SRY-Box Transcription Factor 9 regulates 3’-Phosphoadenosine 5’-Phosphosulfate Synthase 2 mRNA expression through derepression of the transcriptional repressor, CCAAT/enhancer-binding protein beta

Previously, we showed that Papss2 expression is regulated through a Sox9-dependent pathway. Here we explore molecular mechanisms whereby Sox9 regulates mouse Papss2 . A 509bp Sox9-responsive DNA element (Region C) was identified upstream of the Papss2 second start site using co-transfection and luciferase reporter assays. A Sox9 responsive element was narrowed down to 32bps within Region C (Sox9RE). Putative SoxE and C/EBPβ binding sites were identified within S9RE. C/EBPβ was identified as a repressor for Sox9-mediated activity. In cells transfected with expression vectors for C/EBPβ and Sox9, increasing amounts of C/EBPβ resulted in attenuation of Sox9-mediated activation of Region C while increasing amounts of Sox9 activated transcription in the presence of C/EBPβ. Using electromobility shift assays, three protein complexes were identified on S9RE after incubation with nuclear extracts from ATDC5 cells. Super shift assays indicated that under basal conditions C/EBPβ was present in the DNA-protein complexes observed. Unlabeled S9RE with point mutations in the predicted SoxE binding site competed with protein complex formation on the S9RE while excess oligo corresponding to the predicted SoxE binding site did not, suggesting that proteins do not bind to SoxE motiff under basal conditions. Under conditions of high Sox9 expression, the formation of protein-DNA complexes on S9RE was inhibited. We then showed by western blot that increasing Sox9 protein resulted in reduced C/EBPβ protein levels. Co-immunoprecipitation indicated interaction of Sox9 and C/EBPβ proteins. We propose that Sox9 acts to derepress C/EBPβ-inhibited transcription of Papss2 by first interacting with C/EBPβ to prevent it from binding DNA, then reducing C/EBPβ expression.