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Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

ABSTRACT: 20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5'-flanking region of the mouse 20alpha-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5'-flanking region, revealed that the region between -83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wild-type Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20alpha-HSD gene.

SUBMITTER: Hirabayashi K

PROVIDER: S-EPMC1133973 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

Hirabayashi Keiji K Ishida Maho M Suzuki Masatoshi M Yamanouchi Keitaro K Nishihara Masugi M

The Biochemical journal 20040901 Pt 3

20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5'-flanking region of the mouse 20alpha-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of t ...[more]

PMID: 15222881

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Project description:Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5'-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5'-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5'-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (-540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5'-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the -462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5'-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.

Dataset Information

Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

Publications

Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

Similar Datasets

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