Hormone Receptors and Genes Regulating Gonadal and mammary function

调节性腺和乳腺功能的激素受体和基因

• 批准号: 8553826
• 负责人: MARIA DUFAU
• 金额: $ 152.03万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553826
• 关键词: 5' Flanking Region; Acetylation; Address; Adjuvant Therapy; Androgens; Antithymoglobulin; Aromatase Inhibitors; Beryllium; Binding; Biogenesis; Biological Preservation; Boxing; Breast; Breast Cancer Cell; Cancer Patient; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Chromatin; Chromatin Structure; Code; Codon Nucleotides; Complex; Cytokine Receptors; Cytoplasm; DNA; DNA Methylation; DNA Polymerase II; Development; Distal; Elements; Endocrine; Epigenetic Process; Epithelium; Estradiol; Estrogens; Exons; Failure; Family; Functional RNA; Gametogenesis; Generic Drugs; Genes; Genetic Transcription; Germ Cells; Gonadal structure; Gonadotropins; Growth; Histone Deacetylase; Histones; Hormonal; Hormone Receptor; Human; Human Chorionic Gonadotropin; In Vitro; Individual; Investigation; LH Receptors; Laboratories; Lactation; Link; MCF7 cell; Mammary gland; Maps; Mediating; Meiosis; Messenger RNA; Methylation; MicroRNAs; Molecular; Molecular Profiling; Mus; Mutagenesis; Nuclear Orphan Receptor; Ovarian; Pathway Analysis; Pathway interactions; Pattern; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Polyribosomes; Population; Process; Production; Prolactin Receptor; Protein Biosynthesis; Protein Export Pathway; Protein Isoforms; Proteins; RNA Helicase; RNASE3L gene; Receptor Gene; Recruitment Activity; Regulation; Regulatory Pathway; Resistance; Role; SP1 gene; Second Messenger Systems; Signal Transduction; Site; Spermatids; Spermatogenesis; Spermiogenesis; Staging; Sterility; Steroids; Structure; Testis; Testosterone; Time; Transcription Factor TFIIB; Transcriptional Activation; Transcriptional Regulation; Transgenic Mice; Translating; Translations; autocrine; base; cofactor; demethylation; derepression; dimer; gonad function; helicase; human PRM2 protein; in vivo Model; insight; leydig interstitial cell; mRNA Expression; male; malignant breast neoplasm; member; novel; peptide hormone; prevent; promoter; protein expression; receptor; receptor expression; second messenger; sperm cell; steroidogenic acute regulatory protein

The Luteinizing Hormone Receptor (LHR): The LHR is expressed primarily in the gonads where it mediates LH signals that regulate ovarian and testicular function. The LHR gene transcription is regulated by complex and diverse networks, in which coordination and interactions between regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the promoter recruits histone (H) deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcription. Site specific acetylation/methylation-induced phosphatase release serves as an on switch for Sp1 phosphorylation at Ser641 that causes p107 repressor release from Sp1, recruitment of TFIIB and Pol II and transcriptional activation. Maximal derepression of the gene is dependent on DNA demethylation of the promoter, H3/H4 acetylation and HDAC/Sin3A release. Positive Cofactor 4 (PC4) has an important role in the formation/assembly of PIC in TSA-mediated LHR transcription. It is recruited by Sp1 following TSA treatment and acts as its coactivator. However, PC4 does not participate in TSA release of phosphatases, Sp1 phosphorylation or release repressor/complexes. Although TFIIB recruitment is dependent on PC4 we have ruled out TFIIB as its direct target and acetylation of PC4 in the activation process. However, TSA induced acetylation of a PC4 interacting proteins (16 kDa), identified as Acetylated H3 by MS, and its presence in the complex in association to chromatin at the promoter was demonstrated by ChiP/reChiP. The role of these interactions on chromatin structure and their participation in the assembly of the PIC and transcriptional activation are under investigation. Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25): GRTH is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ cells. It is a multi-functional protein essential for the completion of spermatogenesis. Males lacking GRTH are sterile due to the absence of sperm resulting from failure of round spermatids to elongate. In addition, to its intrinsic RNA helicase activity, GRTH is a shuttling protein that exports specific mRNAs from the nucleus to cytoplasmic sites Our studies have demonstrated the essential participation of the GRTH export/transport of mRNAs in the structural integrity of the Chromatoid Body (storage/processing of mRNAs) and their transit/association to actively translating polyribosomes where it may regulate translational initiation of genes. We have identified mRNAs which are associated with GRTH and regulated at polysomal sites of cell populations of the mouse testis. The reduction in mRNAs associated at polysomal sites in the differential studies (KO vs WT) not detected at total cellular level but in the