Sumoylation and its regulation in testicular Sertoli cells

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The molecular regulation of Sertoli cells and their crosstalk with germ cells has not been fully characterized. SUMO proteins are essential for normal development and are expressed in mouse and human Sertoli cells; However, the cell-specific role of sumoylation in those cells has only started to be elucidated. In granulosa cells, sumoylation is regulated by the E3-SUMO ligase KAP1/TRIM28 which is required for granulosa cell differentiation. TRIM28 is expressed in mouse and human Sertoli cells, and its inactivation causes subfertility and testicular hypoplasia. We have shown that TRIM28 interacts and co-localizes with SUMO in Sertoli cells and that its down-regulation causes a decrease in sumoylated proteins in a Sertoli-derived cell line, suggesting the E3-SUMO ligase activity of TRIM28. The analysis revealed a significant reduction in testes size, disorganized seminiferous tubules, and shedding of the germ cells, all of which are defects similar to these seen after the conditional inactivation of TRIM28 in Sertoli cells. However, the molecular changes leading to these defects in both models are not known. Sumoylation is known to regulate many transcription factors (TFs), including those in granulosa cells. Therefore, we hypothesize that some of the Sertoli TFs, when unsumoylated, are not recruited to their specific gene promotors, affecting the transcriptional fate of the cells, and contributing to the observed phenotype. Therefore, to better characterize TRIM28-dependent and -independent sumoylation in Sertoli cells and to understand the mechanism of the molecular changes, we propose to pursue the following specific aims. 1) To compare inactivation of TRIM 28-dependent sumoylation with global inactivation of sumoylation in Sertoli cells. Detailed morphological and marker analysis will assess proliferation, differentiation, and junction complex formation of the Sertoli cells, and determine the exact stages of the spermatogenic defects. 2) To identify transcription factors (TFs) regulated by TRIM28- dependent sumoylation that, when mis-regulated, contributing to the obtained phenotype. These studies will identify both the promoter regions associated with SUMO in control versus TRIM28- deficient Sertoli cells and the genes regulated by TRIM28-dependent sumoylation. A significant part of the proposed project will be conducted by undergraduate students who will have the opportunity to use state-of-the-art equipment, learn about hypothesis generation, and practice various laboratory techniques.