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Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of

Alejandra S Guberman1, Maria E Scassa, Luciana E Giono

  • 1Laboratorio de Biologia Molecular, Departamento de Quimica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II Piso 4, Ciudad Universitaria, Argentina.

The Journal of Biological Chemistry
|November 16, 2002
PubMed

Insights

12-O-tetradecanoylphorbol-13-acetate (TPA) represses 5-aminolevulinate synthase (ALAS) gene expression by inhibiting transcription factor AP-1 binding to the TRE-ALAS site, impacting heme biosynthesis.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biochemistry

Background:

  • Activation protein-1 (AP-1) transcription factors regulate diverse cellular processes.
  • 12-O-tetradecanoylphorbol-13-acetate (TPA) is a common inducer of AP-1 activity.
  • 5-aminolevulinate synthase (ALAS) is the rate-limiting enzyme in heme biosynthesis.

Purpose of the Study:

  • To elucidate the molecular mechanisms of TPA regulation on ALAS gene expression.
  • To identify the specific DNA elements and transcription factors involved in TPA's effect on ALAS.

Main Methods:

  • Subcloning of the rat ALAS 5'-flanking region into a CAT reporter vector.
  • Transient transfection assays in HepG2 cells to measure reporter gene activity.
  • Sequence and deletion analysis to identify regulatory elements (TRE-ALAS).
  • Supershift analysis and co-transfection assays to determine transcription factor involvement (c-Fos, c-Jun, JunD, CBP, p300).

Main Results:

  • TPA repressed ALAS promoter activity in HepG2 cells.
  • A TPA response element (TRE-ALAS) between -261 and -255 was identified as critical for TPA repression.
  • c-Fos, c-Jun, and JunD binding to TRE-ALAS mediated the TPA-induced repression.
  • Ectopic expression of CRE-binding protein or p300 relieved TPA-mediated repression.
  • The TRE-ALAS site acted as a transcriptional enhancer when spatially separated from CRE sites.

Conclusions:

  • TPA inhibits ALAS gene expression via AP-1 factors binding to TRE-ALAS, potentially by sequestering CREB-binding protein.
  • The transcriptional role of the AP-1 site in ALAS regulation is context-dependent on its spatial relationship with CRE sites.
  • This study reveals a novel mechanism of heme biosynthesis regulation impacting cellular processes.

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