Sumoylation and its regulation in testicular Sertoli cells

睾丸支持细胞的苏酰化及其调控

• 批准号: 10654204
• 负责人: Margarita Vigodner
• 金额: $ 45万
• 依托单位: YESHIVA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-05 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654204
• 关键词: Address; Adult; Affect; Apoptotic; Authorship; Binding; Biological Process; Breeding; Cell Differentiation process; Cell Line; Cell physiology; Cells; Cellular biology; Chromatin; Collaborations; Complex; Computer Analysis; Couples; Data; Defect; Development; Developmental Biology; Developmental Process; Down-Regulation; Embryo; Enzymes; Equipment; Failure; Generations; Genes; Genetic Transcription; Genotype; Germ Cells; Healthcare; Human; Infertility; Knock-out; Laboratories; Learning; Ligase; Male Infertility; Mediating; Methods; Microscopy; Modeling; Modification; Molecular; Morphology; Mus; Mutate; Paper; Pathway interactions; Peer Review; Phenotype; Proliferating; Promoter Regions; Proteins; Proteome; Regulation; Reproduction; Research; Role; SUMO1 gene; Seminiferous tubule structure; Signal Transduction; Somatic Cell; Spermatogenesis; Stress; Sumoylation Pathway; TRIM Gene; Techniques; Testis; Tissues; Training; Ubiquitin; Western Blotting; Work; differential expression; experience; granulosa cell; improved; in vivo; male; meetings; mouse model; mutant; novel; promoter; response; sertoli cell; sex; subfertility; transcription factor; transcriptome sequencing; trying to conceive; ubiquitin-protein ligase; undergraduate student

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.

支持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。 表征了SUMO蛋白是正常发育所必需的，并在哺乳动物中表达。 小鼠和人类支持细胞;然而，在这些细胞中，类小泛素化的细胞特异性作用， 才刚刚开始被阐明在颗粒细胞中，SUMO化由E3-SUMO调节， 连接酶KAP 1/TRIM 28是颗粒细胞分化所必需的。TRIM 28表达于 小鼠和人类支持细胞，其失活导致生育力低下和睾丸发育不全。 我们已经证明TRIM 28与SUMO在Sertoli细胞中相互作用并共定位，并且TRIM 28在Sertoli细胞中与SUMO相互作用。 下调导致Sertoli衍生细胞系中类小泛素化蛋白的减少， 表明TRIM 28的E3-SUMO连接酶活性。分析显示， 睾丸大小减少，曲细精管紊乱，生殖细胞脱落，所有 其中的缺陷类似于在Sertoli中TRIM 28条件性失活后所见的缺陷 细胞然而，导致这两种模型中这些缺陷的分子变化尚不清楚。 SUMO化是已知的调节许多转录因子（TF），包括颗粒细胞中的那些 细胞因此，我们假设一些Sertoli TF，当未被sumoylated时， 募集到其特定基因启动子，影响细胞的转录命运，和 有助于观察到的表型。因此，为了更好地表征TRIM 28依赖性 和非依赖性的SUMO化，并了解其机制。 分子的变化，我们建议追求以下具体目标。1)比较 睾丸支持细胞TRIM 28依赖性类小泛素化的失活和类小泛素化的整体失活 细胞详细的形态学和标记物分析将评估增殖、分化和细胞周期。 支持细胞的连接复合体的形成，并确定的确切阶段， 生精缺陷2)为了鉴定受TRIM 28调控的转录因子（TF）， 依赖性类小泛素化，当错误调节时，有助于获得表型。这些 研究将鉴定与对照组和TRIM 28相关的SUMO启动子区域， 缺陷型Sertoli细胞和受TRIM 28依赖性类小泛素化调节的基因。 拟议项目的一个重要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， 实践各种实验室技术。