THE CARDIAC-MUSCLE SPECIFIC ENHANCER-PROMOTER OF SLOW CARDIAC TROPONIN-C BINDS HMG-2/

The cardiac muscle-specific enhancer-promoter of the slow/cardiac trop onin C (cTnC) gene contains five protein binding regions, four of whic h bind cardiac-myocyte specific proteins. We screened a stage II chick embryo expression library with a double-stranded oligonucleotide prob e consisting of one of these regions, CEF-1. One of the clones obtaine d was the chicken high mobility group protein, HMG-2. An electrophoret ic gel mobility shift assay (EMSA) showed a specific binding interacti on between the HMG-2 protein and the dsDNA CEF-1 probe. The cardiac-sp ecific enhancer region of cTnC contains at least one possible HMG bind ing region and it is in the CEF-1 sequence overlapping a known GATA-4 binding site. Mutation of the nucleotide sequence of this HMG binding region diminishes its protein binding ability and markedly decreases i ts cardiac specific transcriptional activity. HMG-2 is a DNA bending p rotein that is predominantly found in the nucleus in proliferating cel ls and in the cytoplasm of terminally differentiated cells. It is an i ntegral and stabilizing factor in the transcription activation nucleop rotein complex and is often described as an 'architectural transcripti on factor'. It markedly stimulates the transcription of many genes, of ten in association with tissue-specific transcription factors. We beli eve that the presence of HMG-2 in the enhancer-promoter binding protei n complex of cTnC augments DNA bending and facilitates the DNA binding and interaction of other tissue-specific factors (e.g. GATA-4, which also binds to this region). This would result in increased transcripti on of the cTnC gene during the proliferation phase of embryonic cardia c myocyte development.