Abstract
The CT element of the c-myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persisted following Sp1 immunodepletion, EGTA totally abolished transactivation, thus implicating another metal requiring factor in CT element activity. As hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differed in their relative binding to the CT- or sterol-response elements. CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element.
Highlights
Proper regulation of the c-myc gene has been shown to be important for the execution of several aspects of cellular metabolism
A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes
CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an API-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element
Summary
Vol 270, No 16, Issue of April 21, pp. 9494-9499, 1995 Printed in U.S.A. Cellular Nucleic Acid Binding Protein Regulates the CT Element of the Human c-myc Protooncogene*. The CT element of the e-myc gene is required for promoter PI usage and can drive expression of a heterologous promoter Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Studies of c-myc identified six candidate regulatory regions hypersensitive to DNase I digestion (11, 12), In several cell lines, the hypersensitivity to DNase I digestion within three ofthese regions correlates with active transcription ofthe c-myc gene One of these three sites, termed 1111, is situated 125 base pairs upstream of PI and consists of five imperfect direct repeats of the sequence CCCTCCCCA (termed the CT element); four repeats are in tandem, while the fifth downstream repeat is separated by nine base pairs. If hnRNP K binds to the pyrimidine-rich strand, are there factors that bind to the purine-rich strand? We present here the purification and cloning of such a binding activity, and identify it as the previously studied cellular nucleic acid binding protein (16)
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