A novel cyclic AMP response element, CACTTGATC, mediates forskolin induction of the myelin basic protein promoter in the rat Schwannoma line, D6P2T.
The rat Schwannoma cell line D6P2T constitutively expresses the mRNA encoding the major myelin protein, P0, but only expresses the mRNA encoding myelin basic protein (MBP) after exposure to forskolin or other substances that raise the levels of intracellular cyclic AMP. In this study we have investigated the molecular basis for forskolin induction of MBP transcription in D6P2T cells. We have found that a 9-bp sequence element, CACTTGATC, located between nucleotides -85 and -77 in the MBP promoter, is necessary for forskolin induction of chloramphenicol acetyltransferase (CAT) expression after transient transfection of MBP promoter-CAT fusion constructs into D6P2T cells. Although similar DNase I footprints, one of which is located within the above 9-bp sequence element, are produced by nuclear extracts prepared from both forskolin-treated and untreated cells, this same sequence can be shown to interact with a forskolin-inducible protein complex using an electrophoretic mobility shift assay. In addition, mutation of this 9-bp sequence abolishes both formation of this new protein--DNA complex and forskolin-inducible CAT expression from the heterologous SV40 promoter. Finally, we have shown that the appearance of this forskolin-inducible protein--DNA complex precedes that of MBP mRNA. Taken together, these data strongly support the notion that the induction of MBP transcription by forskolin in D6P2T cells is mediated by the binding of a forskolin-inducible protein complex to the MBP promoter sequence CACTTGATC.
Li
X
X
Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia.
Wrabetz
L
L
Cheng
Y
Y
Kamholz
J
J
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000502:physiology; D003850:Deoxyribonuclease I / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D008969:Molecular Sequence Data; D009154:Mutation; D004676:Myelin Basic Protein / Q000235:genetics* / Q000502:physiology; D009442:Neurilemmoma / Q000737:chemistry / Q000235:genetics* / Q000473:pathology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D004676:Myelin Basic Protein; D012333:RNA, Messenger; D005576:Colforsin; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I