Single-stranded DNA-binding proteins PURalpha and PURbeta bind to a purine-rich negative regulatory element of the alpha-myosin heavy chain gene and control transcriptional and translational regulation of the gene expression. Implications in the repression of alpha-myosin heavy chain during heart failure.
The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells.
Gupta
Madhu
M
Hope Children's Hospital, University of Illinois, Chicago, Illinois 60612, USA.
Sueblinvong
Viranuj
V
Raman
Jai
J
Jeevanandam
Valluvan
V
Gupta
Mahesh P
MP
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006331:Heart Diseases / Q000503:physiopathology; D006801:Humans; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics*; D009419:Nerve Tissue Proteins; D015513:Oncogene Proteins / Q000378:metabolism; D014176:Protein Biosynthesis; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000032:analysis; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C086924:Elk3 protein, mouse; D009419:Nerve Tissue Proteins; D015513:Oncogene Proteins; C501130:PURA protein, human; C491579:PURB protein, human; D050783:Proto-Oncogene Proteins c-ets; C121485:Pura protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D018995:Myosin Heavy Chains