine fashion to maintain homeostasis of this tissue during the reproductive cycle. These factors are also responsible for preparing the uterine environment for successful establishment of pregnancy. Disruption of the uterine stromal–epithelial crosstalk involving cytokines and growth factors often results in altered uterine physiology, leading to pathological conditions or unsuccessful pregnancy outcomes. An in depth understanding of the role of these myriad cytokines and growth factors has helped to advance the understanding of mechanisms underlying uterine physiology and reproductive medicine.