Sequence-specific DNA binding activity of RNA helicase A to the p16INK4a promoter.
p16(INK4a) is frequently altered in human cancer, often through epigenetically mediated transcriptional silencing. However, little is known about the transcriptional regulation of this gene. To learn more about such control, we initiated studies of proteins that bind to the promoter in cancer cells that do, and do not, express the gene. We identify RNA helicase A (RHA) as a protein that binds much better to the p16(INK4a) promoter in the expressing cells. RHA has not previously been characterized to manifest sequence-specific DNA interaction but does so to the sequence 5' CGG ACC GCG TGC GC 3' in the p16(INK4a) promoter. The Drosophila homologue to RHA, maleless (Mle), functions in the fly for 2-fold activation of male X-chromosome genes. In our experimental setting, RHA induces a similar modest up-regulation of the p16(INK4a) promoter that is dependent upon its sequence-specific interaction. Mle colocalizes with hyperacetylated H4Ac16 on the X-chromosome and some autosomal loci. The decreased binding of RHA to p16(INK4a) in our cells, where the gene is transcriptionally inactive, is associated with decreased amounts of RHA that immunoprecipitate with acetylated lysine antibodies. Finally, we show RHA to be a cellular substrate for caspase-3, which decreases its sequence-specific binding to p16(INK4a) by cleavage of the N terminus. Thus, we have identified a new protein interaction with the p16(INK4a) promoter that involves an important protein for transcriptional modulation. This interaction is decreased in cancer cells, where this gene is aberrantly transcriptionally silent.
Myöhänen
S
S
Johns Hopkins Oncology Center, Baltimore, Maryland 21231, USA.
Baylin
S B
SB
D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018288:Carcinoma, Small Cell; D002294:Carcinoma, Squamous Cell; D002467:Cell Nucleus / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D053487:DEAD-box RNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D005786:Gene Expression Regulation; D016147:Genes, Tumor Suppressor; D006801:Humans; D008175:Lung Neoplasms; D008297:Male; D009363:Neoplasm Proteins; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014960:X Chromosome
D001324:Autoantigens; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases