Sumoylation and its regulation in testicular Sertoli cells

Sertoli细胞的分子调控及其与生殖细胞的相互作用尚未完全阐明 特色化的。相扑蛋白是正常发育所必需的，在 小鼠和人的支持细胞；然而，在这些细胞中苏莫化的细胞特异性作用 只是刚刚开始被阐明。在颗粒细胞中，和甲基化受E3-SUMO的调节。 颗粒细胞分化所需的连接酶KAP1/TRIM28。TRIM28表示为 小鼠和人类的Sertoli细胞，其失活会导致生育不足和睾丸发育不良。 我们已经证明TRIM28在Sertoli细胞中与相扑相互作用并共定位，并且它的 下调导致Sertoli来源的细胞系中总和蛋白的减少， 提示TRIM28具有E3-相扑连接酶活性。这项分析揭示了显著的 睾丸体积缩小，曲细精管结构紊乱，生殖细胞脱落，这些都是 其中的缺陷类似于在Sertoli中TRIM28条件失活后看到的缺陷 细胞。然而，在这两个模型中，导致这些缺陷的分子变化尚不清楚。 已知相思甲基化可以调节许多转录因子，包括颗粒细胞中的转录因子。 细胞。因此，我们假设，一些Sertoli TF，当未被求和时，不是 被招募到它们特定的基因启动子，影响细胞的转录命运，以及 对观察到的表型有贡献。因此，为了更好地表征TRIM28依赖 和不依赖于Sertoli细胞的总和甲基化，并了解其机制 对于分子变化，我们建议追求以下具体目标。1)比较 Sertoli中TRIM 28依赖的总合甲基化失活与总合甲基化失活 细胞。详细的形态和标记分析将评估细胞的增殖、分化和 Sertoli细胞的连接复合体的形成，并确定 生精缺陷。2)鉴定受TRIM28-调控的转录因子(TF) 依赖的和甲基化，当被错误调节时，导致获得的表型。这些 研究将确定与控制相扑相关的启动子区域与TRIM28- 支持细胞缺陷及其依赖于TRIM28的总甲基化调控基因。 拟议项目的很大一部分将由本科生进行，他们将 有机会使用最先进的设备，了解假设生成，以及 练习各种实验室技术。

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.