Decreased Recognition of SUMO-Sensitive Target Genes following Modification of SF-1 (NR5A1)

Abstract

SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), 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 DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibin␣; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibin␣ promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.Posttranslational modification with ubiquitin-like proteins has emerged as an important regulatory mechanism in many aspects of cellular function (29). Indeed, the small ubiquitinlike modifier (SUMO) conjugate modifies many transcription factors and results in marked transcriptional repression (27). Sumoylation occurs on lysines within consensus KxE sites through an enzymatic mechanism analogous to ubiquitination (6). While the requirement for an obligate SUMO E3 ligase is still debated, several proteins exhibit SUMO E3 ligase activity in cells, with the largest group belonging to the protein inhibitors of activated STATs (PIAS) family (47). Sumoylation is easily reversible by the action of SUMO isopeptidases (SUSPs or SENPs), with seven members identified in humans thus far (40).While recent structural studies have helped to elucidate the enzymatic details of SUMO conjugation and substrate recognition (44, 45), the mechanisms underlying transcriptional repression by sumoylation are less clear. Identification of a SUMO-interacting motif found in the PML protein and transcriptional repressors such as Daxx has helped to establish how the SUMO conjugate might function as a passive molecular mark to attenuate gene expression (28,39). However, it is also plausible that the SUMO conjugate functions as an active mark to modify either protein-protein or protein-DNA interactions. Evidence for this latter hypothesis remains controversial. For instance, sumoylation of thymine-...

Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

“…SF-1 is SUMOylated at two conserved lysines, Lys119 and Lys194, which reside adjacent to the DBD and LBD, respectively, and this modification clearly represses SF-1 transactivation (5,22,26,67). This was attributed to the SUMOylation on Lys194, which was shown to reduce Ser203 phosphorylation, decrease coregulator binding, and cause a selective loss of binding to certain target genes (5,67). In contrast, we found that SF-1 SUMOylation was not affected by S203 phosphorylation and that SUMOylation on Lys194 is a prerequisite for SUMOylation on Lys119, which has been confirmed in other reports (22,69).…”

“…We have demonstrated that ubiquitin targets SF-1 on Lys119, which is also targeted by SUMO. SF-1 is SUMOylated at two conserved lysines, Lys119 and Lys194, which reside adjacent to the DBD and LBD, respectively, and this modification clearly represses SF-1 transactivation (5,22,26,67). This was attributed to the SUMOylation on Lys194, which was shown to reduce Ser203 phosphorylation, decrease coregulator binding, and cause a selective loss of binding to certain target genes (5,67).…”

“…However, SF-1 is subject to additional posttranslational modifications which alter its activity, and it can be SUMOylated at two conserved lysines, Lys119 and Lys194, which are adjacent to the DNA-binding domain (DBD) and LBD, respectively. SUMOylation at Lys194 was seen to inhibit Ser203 phosphorylation and was also associated with reduced transactivation of various SF-1 target genes (5,67). In gonadotropes, SF-1 was seen also to be ubiquitinated, and this form was associated with the LH␤ gene promoter (60).…”

Coactivation of SF-1-Mediated Transcription of Steroidogenic Enzymes by Ubc9 and PIAS1

“…Furthermore, a recent study identified androgen receptor interacting protein 4, a member of the Rad54 chromatin remodeling family, as a factor that interacts with sumoylated SF-1, and androgen receptor interacting protein 4 may be a cofactor that modulates SUMO-mediated fine-tuning of transcriptional repression (28). In contrast, sumoylation of SF-1 was shown to result in coordinated protein modification, such as reducing CDK7-mediated serine 203 phosphorylation (47) and reduction of DNA binding activity of SF-1 (26).…”

Novel Insights into 46,XY Disorders of Sex Development due to <b><i>NR5A1</i></b> Gene Mutation

“…Electrophoretic mobility shift assays (EMSA) were performed using the Light Shift Chemiluminescent EMSA Kit according to the recommendations of the manufacturer (Thermo Scientific). A 5 ′ -biotin-labelled oligonucleotide comprising an SF-1-responsive element of the mouse Amh promoter as well as mutated oligonucleotides of this motif previously described by Campbell et al [2008] were used. Twenty nanograms of 5 ′ -biotin-labelled probe were incubated with nuclear extracts of transfected HeLa cells with or without either a 200-fold excess of unlabelled target DNA or unlabelled mutated target DNA (see fig.…”