Modifying SF-1 Transcriptional Programs by Sumoylation

描述（由申请方提供）：类固醇生成因子-1（SF-1）是一种组成型活性核受体，调节肾上腺功能和下丘脑-垂体-性腺轴的关键方面。该提案的目标是了解sumoylation，一种翻译后修饰，如何调节SF-1的体内功能。为此，我们的实验室已经产生了一个SUMO缺陷突变体（2KR）SF-1基因敲入小鼠（SF-1 2KR/2KR）。我们的初步研究揭示了纯合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SUMO缺陷型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，我建议调查两个问题，在体内SF-1类小泛素化：1）什么早期发育过程被颠覆的SF-1的2KR突变，可能会导致观察到的胚胎致死性？2)哪些基因和特定的启动子元件在早期发育中受到2KR SF-1突变的不适当调控？通过回答这些问题，我希望提供第一个在体内机制的见解，sumoylation如何调节转录活性。目的1是确定SF-1 2KR/2KR胚胎致死的发育阶段和原因。我们将进行定时交配，并通过苏木精和伊红（H&E）检查固定的胎盘和胚胎。将采用原位和免疫组织化学研究来监测滋养层分化并鉴定胎盘中SF-1表达的细胞谱系。这些研究有望揭示SF-1的新的发育作用。目的2是通过基因芯片分析鉴定SF-1中SUMO缺陷型2KR突变不适当调控的基因。本人将通过定量实时PCR验证已鉴定的SUMO依赖性SF-1靶基因，并通过免疫细胞化学测定确认直接靶点。由于我们对SF-1类小泛素化影响的反应元件的初步调查似乎是非典型的，低亲和力的网站，我将使用染色质免疫沉淀来确定精确的网站内的反应基因，使他们敏感的类小泛素化。目标2的工作将确定SF-1的发育基因靶点，并阐明类小泛素化修饰转录活性的机制。有超过20种已知的人SF-1突变导致肾上腺功能不全和/或性腺发育不全。对SUMO修饰敏感的SF-1靶基因的鉴定可以提供有关人SF-1中部分功能丧失突变如何介导完全功能丧失表型的见解。

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.