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. ? ? ?
|Lee, Florence Y; Faivre, Emily J; Suzawa, Miyuki et al. (2011) Eliminating SF-1 (NR5A1) sumoylation in vivo results in ectopic hedgehog signaling and disruption of endocrine development. Dev Cell 21:315-27|