INVESTIGADORES
CANEPA Eduardo Tomas
artículos
Título:
Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator
Autor/es:
ALEJANDRA S. GUBERMAN; MARÍA E. SCASSA; LUCIANA E. GIONO; CECILIA L. VARONE; EDUARDO T. CÁNEPA
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
The American Society for Biochemistry and Molecular Biology, Inc
Referencias:
Lugar: Bethesda, EEUU; Año: 2003 vol. 278 p. 2317 - 2326
ISSN:
0021-9258
Resumen:
Activation protein-1 (AP-1) transcription factors are
early response genes involved in a diverse set of transcriptional
regulatory processes. The phorbol ester 12-
O-tetradecanoylphorbol-13-acetate (TPA) is often used
to induce AP-1 activity. The purpose of this work was
to explore the molecular mechanisms involved in the
TPA regulation of ubiquitous 5-aminolevulinate synthase
(ALAS) gene expression, the first and ratecontrolling
step of the heme biosynthesis. Previous
analysis of the 5-flanking sequence of ALAS revealed
the existence of two cAMP-response elements (CRE)
required for basal and cAMP-stimulated expression.
The fragment 833 to 42 in the 5-flanking region of
rat ALAS gene was subcloned into a chloramphenicol
acetyltransferase (CAT) reporter vector. The expression
vector pALAS/CAT produced a significant CAT
activity in transiently transfected HepG2 human hepatoma
cells, which was repressed by TPA. Sequence
and deletion analysis detected a TPA response element
(TRE), located between 261 and 255 (TRE-ALAS),
that was critical for TPA regulation. We demonstrated
that c-Fos, c-Jun, and JunD are involved in TPA inhibitory
effect due to their ability to bind TRE-ALAS,
evidenced by supershift analysis and their capacity to
repress promoter activity in transfection assays. Repression
of ALAS promoter activity by TPA treatment
or Fos/Jun overexpression was largely relieved when
CRE protein-binding protein or p300 was ectopically
expressed. When the TRE site was placed in a different
context with respect to CRE sites, it appeared to act as
a transcriptional enhancer. We propose that the decrease
in ALAS basal activity observed in the presence
of TPA may reflect a lower ability of this promoter to
assemble the productive pre-initiation complex due to
CRE protein-binding protein sequestration. We also
suggest that the transcriptional properties of this AP-1
site would depend on a spatial-disposition-dependent
manner with respect to the CRE sites and to the transcription
initiation site.-tetradecanoylphorbol-13-acetate (TPA) is often used
to induce AP-1 activity. The purpose of this work was
to explore the molecular mechanisms involved in the
TPA regulation of ubiquitous 5-aminolevulinate synthase
(ALAS) gene expression, the first and ratecontrolling
step of the heme biosynthesis. Previous
analysis of the 5-flanking sequence of ALAS revealed
the existence of two cAMP-response elements (CRE)
required for basal and cAMP-stimulated expression.
The fragment 833 to 42 in the 5-flanking region of
rat ALAS gene was subcloned into a chloramphenicol
acetyltransferase (CAT) reporter vector. The expression
vector pALAS/CAT produced a significant CAT
activity in transiently transfected HepG2 human hepatoma
cells, which was repressed by TPA. Sequence
and deletion analysis detected a TPA response element
(TRE), located between 261 and 255 (TRE-ALAS),
that was critical for TPA regulation. We demonstrated
that c-Fos, c-Jun, and JunD are involved in TPA inhibitory
effect due to their ability to bind TRE-ALAS,
evidenced by supershift analysis and their capacity to
repress promoter activity in transfection assays. Repression
of ALAS promoter activity by TPA treatment
or Fos/Jun overexpression was largely relieved when
CRE protein-binding protein or p300 was ectopically
expressed. When the TRE site was placed in a different
context with respect to CRE sites, it appeared to act as
a transcriptional enhancer. We propose that the decrease
in ALAS basal activity observed in the presence
of TPA may reflect a lower ability of this promoter to
assemble the productive pre-initiation complex due to
CRE protein-binding protein sequestration. We also
suggest that the transcriptional properties of this AP-1
site would depend on a spatial-disposition-dependent
manner with respect to the CRE sites and to the transcription
initiation site.-flanking sequence of ALAS revealed
the existence of two cAMP-response elements (CRE)
required for basal and cAMP-stimulated expression.
