Expression Analysis Of MTA1 And Preliminary Investigation Of Its Function During Spermatogenesis

The rat metastasis associated gene 1 (mta1) was originally identified by differential cDNA screening using adenocarcinoma cell lines with high metastatic potential by Yasushi Toh in 1994. The molecule was named because of its strong correlation with the metastasis process of malignancies. To date, there are three different known genes (MTA1, MTA2, and MTA3) and six reported isoforms (MTA1, MTA1s, MTA1-ZG29p, MTA2, MTA3, MTA3L). MTA1, MTA2, and MTA3 are components of the nucleosome remodeling and deacetylation complex, which is associated with adenosine triphosphate-dependent chromatin remodeling and transcriptional regulation. MTA proteins, as a part of the NuRD complex (nuclear remodeling and deacetylation complex), are thought to modulate transcription by influencing the status of chromatin remodeling.Subsequent studies found a widespread upregulation of MTA1 in human tumors. In spite of a strong correlation between MTA1 upregulation and cancer, the molecular function of the MTA family remained a mystery until proteomic analyses of the NuRD complex identified MTA1 and MTA2 as integral subunits, thus providing essential clues to the chromatin modifying roles of MTA proteins. Analysis of the primary structure of MTA proteins shows that human MTA2 and MTA3 are 68% and 73% identical to human MTA1, respectively, with the highest homology concentrated in the N-terminal half of the proteins. In contrast, the C-terminal halfs of MTA proteins are divergent. The MTA proteins (with the exception of the ZG29p variant) contain one BAH domain, one ELM domain, and one SANT domain that is similar to the DNA binding domain of myb-related proteins. Our understanding of the functional significance of these domains in MTA family is derived from studies of other proteins containing such domains. For example, the BAH domain is involved in protein-protein interactions, whereas the SANT domain binds to histone tails. Also, all MTA family members contain a GATA zinc-finger DNA binding domain; MTA1 contains two bipartite nuclear localization signals and one basic amino acid–rich nuclear localization signal.In addition, the MTA1 complex contains histone deacetylases (HDAC1/2), RbAp46/48, MBD3 and could be further separated, resulting in a core MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity are integral properties of the MTA1 complex.Prof.Wang, a member of our group, has made markable progress on the research of MTA1.He reported that the stimulation of cells with HRG, a ligand of epidermal growth factor receptor (EGFR), is accompanied by suppression of histone acetylation and enhancement of deacetylase activity, and then blocks the ability of oestradiol to stimulate ER-mediated transcription.Besides, estrogen and c-myc could both upregulate the expression of MTA1, resulting in an enhancement of the metastasis. At present, the physiological relevance of MTA1 has not been elucidated. However, quantitative analysis revealed that it could also be expressed in several normal tissues, indicating that its potential role, yet poorly characterized, may take place. For example, forced overexpression of MTA1 in the mouse mammary gland epithelium was accompanied by extensive ductal branching and proliferation in virgin glands, which was mechanistically linked to an altered ratio of progesterone receptor A and B isoforms. It should be noted that MTA1 expression level is generally low in most tissues except mammalian gland and testis. Thus, a more important regulatory role during organgenesis in these two kinds of tissues comes to the fore.DNA is packaged into chromatin in eukaryotes. Formation of closed chromatin precludes the access of transcription factors to DNA and inevitably leads to transcriptional repression. It is dynamic changes in the chromatin structure that, therefore, lead to either transcriptional activation or transcriptional repression. Alteration of the chromatin structure utilizing energy derived from ATP hydrolysis is called "chromatin remodeling" and is one of the two transcriptional regulatory mechanisms at the chromatin level. The other mechanism involves covalent modifications of nucleosomes, the basic units of chromatin. The N termini of core histones are exposed and unstructured; these exposed tails are thought to contact neighboring core histones as well as DNA and are critical in mediating higher order structures of chromatin. As a result, covalent modifications of nucleosomes also lead to changes in the chromatin structure. One of the most well studied nucleosomal modifications is acetylation and deacetylation of histone proteins at the unstructured N-terminal tails. Acetylation of histone proteins opens up the chromatin structure and leads to transcriptional activation. Conversely, deacetylation of histone proteins condenses the chromatin structure and is associated with transcriptional repression. Interestingly, the MTA1 complex contains histone deacetylases (HDAC1/2) and can be further separated, resulting in a core MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity are integral properties of the MTA1 complex.Overall, it could be concluded that MTA1 might play an important role during the regulation of spermatogenesis and the further proposed analysis would enable us to characterize in detail the developmental regulation of Mta1 expression in mammalian testis, thus paving the way for a better understanding of the potential physiological role of this newly discovered molecule. The present work consists of five parts:Part1 Immunolocalization of MTA1 in human testisPart2 Expression analysis of MTA1 during the development of mouse testisPart3 Expression analysis of MTA1 in the isolated germ cellsPart4 Expression analysis of MTA1 in the pachytene-spermatocyte-deleted mouse testisPart5 Specific removal of Mta1 and subsequent investigation of its role during mouse spermatogenesis by RNA interference.Taken together, our observations revealed that Mta1 protein accumulation during spermatogenesis occurs specifically in primary spermatocytes and spermatogonia and spermatocyte-Sertoli cell interactions; the most intense immnoreactant was located in pachytene spermatocytes, suggesting that the protein might have a more important role to play in the meiotic stage of spermatogenesis.Finally, the pSUPER RNAi plasmid, which has successfully constructed, may facilitate the following study the physiological aspects of Mta1.