Modifying SF-1 Transcriptional Programs by Sumoylation

描述(申请人提供)：类固醇生成因子-1(SF-1)是一种结构性活性核受体，调节肾上腺功能和下丘脑-垂体-性腺轴的关键方面。这项建议的目的是了解翻译后修饰的SUMO化如何调节SF-1的体内功能。为此，我们实验室培育了相扑缺陷突变(2KR)SF-1敲入小鼠(SF-12 2KR/2KR)。我们的初步研究揭示了纯合子2KR突变SF-1动物的胚胎致死表型(E9.5-13.5)。我们强烈怀疑这些相扑缺陷的SF-1突变小鼠死于胎盘缺陷，并假设胎盘中的正常转录程序受到2KR SF-1突变的不适当调控。在此，我建议研究两个与体内SF-1苏莫化有关的问题：1)SF-1的2KR突变会颠覆哪些早期发育过程，从而可能导致观察到的胚胎死亡？2)在早期发育过程中，哪些基因和特定的启动子元件会受到2KR SF-1突变的不适当调控？通过回答这些问题，我希望为苏莫化如何调节转录活性提供第一个活体机制方面的见解。目标1的目的是确定SF-1 2KR/2KR胚胎致死的发育阶段和原因。我们将进行定时配对，并用血毒素和曙红(H&E)检查固定的胎盘和胚胎。将利用原位和免疫组织化学研究来监测滋养层细胞的分化，并鉴定胎盘中SF-1表达的细胞谱系。这些研究有望揭示SF-1的一个新的发育作用。目标2的目的是通过微阵列分析来识别SF-1中受相扑缺陷2KR突变不适当调控的基因。我将用实时定量聚合酶链式反应验证已鉴定的相扑依赖的SF-1靶基因，并用免疫细胞化学方法确认直接靶基因。由于我们对SF-1相加作用影响的反应元件的初步调查似乎是非规范的、低亲和力的位点，我将使用染色质免疫沉淀来确定使它们对相加作用敏感的反应基因内的精确位点。目标2中的工作将确定SF-1的发育基因靶点，并解决苏莫化修饰转录活性的机制。已知的人类SF-1基因突变有20多种，可导致肾上腺功能不全和/或性腺发育不全。识别对相扑修饰敏感的SF-1靶基因可能有助于深入了解人类SF-1部分功能缺失突变是如何介导完全功能丧失表型的。

DESCRIPTION (provided by applicant): Steroidogenic Factor-1 (SF-1) is a constitutively active nuclear receptor that regulates critical aspects of adrenal function and the hypothalamic-pituitary-gonadal axis. The goal of this proposal is to understand how sumoylation, a post-translation modification, regulates the in vivo function of SF-1. To this end, our laboratory has generated a SUMO-deficient mutant (2KR) SF-1 knock-in mouse (SF-1 2KR/2KR). Our preliminary studies reveal an embryonic lethal phenotype (E9.5-13.5) for homozygous 2KR mutant SF-1 animals. We strongly suspect that these SUMO-deficient SF-1 mutant mice die from a placental defect, and also hypothesize that the normal transcriptional programs in the placenta are inappropriately regulated by the 2KR SF-1 mutation. Herein, I propose to investigate two questions relating to in vivo SF-1 sumoylation: 1) What early developmental processes are subverted by the 2KR mutation in SF-1 that might lead to the observed embryonic lethality? 2) What genes and specific promoter elements are inappropriately regulated by the 2KR SF-1 mutation in early development? By answering these questions, I hope to provide the first in vivo mechanistic insight to how sumoylation regulates transcriptional activity. The goal of Aim 1 is to determine the developmental stage and cause of SF-1 2KR/2KR embryonic lethality. We will conduct timed matings and examine fixed placenta and embryos by haemotoxylin and eosin (H&E). In situ and immunohistochemistry studies will be employed to monitor trophoblast differentiation and identify the cell lineage of SF-1 expression in the placenta. These studies are expected to reveal a novel developmental role of SF-1. The goal of Aim 2 is to identify genes inappropriately regulated by SUMO-Deficient 2KR mutation in SF-1 by microarray analysis. I will validate the identified SUMO- dependent SF-1 target genes by Quantitative Real-Time PCR and confirm direct targets by immunocytochemistry assays. Because our preliminary survey of response elements affected by SF-1 sumoylation appear to be noncanonical, low-affinity sites, I will use chromatin Immunoprecipitation to define the precise sites within the responsive genes that sensitize them to sumoylation. The work entailed in aim 2 will identify developmental gene targets of SF-1 and address the mechanism by which sumoylation modifies transcription activity. There are over 20 known human SF-1 mutations that cause adrenal insufficiency and/or gonadal dysgenesis. Identification of SF-1 target genes sensitive to SUMO-modification may provide insight as to how partial loss-of function mutations in human SF-1 mediate a complete loss of function phenotype.