Hormone Receptors and Enzymes Regulating Gonadal and Mam

LH受体：促黄体生成素受体（LHR）是一个由Sp1/Sp3驱动转录的TATAless基因。我们观察到调节LHR基因转录的沉默机制涉及组蛋白去乙酰化酶/mSin3A复合物与176 bp启动子的近端Sp1位点的关联。组蛋白乙酰化的能力？LHR基因启动子染色质变化介导的转录抑制表明表观遗传调控对LHR基因表达至关重要（Zhang Y., Dufau ML . J Biol Chem 277:33431, 2002）。随后的研究表征了JAR细胞中LHR基因启动子的甲基化状态，并探索了其与组蛋白修饰在调控LHR基因转录中的功能联系。启动子高度甲基化，TSA（组蛋白去乙酰化酶抑制剂）和AzaC （DNA去甲基化剂）在LHR转录激活中表现出明显的协同作用。这种效应定位于176 bp启动子，表明其DNA甲基化和染色质状态控制着LHR基因的表达。在TSA和AzaC的联合作用下，LHR基因启动子活性的最大降低是由于CpG岛（启动子/邻近3'编码域）的完全去甲基化，以及启动子区域组蛋白修饰的改变。组蛋白乙酰化和甲基化都有助于染色质结构的改变。这包括TSA或AzaC在第4赖氨酸处引起组蛋白H3甲基化，并在第9赖氨酸处进行去甲基化和乙酰化，以及TSA诱导的H4乙酰化。虽然H3和H4乙酰化和去甲基化可以单独在TSA存在时发生，但当LHR的转录比单独TSA增加2倍（TSA增加40倍；AzaC和TSA增加120倍）时，CpG岛的去甲基化需要两种药物的参与。LHR启动子中的去甲基化和组蛋白修饰的联合作用诱导了LHR基因表达更有利的染色质环境，抑制hdac /mSin3A复合物和DNMT1的解离以及RNA Pol II募集到启动子的增强。这些研究已经证明了一种连接DNA甲基化和组蛋白修饰以控制LHR基因表达的机制。

The LH receptor: The luteinizing hormone receptor(LHR)is expressed as a TATAless gene whose transcription is driven by Sp1/Sp3. We observed that a silencing mechanism regulating transcription of the LHR gene involved association of a histone deacetylase/mSin3A complex with the proximal Sp1 site of the 176 bp promoter. The ability of histone acetylation?mediated chromatin changes at the LHR gene promoter to cause derepression of transcription revealed that epigenetic regulation was critical for LHR gene expression (Zhang Y., Dufau ML J Biol Chem 277:33431, 2002). Subsequent studies have characterized the methylation status of the LHR gene promoter in JAR cells and explored its functional connection with histone modifications in the regulation of LHR gene transcription. The promoter is highly methylated, and TSA (histone deacetylase inhibitor) and AzaC (DNA demethylating agent)exhibit marked synergism in the activation of LHR transcription. This effect was localized to the 176 bp promoter, implying that its DNA methylation and chromatin status control LHR gene expression. Maximal derepression of LHR gene promoter activity by the combined effect of TSA and AzaC resulted from complete demethylation of a CpG island (promoter/adjacent 3' coding domains), and also from changes of histone modifications at the promoter region. Both histone acetylation and methylation contributed to the alteration of chromatin structure. These include methylation of histone H3 at Lys 4 caused by TSA or AzaC with demethylation and acetylation at Lys 9, and acetylation of H4 induced by TSA. Although H3 and H4 acetylation and demethylation can occur solely in presence TSA, demethylation of the CpG island requires the participation of both drugs when transcription of the LHR is increased by 2-fold over TSA alone (40-fold by TSA; 120-fold over basal by AzaC and TSA). The combined effects of demethylation, and histone modifications in the LHR promoter induced a more favorable chromatin environment for LHR gene expression, with dissociation of the repressive HDACs/mSin3A complex and DNMT1 and enhancement of RNA Pol II recruitment to the promoter. These studies have demonstrated a mechanism linking DNA methylation and histone modifications for the control of LHR gene expression. Control of testicular function: Spermatogenesis is a complex process that depends on the integrated expression of an array of genes that must operate in a precise temporal sequence to produce normal mature spermatozoa. Translation of stored mRNAs associated with protein (mRNP) in the cytoplasm of spermatids at specific times is essential for the completion of spermatogenesis. However, knowledge about the functional involvement of DEAD-box proteins in testicular germ cells is limited in mammals. We have discovered a novel gonadotropin-regulated testicular RNA helicase(GRTH/DdX25)that is a male-specific protein expressed in the rat, mouse and human testis in Leydig cells and germ cells (meiotic spermatocytes and round spermatids. GRTH is developmentally regulated, and is increased by gonadotropin and androgen at the transcriptional and translation levels. Recent studies have demonstrated that GRTH is present in the nucleus, cytoplasm and chromatoid body of