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.
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