NF-I proteins from brain interact with the proenkephalin cAMP inducible enhancer.
A short region of the human proenkephalin promoter has been shown previously to mediate transcriptional regulation in response to activation of the cAMP, TPA, and Ca+ + dependent intracellular signalling pathways. Two adjacent DNA elements, CRE-1 and CRE-2, are essential for this regulation although neither element alone is sufficient for inducible expression. The CRE-2 element consists of overlapping binding sites for the transcription factors AP-1 and AP-4. The CRE-1 element has been shown to interact with a DNA binding factor called ENKTF-1. Here we characterize proteins from bovine brain which bind the CRE-1 element of the human proenkephalin gene. Interactions between proteins binding the CRE-1 and CRE-2 elements are characterized in vitro using affinity purified DNA binding proteins. We demonstrate that CRE-1 binding proteins from bovine brain consist of three different polypeptides each belonging to the NF-I family of transcription factors. Point mutation analysis of the contacts of these proteins with the CRE-1 element indicate that NF-I proteins contact the inducible enhancer at the sequence CTGGCxxxxxxCCT which overlaps the CRE-1 element (underlined) defined by in vivo point mutation analysis. Cotransfection of one of the three NF-I proteins purified from bovine brain, NF-I/Red1, together with a proenkephalin/bacterial chloramphenicol acetyl transferase (CAT) fusion gene repressed protein kinase A or forskolin stimulated CAT expression.
Chu
H M
HM
Laboratory of Molecular Neurobiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.
Fischer
W H
WH
Osborne
T F
TF
Comb
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins; D002417:Cattle; D017362:Cyclic AMP Response Element-Binding Protein; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004745:Enkephalins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011498:Protein Precursors / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004745:Enkephalins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011498:Protein Precursors; D012097:Repressor Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; C029992:proenkephalin; D004247:DNA; D003850:Deoxyribonuclease I