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Related Experiment Videos

3 alpha-hydroxysteroid dehydrogenase: three dimensional structure and gene regulation

T M Penning1

  • 1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104 6084, USA.

The Journal of Endocrinology
|September 1, 1996
PubMed
Summary

Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) regulate steroid hormone levels. Glucocorticoids positively regulate rat 3 alpha-HSD gene transcription via steroid response units in the promoter.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) are crucial for regulating steroid hormone levels.
  • Rat liver 3 alpha-HSD, belonging to the aldo-keto reductase (AKR) superfamily, shares high sequence identity with human isoforms.
  • Its crystal structure reveals an (alpha/beta)8-barrel scaffold with key catalytic residues and cofactor binding sites.

Purpose of the Study:

  • To investigate the gene structure and regulatory elements of rat 3 alpha-HSD.
  • To elucidate the mechanisms of transcriptional regulation, particularly the role of glucocorticoids.

Main Methods:

  • cDNA cloning and sequence analysis.
  • X-ray crystallography to determine enzyme structure.

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  • Gene cloning, promoter analysis using reporter gene assays (chloramphenicol acetyltransferase, CAT).
  • Band-shift and supershift assays to identify transcription factor binding.

    • Main Results:

    • The rat 3 alpha-HSD gene is 47 kb with 9 conserved exon-intron boundaries.
    • The 5'-flanking regions contain steroid hormone response elements (REs) and binding sites for transcription factors like Oct-1.
    • Reporter gene assays identified promoter enhancers and a silencer region where Oct-1 binds.
    • Glucocorticoid stimulation robustly activated CAT activity, indicating positive transcriptional regulation via steroid response units.

    Conclusions:

    • Glucocorticoids positively regulate rat 3 alpha-HSD gene transcription.
    • Steroid response units within the promoter play a significant role in this regulation.
    • The findings provide insights into the molecular mechanisms controlling steroid hormone metabolism.