The cardiac-muscle specific enhancer-promoter of slow/cardiac troponin C binds HMG-2.

The cardiac muscle-specific enhancer-promoter of the slow/cardiac troponin C (cTnC) gene contains five protein binding regions, four of which bind cardiac-myocyte specific proteins. We screened a stage 11 chick embryo expression library with a double-stranded oligonucleotide probe consisting of one of these regions, CEF-1. One of the clones obtained was the chicken high mobility group protein, HMG-2. An electrophoretic gel mobility shift assay (EMSA) showed a specific binding interaction between the HMG-2 protein and the dsDNA CEF-1 probe. The cardiac-specific enhancer region of cTnC contains at least one possible HMG binding 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 its cardiac specific transcriptional activity. HMG-2 is a DNA bending protein that is predominantly found in the nucleus in proliferating cells and in the cytoplasm of terminally differentiated cells. It is an integral and stabilizing factor in the transcription activation nucleoprotein complex and is often described as an 'architectural transcription factor'. It markedly stimulates the transcription of many genes, often in association with tissue-specific transcription factors. We believe that the presence of HMG-2 in the enhancer-promoter binding protein 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 transcription of the cTnC gene during the proliferation phase of embryonic cardiac myocyte development.