Modifying SF-1 Transcriptional Programs by Sumoylation

描述（由申请人提供）：甾体生成因子-1 （SF-1）是一种构成活性的核受体，调节肾上腺功能和下丘脑-垂体-性腺轴的关键方面。本研究的目的是了解翻译后修饰（sumoylation）如何调节SF-1的体内功能。为此，我们实验室培育了sumo缺陷突变体（2KR） SF-1敲入小鼠（SF-1 2KR/2KR）。我们的初步研究揭示了纯合子2KR突变体SF-1动物的胚胎致死表型（E9.5-13.5）。我们强烈怀疑这些sumo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突变的不适当调节。在此，我建议研究与体内SF-1聚合化有关的两个问题：1)SF-1中2KR突变破坏了哪些早期发育过程，从而可能导致观察到的胚胎致死？2)在发育早期，有哪些基因和特定的启动子元件受到2KR SF-1突变的不适当调控？通过回答这些问题，我希望提供第一个体内机制的见解如何sumo化调节转录活性。Aim 1的目的是确定SF-1 2KR/2KR胚胎致死性的发育阶段和原因。我们将进行定时配种，并用血红素和伊红（H&E）检查固定胎盘和胚胎。原位和免疫组织化学研究将用于监测滋养细胞分化和鉴定胎盘中SF-1表达的细胞系。这些研究有望揭示SF-1在发育中的新作用。Aim 2的目的是通过微阵列分析确定SF-1中sumo缺陷2KR突变不适当调控的基因。我将通过定量实时PCR验证鉴定的SUMO依赖性SF-1靶基因，并通过免疫细胞化学分析确认直接靶基因。由于我们对受SF-1 sumoylation影响的应答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应答基因中使它们对sumoylation敏感的精确位点。目标2所涉及的工作将确定SF-1的发育基因靶点，并解决sumoylation修饰转录活性的机制。有超过20种已知的人类SF-1突变导致肾上腺功能不全和/或性腺发育障碍。鉴定对sumo修饰敏感的SF-1靶基因可能有助于了解人类SF-1中部分功能缺失突变如何介导完全功能缺失表型。

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.