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PMID: 7823934 Published · ppublish English Comparative Study Journal Article

Transcriptional regulation of the mouse alpha A-crystallin gene: activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a Pax-6-binding site.

Molecular and cellular biology ·Vol. 15 ·No. 2 ·1995-02-00 ·Pages 653-60

Cvekl A, Kashanchi F, Sax CM, Brady JN, Piatigorsky J

Abstract

Two cis-acting promoter elements (-108 to -100 and -49 to -33) of the mouse alpha A-crystallin gene, which is highly expressed in the ocular lens, were studied. Here we show that DE1 (-108 to -100; 5'TGACGGTG3'), which resembles the consensus cyclic AMP (cAMP)-responsive element sequence (CRE; 5'TGACGT[A/C][A/G]3'), behaves like a functional CRE site. Transfection experiments and electrophoretic mobility shift assays (EMSAs) using site-specific mutations correlated a loss of function with deviations from the CRE consensus sequence. Results of EMSAs in the presence of antisera against CREB, delta CREB, and CREM were consistent with the binding of CREB-like proteins to the DE1 sequence. Stimulation of alpha A-crystallin promoter activity via 8-bromo-cAMP, forskolin, or human T-cell leukemia virus type I Tax1 in transfections and reduction of activity of this site in cell-free transcription tests by competition with the somatostatin CRE supported the idea that DE1 is a functional CRE. Finally, Pax-6, a member of the paired-box family of transcription factors, activated the mouse alpha A-crystallin promoter in cotransfected COP-8 fibroblasts and bound to the -59 to -29 promoter sequence in EMSAs. These data provide evidence for a synergistic role of Pax-6 and CREB-like proteins for high expression of the mouse alpha A-crystallin gene in the lens.

MeSH Terms
8-Bromo Cyclic Adenosine Monophosphate/pharmacology Animals Base Sequence Binding Sites Cell Line Cell Nucleus Cloning, Molecular Colforsin/pharmacology Consensus Sequence Crystallins/biosynthesis,genetics Cyclic AMP Response Element Modulator Cyclic AMP Response Element-Binding Protein/metabolism DNA Primers DNA-Binding Proteins/metabolism Eye Proteins Gene Expression Regulation Gene Products, tax/metabolism Homeodomain Proteins Human T-lymphotropic virus 1/metabolism Humans Lens, Crystalline/metabolism Mice/genetics Molecular Sequence Data Mutagenesis, Site-Directed PAX6 Transcription Factor Paired Box Transcription Factors Polymerase Chain Reaction Promoter Regions, Genetic Rabbits Regulatory Sequences, Nucleic Acid Repressor Proteins/metabolism Sequence Homology, Nucleic Acid Transcription Factors/metabolism Transcription, Genetic Transcriptional Activation Transfection
Chemicals
Crystallins Cyclic AMP Response Element-Binding Protein DNA Primers DNA-Binding Proteins Eye Proteins Gene Products, tax Homeodomain Proteins PAX6 Transcription Factor PAX6 protein, human Paired Box Transcription Factors Pax6 protein, mouse Repressor Proteins Transcription Factors Cyclic AMP Response Element Modulator Colforsin 8-Bromo Cyclic Adenosine Monophosphate
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Cvekl A
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 20892-2730.
Kashanchi F
Sax C M
Brady J N
Piatigorsky J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.15.2.653
Published
1995-02-00
Pages
653-60
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC231924
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.15.2.653 PMC Full Text View journal details
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