YY1 binding within the human HSD3B2 gene intron 1 is required for maximal basal promoter activity: identification of YY1 as the 3beta1-A factor

Abstract

The oxidation and isomerization of 3 -hydroxy-5-ene steroids into keto-4-ene steroids, a pivotal step in the synthesis of all hormonal steroids, is catalyzed by several isoforms of 3 -hydroxysteroid dehydrogenase. In humans, two highly homologous isoforms exist, type I expressed by the HSD3B1 gene in peripheral tissues, and type II expressed by the HSD3B2 gene in steroidogenic organs. Previously, it was shown that the HSD3B1 gene 3 1-A element, encompassing 24 nucleotides of intron 1 not perfectly conserved between the two genes and overlapping with a conserved TG box, contributes to maximal basal promoter activity by binding the ubiquitous and unidentified 3 1-A transcription factor. In this study for the first time we report that similarly, the HSD3B2 gene intron 1 is required for maximal basal promoter activity in reporter gene analyses, as lack of intron 1 results in a 4-to 10-fold reduction in promoter activity. Mutational analysis in gel shift assays revealed that the 3 1-A factor binds both the HSD3B2 and HSD3B1 gene intron 1 by requiring only seven nucleotides of a conserved segment within the 3 1-A element. By competition analysis and use of anti-YY1 antibody in both gel shift and Western blot experiments, we identified the 3 1-A protein as the ubiquitous transcription factor YY1. In addition, we have characterized another similar YY1 binding site differently located with respect to the 3 1-A element in both genes. Deletion and mutational analysis in transient transfections experiments revealed that contrarily to as previously shown for the HSD3B1 gene, lack of YY1 binding to the type II 3 1-A element only results in a marginal reduction of basal promoter activity. Instead, YY1 binding to the second site, placed 35 bp downstream from the 3 1-A element, strongly activates the HSD3B2 gene basal promoter activity, as preventing YY1 binding to this region caused a 50% decrease of basal transcription. Complete abrogation of YY1 binding within type II intron 1 resulted in a gene reporter activity identical to a reporter construct lacking the whole intron 1. These results designate YY1 as the factor responsible for the intron 1-mediated boost of the HSD3B2 gene basal promoter activity. Similarities and dissimilarities between YY1 binding within the HSD3B1 and HSD3B2 gene intron 1 are discussed involving the conserved intron 1 TG box, that suggests different mechanisms are implicated in the YY1-mediated stimulation of these two genes basal promoter activity.

Yin Yang 1 promotes hepatic steatosis through repression of farnesoid X receptor in obese mice

“…We further found that YY1 suppressed FXR expression via interaction with the YY1 binding site in the first intron of the FXR gene. Similar regulatory models were also reported previously regarding CD21 and HSD3B2 expression regulated by YY1 29 30…”

Pioglitazone Inhibits Androgen Production in NCI-H295R Cells by Regulating Gene Expression of CYP17 and HSD3B2

“…Because artificial "promoterreporter" assays allow the use of chosen parts of promoters only, essential elements, which are modulated by pioglitazone, may be missed. Recently, a specific intronic sequence of the HSD3B gene has been reported to be important for basic promoter activity of both HSD3B2 and HSD3B1 genes (Foti and Reichardt, 2004), corroborating our hypothesis. Alternatively, pioglitazone might affect the stability of HSD3B2 mRNA.…”

Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression

“…YY1 has been implicated in the regulation of a large number of mammalian genes [41]. Moreover, YY1 was also found to affect gene expression by interaction with binding motifs positioned downstream of the transcriptional start site, within the coding region [42] or the intron sequences of target genes [43], [44].…”