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Nucleic Acids Research, 1991, Vol. 19, No. 10 2721-2728
© 1991

MOLECULAR BIOLOGY

NF-I proteins from brain interact with the proenkephalin cAMP inducible enhancer

Hung-Ming Chu, Wolfgang H. Fischer1, Timothy F. Osborne and Michael J. Comb*

Laboratory of Molecular Neurobiology, Massachusetts General Hospital, The Program in Neuroscience, Harvard Medical School Boston, MA 02114 1The Salk Institute for Biological Studies La Jolla, CA 92037 2Department of Molecular Biology & Biochemistry, University of California Irvine, CA 92715, USA

* To whom correspondence should be addressed

Received January 23, 1991. Revised April 15, 1991. Accepted April 15, 1991.

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 neitherelement 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.


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