germ cells, and is an integral component of mRNP particles. Male mice with a null mutation in the GRTH gene have normal gonadotropin and androgen profiles. However, they are sterile due to azoospermia caused by a complete arrest of spermiogenesis at step 8 of round spermatids, with failure to elongate. Electron microscopy studies on round spermatids of the null mice showed marked diminution in the size (by 90%) of chromatoid bodies (cytoplasmic organelles, viewed as scaffolds of storage of mRNP). The transcription of relevant messages was not altered, but their translation was abrogated in a selective manner. Protein expression of transition protein 1 and 2, and angiotensin-converting enzyme, was completely absent, whereas that of the transcriptional activator cAMP responsive modulator (CREM) was intact. Thus,the GRTH protein may serve as a master translational regulator of a selective panel or cascade of genes that are crucial for spermiogenesis. Although significant apoptosis was present at the metaphase of meiosis in the GRTH-null mice, spermatogenesis proceeded to step 8 of spermiogenesis, when complete arrest occurred. This progression may relate to compensatory gene functions and/or the observed up-regulation of DNA repair proteins Rad51 and Dmc1. From these studies we deduce that GRTH protein functions as a component of mRNP and/or may be required for the formation of chromatoid bodies. GRTH is important in the translation of crucial genes at specific times during spermatogenesis. It could also affect transport of poly(A)+ mRNA to the cytoplasm for storage in chromatoid bodies of spermatids, to be released for translation in a time-specific manner during spermiogenesis. Furthermore, GRTH associated with polyribosomes could influence the translation of genes. In summary, our studies have demonstrated that GRTH is essential for spermatid development (elongation) and completion of spermatogenesis, providing insights into intrinsic requirements for spermiogenesis, and has established a model for studies of male infertility and contraception. Prolactin receptors in human breast cancer: Prolactin acts through the long form of the receptor (LF) to cause differentiation of mammary epithelial cells through activation of the Jak2/Stat5 pathway and subsequent transcriptional events. Two novel short forms (SF) with abbreviated cytoplasmic domains (S1a, S1b)identified in this laboratory can inhibit the activation induced by prolactin through the long form. Current evidence strongly suggests that prolactin has a role in the development of human breast tumors, but the significance of the prolactin receptor variants in breast cancer is unknown. This year the first phase of an evaluation of the expression of PRL receptors in breast tumors and adjacent normal tissue, including the long receptor form (LF; stimulatory) and two SFs (S1a and S1B; inhibitory) has been completed. Southern analysis of breast cancer profiling arrays and quantification of hPRLR variants by real-time PCR in human normal and breast tumor matched tissues revealed a significant decrease in the ratio of SF to LF in the tumor tissue. There is no specific correlation in the change of hPRLR variant levels or SF/LF ratio with the type of breast tumor. Further evidence linking the low SF/LF to breast tumor is provided by their relative expression in normal versus mammary cancer cell lines of several origins. These finding in cells support the SF/LF findings in breast tumor tissues and provides useful information to select specific cells lines for PRLR variant-related projects. In summary, we have observed a general pattern of low ratio of the individual short forms to long form (SFs/LF) associated with both breast tumor tissues and cancerous cell lines when compared with normal samples. These observations provide an additional index for evaluation of human breast cancer. The decreased ratio of SF to LF in tumors suggests that a loss of the inhibitory regulation of SF to LF may accelerate abnormal cell proliferation and differentiation.