The fragment 833 to 42 in the 5-flanking region of
rat ALAS gene was subcloned into a chloramphenicol
acetyltransferase (CAT) reporter vector. The expression
vector pALAS/CAT produced a significant CAT
activity in transiently transfected HepG2 human hepatoma
cells, which was repressed by TPA. Sequence
and deletion analysis detected a TPA response element
(TRE), located between 261 and 255 (TRE-ALAS),
that was critical for TPA regulation. We demonstrated
that c-Fos, c-Jun, and JunD are involved in TPA inhibitory
effect due to their ability to bind TRE-ALAS,
evidenced by supershift analysis and their capacity to
repress promoter activity in transfection assays. Repression
of ALAS promoter activity by TPA treatment
or Fos/Jun overexpression was largely relieved when
CRE protein-binding protein or p300 was ectopically
expressed. When the TRE site was placed in a different
context with respect to CRE sites, it appeared to act as
a transcriptional enhancer. We propose that the decrease
in ALAS basal activity observed in the presence
of TPA may reflect a lower ability of this promoter to
assemble the productive pre-initiation complex due to
CRE protein-binding protein sequestration. We also
suggest that the transcriptional properties of this AP-1
site would depend on a spatial-disposition-dependent
manner with respect to the CRE sites and to the transcription
initiation site.833 to 42 in the 5-flanking region of
rat ALAS gene was subcloned into a chloramphenicol
acetyltransferase (CAT) reporter vector. The expression
vector pALAS/CAT produced a significant CAT
activity in transiently transfected HepG2 human hepatoma
cells, which was repressed by TPA. Sequence
and deletion analysis detected a TPA response element
(TRE), located between 261 and 255 (TRE-ALAS),
that was critical for TPA regulation. We demonstrated
that c-Fos, c-Jun, and JunD are involved in TPA inhibitory
effect due to their ability to bind TRE-ALAS,
evidenced by supershift analysis and their capacity to
repress promoter activity in transfection assays. Repression
of ALAS promoter activity by TPA treatment
or Fos/Jun overexpression was largely relieved when
CRE protein-binding protein or p300 was ectopically
expressed. When the TRE site was placed in a different
context with respect to CRE sites, it appeared to act as
a transcriptional enhancer. We propose that the decrease
in ALAS basal activity observed in the presence
of TPA may reflect a lower ability of this promoter to
assemble the productive pre-initiation complex due to
CRE protein-binding protein sequestration. We also
suggest that the transcriptional properties of this AP-1
site would depend on a spatial-disposition-dependent
manner with respect to the CRE sites and to the transcription
initiation site.261 and 255 (TRE-ALAS),
that was critical for TPA regulation. We demonstrated
that c-Fos, c-Jun, and JunD are involved in TPA inhibitory
effect due to their ability to bind TRE-ALAS,
evidenced by supershift analysis and their capacity to
repress promoter activity in transfection assays. Repression
of ALAS promoter activity by TPA treatment
or Fos/Jun overexpression was largely relieved when
CRE protein-binding protein or p300 was ectopically
expressed. When the TRE site was placed in a different
context with respect to CRE sites, it appeared to act as
a transcriptional enhancer. We propose that the decrease
in ALAS basal activity observed in the presence
of TPA may reflect a lower ability of this promoter to
assemble the productive pre-initiation complex due to
CRE protein-binding protein sequestration. We also
suggest that the transcriptional properties of this AP-1
site would depend on a spatial-disposition-dependent
manner with respect to the CRE sites and to the transcription
initiation site.