cytoplasm with abolition of protein expression are reflective of the importance of the transport function of GRTH to relevant sites and underscore its impact in protein synthesis. This differential study has revealed messages regulated in different testicular cells associated with GRTH at polysomes (Geo #GSE38860). Network analysis has provided information about regulatory pathways that link to GRTH function, and fertile ground for exploration of GRTH regulation in the progress of spermatogenesis. In other studies we determine GRTH regulation of miRNAs in round spermatids. Differential expression profiles from WT and GRTH KO mice revealed a panel of miRNAs and their primary miRNAs significantly increased in KO (Geo#GSE 33969). The testis specific upregulated miRNA-469 repressed the translation of Transition Protein 2 (TP2) and Protamine 2 (Prm2) through binding to their mRNA coding region. This is consistent with the preservation of TP2 and Prm2 mRNA expression and failure of their protein expression in KO. Thus, GRTH has an important role in microRNA regulation acting as a negative regulator miRNA-469 biogenesis and expression levels of DROSHA/DGCR8 (mRNA/protein). miRNA-469 silencing of TP2 and Prm2 (chromatin remodelers) is essential for their timely translation at later stages of spermiogenesis, which is critical to attain mature sperm. GRTH is regulated by LH through androgen (A) at the transcriptional level in LCs (direct) and germ cells (presumably indirect) of the testis where its expression is both cell- and stage specific. This helicase displays a novel negative autocrine control of androgen production in LCs by preventing overstimulation of the LH-induced androgen pathway through enhanced degradation of StAR protein. Our initial studies using transgenic mice carrying sequential deletions of 5' flanking sequences define an A responsive l.4 kb region adjacent to ATG codon that contains an ARE half-site at -827. The transcriptional regulation/expression of GRTH by androgen (A) was studied using in vitro and in vivo models where its expression induced by A or hCG (via A) was inhibited by AR antagonist. Deletion mapping and mutagenesis demonstrated that the ARE was critical for DHT induced activation and recruited AR in a DHT dependent manner. ChiP analysis revealed recruitment of AR, steroid coactivator 1 (SRC-1), mediator1 (Med-1), TFII and Pol II to both the ARE and a region around the initiator sites at the promoter. ChiP-3C revealed short-range chromosomal looping between the distal AR/ARE and the core transcriptional machinery at the promoter. Med-1 and SRC-1-were not require for looping but these were essential for the activation of the complex. These findings have provided new insights on the molecular mechanism of AR-regulated transcription of the GRTH gene in LCs. Prolactin receptor (PRLR): The PRLR is a member of the lactogen/cytokine receptor family which mediates the diverse cellular actions of PRL. PRL is a major factor in the proliferation and differentiation of breast epithelium and is essential for lactation. It has been also implicated in the development of breast cancer, tumoral growth and chemoresistance. hPRLR expression is controlled at the transcriptional level by multiple promoters (one generic, PIII, and five human specific hPN1-hPN5) that were defined and characterized in our laboratory. Each promoter directs transcription/expression of a specific non-coding Exon 1 (E1-3, hEN1-hEN5), a common non-coding exon 2 and coding exons (E3-E11). The transcription of PRLR in breast cancer cells by the preferentially utilized PIII which lacks an estrogen responsive element is directed by Estradiol (E2)/ERa through complex formation with SP1 and C/EBPb that associate with cognate elements induces TFIIB and Pol II recruitment. BRET revealed ERa constitutive homodimers. E2-enhanced complex formation of the ERa dimer with SP1 and C/EBPb dimers at the PIII promoter has an essential role in the transcriptional activation of the hPRLR gene in breast cancer cells. In further studies we investigated the effect of E2 and Testosterone on the expression of various E1 isoforms (hE1-3, hEN1-hEN5) transcribed under the control their specific promoters in MCF-7 cells. Real-time PCR of exons 1 showed that E2 stimulated the expression of all exon 1 isoforms except hE1N2. This effect was prevented by ER antagonist. Exogenous testosterone showed comparable stimulation that was prevented by an aromatase inhibitor confirming the endogenous estrogen action in PRLR expression. Changes on PRLR protein expression and cell proliferation displayed similar stimulatory patterns. Current studies are addressing homologous effect of PRL on PRLR through individual promoters. Investigations on PRLR promoter usage and their regulation under hormonal treatment may provide insights into resistance states of breast cancer patients under adjuvant therapies.

