Association of USF and c-Myc with a helix-loop-helix-consensus motif in the core promoter of the murine type II beta regulatory subunit gene of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase.
Previous studies showed that the core promoter of the mouse cAMP-dependent protein kinase regulatory subunit type II beta (RII beta) gene was composed of two functional elements. One element was GC rich and bound the Sp1 transcription factor. The second element contained a helix-loop-helix (HLH)-motif. Each element conferred transcriptional activity when inserted upstream of a reporter gene, chloramphenicol acetyltransferase and transfected into mouse NB2a neuroblastoma cells and Chinese hamster ovary (CHO) cells. The core promoter was further characterized by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factors and reduced the transcriptional activity of constructs containing the chloramphenicol acetyltransferase reporter when transfected into these cells. The results showed that the central nucleotides as well as the adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and interference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription factors were forming complexes with the HLH-consensus motif. The results suggest that RII beta transcription may be regulated, in part, by USF and c-Myc in NB2a and CHO cells.
Singh
I S
IS
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Luo
Z
Z
Kozlowski
M T
MT
Erlichman
J
J
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins; D005796:Genes; D018257:Helix-Loop-Helix Motifs; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000473:pathology; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D004268:DNA-Binding Proteins; C517838:Prkar2b protein, mouse; C517839:Prkar2b protein, rat; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497182:Usf1 protein, rat; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases