Characterization of the Cyclic Adenosine 3′,5′-Monophosphate Response Element of the Rabbit Surfactant Protein-A Gene

Evidence for Transactivators Distinct from CREB/ATF Family Members

Laura F. Michael, Joseph L. Alcorn, Erwei Gao, Carole R. Mendelson

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5′-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an ∼43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREsp-a, as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

Original languageEnglish (US)
Pages (from-to)159-170
Number of pages12
JournalMolecular Endocrinology
Volume10
Issue number2
StatePublished - 1996

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Pulmonary Surfactant-Associated Protein A
Trans-Activators
Response Elements
Leucine Zippers
Adenosine
Carrier Proteins
Electrophoretic Mobility Shift Assay
Rabbits
Lung
Nuclear Proteins
Genes
Steroid Receptors
Reporter Genes
Gene Expression
Protein Binding
Mutagenesis
Peptides
Cyclic AMP Response Element Modulator
Activating Transcription Factor 1
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{460ceaf487f24e35935306c703281489,
title = "Characterization of the Cyclic Adenosine 3′,5′-Monophosphate Response Element of the Rabbit Surfactant Protein-A Gene: Evidence for Transactivators Distinct from CREB/ATF Family Members",
abstract = "Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5′-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an ∼43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREsp-a, as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.",
author = "Michael, {Laura F.} and Alcorn, {Joseph L.} and Erwei Gao and Mendelson, {Carole R.}",
year = "1996",
language = "English (US)",
volume = "10",
pages = "159--170",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
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T1 - Characterization of the Cyclic Adenosine 3′,5′-Monophosphate Response Element of the Rabbit Surfactant Protein-A Gene

T2 - Evidence for Transactivators Distinct from CREB/ATF Family Members

AU - Michael, Laura F.

AU - Alcorn, Joseph L.

AU - Gao, Erwei

AU - Mendelson, Carole R.

PY - 1996

Y1 - 1996

N2 - Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5′-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an ∼43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREsp-a, as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

AB - Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5′-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an ∼43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREsp-a, as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

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