黄体生成素受体 (LHR)：LHR 主要在性腺中表达，介导调节卵巢和睾丸功能的 LH 信号。 LHR 基因转录受到复杂多样的网络调节，其中调节效应器之间的协调和相互作用对于 LHR 表达的沉默/激活至关重要。启动子的近端 Sp1 位点募集组蛋白 (H) 脱乙酰酶和 Sin3A 辅阻遏物复合物，有助于 LHR 转录的沉默。位点特异性乙酰化/甲基化诱导的磷酸酶释放充当 Sp1 Ser641 磷酸化的开关，导致 Sp1 释放 p107 阻遏物，募集 TFIIB 和 Pol II 以及转录激活。该基因的最大去抑制取决于启动子的 DNA 去甲基化、H3/H4 乙酰化和 HDAC/Sin3A 释放。正辅因子 4 (PC4) 在 TSA 介导的 LHR 转录中的 PIC 形成/组装中发挥重要作用。 它在 TSA 处理后被 Sp1 招募并充当其共激活剂。 然而，PC4 不参与磷酸酶、Sp1 磷酸化或释放阻遏物/复合物的 TSA 释放。 尽管 TFIIB 募集依赖于 PC4，但我们排除了 TFIIB 作为其直接靶标以及在激活过程中 PC4 乙酰化的可能性。然而，TSA 诱导 PC4 相互作用蛋白 (16 kDa) 乙酰化，经 MS 鉴定为乙酰化 H3，并且 ChiP/reChiP 证明了其在启动子处与染色质相关的复合物中的存在。这些相互作用对染色质结构的作用及其参与 PIC 组装和转录激活的作用正在研究中。 促性腺激素调节的睾丸 RNA 解旋酶 (GRTH/DDX25)：GRTH 是睾丸特异性成员，属于睾丸间质细胞 (LC) 和减数分裂生殖细胞中存在的 RNA 解旋酶 DEAD-box 家族。 它是完成精子发生所必需的多功能蛋白质。缺乏 GRTH 的雄性是不育的，因为圆形精子细胞无法伸长，导致缺乏精子。此外，除了其内在的 RNA 解旋酶活性外，GRTH 是一种穿梭蛋白，可将特定的 mRNA 从细胞核输出到细胞质位点。我们的研究表明，GRTH 的 mRNA 输出/运输在类染色质体的结构完整性（存储/mRNA 的加工）及其与主动翻译多核糖体的转运/关联，它可以调节基因的翻译起始。我们已经鉴定出与 GRTH 相关并在小鼠睾丸细胞群的多核糖体位点上受到调节的 mRNA。在差异研究（KO vs WT）中，多核糖体位点相关 mRNA 的减少并未在总细胞水平上检测到，而是在细胞质中检测到，并消除了蛋白质表达，这反映了 GRTH 转运功能至相关位点的重要性，并强调了其影响在蛋白质合成中。这项差异研究揭示了不同睾丸细胞中与多核糖体 GRTH 相关的信息调节 (Geo #GSE38860)。网络分析提供了与 GRTH 功能相关的调控途径的信息，为探索精子发生过程中 GRTH 调控提供了沃土。在其他研究中，我们确定了圆形精子细胞中 miRNA 的 GRTH 调节。 WT 和 GRTH KO 小鼠的差异表达谱揭示了一组 miRNA 及其初级 miRNA 在 KO 中显着增加 (Geo#GSE 33969)。睾丸特异性上调的 miRNA-469 通过与 mRNA 编码区结合来抑制过渡蛋白 2 (TP2) 和鱼精蛋白 2 (Prm2) 的翻译。这与 TP2 和 Prm2 mRNA 表达的保留以及它们的蛋白表达在 KO 中的失败是一致的。因此，GRTH 作为负调节因子 miRNA-469 生物发生和 DROSHA/DGCR8（mRNA/蛋白）表达水平在 microRNA 调节中发挥着重要作用。 