INVESTIGADORES
CANEPA Eduardo Tomas
artículos
Título:
5-aminolevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)
Autor/es:
LUCIANA E. GIONO; CECILIA L. VARONE; EDUARDO T. CÁNEPA
Revista:
BIOCHEMICAL JOURNAL
Editorial:
Biochemical Society
Referencias:
Lugar: Londres; Año: 2001 vol. 353 p. 307 - 316
ISSN:
0264-6021
Resumen:
The ®rst and rate-controlling step of the haem biosynthetic
pathway in mammals and fungi is catalysed by the mitochondrialmatrix
enzyme 5-aminolaevulinate synthase (ALAS). The purpose
of this work was to explore the molecular mechanisms
involved in the cAMP regulation of rat housekeeping ALAS
gene expression. Thus we have examined the ALAS promoter for
putative transcription-factor-binding sites that may regulate
transcription in a cAMP-dependent protein kinase (PKA)-
induced context. Applying both transient transfection assays
with a chloramphenicol acetyltransferase reporter gene driven by
progressive ALAS promoter deletions in HepG2, and electrophoresis
mobility-shift assays we have identi®ed two putative
cAMP-response elements (CREs) at positions ®38 and ®142.
Functional analysis showed that both CRE-like sites were
necessary for complete PKA induction, but only one for basal
expression. Co-transfection with a CRE-binding protein
(CREB) expression vector increased PKA-mediated induction of
ALAS promoter transcriptional activity. However, in the absence
of co-transfected PKA, CREB worked as a speci®c repressor for
ALAS promoter activity. A CREB mutant de®cient in a PKA
phosphorylation site was unable to induce expression of the
ALAS gene but could inhibit non-stimulated promoter activity.
Furthermore, a DNA-binding mutant of CREB did not interfere
with ALAS promoter basal activity. Site-directed-mutagenesis
studies showed that only the nearest element to the transcription
start site was able to inhibit the activity of the promoter.
Therefore, we conclude that CREB, through its binding to CRElike
sites, mediates the effect of cAMP on ALAS gene expression.
Moreover, we propose that CREB could also act as a repressor
of ALAS transcription, but is able to reverse its role after PKA
activation. Dephosphorylated CREB would interfere in a spatialdisposition-
dependent manner with the transcriptional machinery
driving inhibition of gene expression.®38 and ®142.
Functional analysis showed that both CRE-like sites were
necessary for complete PKA induction, but only one for basal
expression. Co-transfection with a CRE-binding protein
(CREB) expression vector increased PKA-mediated induction of
ALAS promoter transcriptional activity. However, in the absence
of co-transfected PKA, CREB worked as a speci®c repressor for
ALAS promoter activity. A CREB mutant de®cient in a PKA
phosphorylation site was unable to induce expression of the
ALAS gene but could inhibit non-stimulated promoter activity.
Furthermore, a DNA-binding mutant of CREB did not interfere
with ALAS promoter basal activity. Site-directed-mutagenesis
studies showed that only the nearest element to the transcription
start site was able to inhibit the activity of the promoter.
Therefore, we conclude that CREB, through its binding to CRElike
sites, mediates the effect of cAMP on ALAS gene expression.
Moreover, we propose that CREB could also act as a repressor
of ALAS transcription, but is able to reverse its role after PKA
activation. Dephosphorylated CREB would interfere in a spatialdisposition-
dependent manner with the transcriptional machinery
driving inhibition of gene expression.