Single-stranded DNA-binding complex involved in transcriptional regulation of mouse mu-opioid receptor gene.
Previously, we reported the presence of dual (distal and proximal) promoters in mouse mu-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is approximately 25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.
Ko
J L
JL
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. koxxx001@maroon.tc.umn.edu
Loh
H H
HH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017450:Receptors, Opioid, mu / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D017450:Receptors, Opioid, mu; D015534:Trans-Activators; D015719:Single-Strand Specific DNA and RNA Endonucleases