TP2 和 Prm2（染色质重塑蛋白）的 miRNA-469 沉默对于它们在精子发生后期的及时翻译至关重要，这对于获得成熟的精子至关重要。 GRTH 在睾丸的 LC（直接）和生殖细胞（可能是间接）的转录水平上由 LH 通过雄激素 (A) 进行调节，其表达具有细胞特异性和阶段特异性。该解旋酶通过增强 StAR 蛋白的降解来防止 LH 诱导的雄激素途径的过度刺激，从而对 LC 中雄激素的产生显示出一种新颖的负自分泌控制。我们的初步研究使用携带 5' 侧翼序列连续缺失的转基因小鼠，定义了与 ATG 密码子相邻的 A 响应性 l.4 kb 区域，该区域在 -827 处包含 ARE 半位点。 使用体外和体内模型研究了雄激素 (A) 对 GRTH 的转录调节/表达，其中由 A 或 hCG（通过 A）诱导的 GRTH 表达被 AR 拮抗剂抑制。缺失定位和诱变表明，ARE 对于 DHT 诱导的激活至关重要，并以 DHT 依赖性方式招募 AR。 ChiP 分析显示 AR、类固醇辅激活因子 1 (SRC-1)、介体 1 (Med-1)、TFII 和 Pol II 被招募到 ARE 和启动子起始位点周围的区域。 ChiP-3C揭示了远端AR/ARE和启动子处核心转录机制之间的短程染色体环。 Med-1 和 SRC-1- 不需要循环，但它们对于复合物的激活是必需的。这些发现为LCs中AR调节GRTH基因转录的分子机制提供了新的见解。 催乳素受体 (PRLR)：PRLR 是催乳素/细胞因子受体家族的成员，介导 PRL 的多种细胞作用。 PRL 是乳腺上皮增殖和分化的主要因素，对于哺乳至关重要。它还与乳腺癌的发生、肿瘤生长和化疗耐药有关。 hPRLR 表达在转录水平上由我们实验室定义和表征的多个启动子（一个通用启动子、PIII 和五个人类特异性 hPN1-hPN5）控制。每个启动子指导特定非编码外显子 1（E1-3、hEN1-hEN5）、常见非编码外显子 2 和编码外显子（E3-E11）的转录/表达。 PRLR 在乳腺癌细胞中的转录由优先利用的缺乏雌激素反应元件的 PIII 进行，由雌二醇 (E2)/ERa 通过与 SP1 和 C/EBPb 形成复合物来指导，SP1 和 C/EBPb 与同源元件相关，诱导 TFIIB 和 Pol II 募集。 BRET揭示了ERa组成型同二聚体。 ERa二聚体与PIII启动子处的SP1和C/EBPb二聚体的E2增强复合物形成在乳腺癌细胞中hPRLR基因的转录激活中具有重要作用。在进一步的研究中，我们研究了 E2 和睾酮对在 MCF-7 细胞中在其特定启动子控制下转录的各种 E1 亚型（hE1-3、hEN1-hEN5）表达的影响。外显子 1 的实时 PCR 显示，E2 刺激除 hE1N2 之外的所有外显子 1 亚型的表达。 ER 拮抗剂可以阻止这种作用。外源睾酮显示出类似的刺激作用，但芳香酶抑制剂可以阻止这种刺激作用，这证实了内源雌激素在 PRLR 表达中的作用。 PRLR 蛋白表达和细胞增殖的变化表现出相似的刺激模式。目前的研究正在解决 PR​​L 通过个体启动子对 PRLR 的同源效应。对 PRLR 启动子的使用及其在激素治疗下的调节的研究可能有助于了解乳腺癌患者在辅助治疗下的耐药状态。