Modulating Nuclear Receptor Activity via Sumoylation

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DESCRIPTION (provided by applicant): Modulating Nuclear Receptor Activity via Sumoylation Steroidogenic factor-1 (SF-1) is a constitutively active nuclear receptor that regulates critical aspects of adrenal function and the hypothalamic-pituitary-gonadal axis. SUMO modification of nuclear receptors including SF-1 and other NR5A receptors is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines Lys119 and Lys194 that reside adjacent to the DBD and LBD, respectively. Surprisingly, while previous loss-of-function studies in cells predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein were identical to non-sumoylated protein; only a decrease in Ser203 phosphorylation was noted. By contrast, a selective loss of DNA binding at non-canonical SF-1 targets was observed using DBD protein sumoylated at Lys119. Moreover, regulating SF-1 function by Lys119 sumoylation must occur prior to DNA binding. We propose that sumoylation of transcription factors is an active posttranslational mark that eliminates recognition of "SUMO-sensitive" target genes and restrains gene expression. While these molecular and biochemical studies suggest that sumoylation restrains the transcriptional output of SF-1, this hypothesis needs to be tested in vivo. To this end, a SUMO-deficient mutant knock-in mouse was created in the SF-1 allele, referred to as SF-12KR/2KR. This mutant mouse exhibits embryonic lethality at mid-gestation (E9.5-13.5), which is surprising considering that SF-1 null mice die postnatally due to adrenal insufficiency. Here we will test the hypothesis that normal transcriptional programs are inappropriately regulated in a sumo-deficient SF-1 mutant using whole animal, genomic, and structural approaches. This competitive renewal will address three remaining questions as to the role of SF-1 sumoylation: 1) What developmental or hormonal pathways are subverted in SF-12KR/2KR mice that lead to mid-gestation lethality. 2) What specific elements and genes are inappropriately regulated by the 2KR SF-1 mutation in the placenta and other endocrine tissues? 3) What are the structural consequences of SF-1 sumoylation on the DNA binding domain. Answers to these questions should provide in vivo and structural mechanistic insights as to how sumoylation modulates gene expression. PUBLIC HEALTH RELEVANCE: Post-translational modifications regulate the activity of many types of cellular proteins. Sumoylation of proteins involves protein conjugation and in general dampens the activity of many transcriptional regulators. In this study we will assess for the first time, how a sumo-deficient mutation of the nuclear receptor affects endocrine tissue development and function. Results from this work will potentially identify important new gene targets that are especially sensitive to the relative pools of sumoylated protein. They will also provide mechanistic insight into how sumoylation restrains and "fine tunes" transcriptional programs of the endocrine system.