Abstract 322 Cardiac Development and Gene Regulation Platform, Sunday, 5/2

Transcription of a number of cardiac- and skeletal-muscle specific genes appears to be regulated in part by members of the TEF-1 (transcription enhancer factor) family of transcription factors. TEF-1 proteins are thought to regulate transcription by binding to a conserved heptameric motif with the sequence CATTCCT. This motif, known as the MCAT element, is found in the promoter regions of the genes encoding a number of cardiac and skeletal muscle-specific proteins, including cardiac troponin T, the α and β myosin heavy chains, and skeletal α actin. MCAT elements have been implicated in tissue-specific expression of these and other genes. Four TEF-1 isoforms, NTEF-1, RTEF-1, DTEF-1, and ETEF have been isolated and characterized. These isoforms differ in their tissue distribution; RTEF-1 is enriched in skeletal muscle, DTEF-1 is found in cardiomyocytes. ETEF-1 is found early in embryonic development, while NTEF is found in most tissues. As a first step in understanding the function of these different isoforms in cell-specific transcription, we compared their DNA binding properties. We used two techniques to study the interaction of TEF-1 proteins with DNA. First, we used hydroxyl radical DNA footprinting to examine the interaction of two chicken TEF-1 isoforms, RTEF-1 and NTEF-1, with the two MCAT elements found in the cardiac troponin T promoter region. In this assay, RTEF-1 and NTEF-1 produced the same footprint on this promoter suggesting that they interact with MCAT elements in the same manner. Second, we performed CASTing (cyclic amplification of selected targets) to determine whether RTEF-1 and NTEF-1 have different binding specificities. Optimal protein binding sites were selected from a pool of random oligonucleotides by six cycles of binding/immunoprecipitation/amplification, then cloned and sequenced. Both of these TEF-1 isoforms selected oligonucleotides containing and 8 nucleotide motif, PuCATT/ACCN, in agreement with the binding site derived from naturally-occurring MCAT elements. However, the two isoforms differed in that while RTEF-1 selected binding sites with one MCAT element, NTEF showed a strong preference for binding sites with two MCAT elements. This result indicates that RTEF-1 binds to MCAT elements as a monomer, while NTEF binds cooperatively as a dimer and suggests that the ability of TEF-1 proteins to bind to their target promoters may be influenced by the configuration of MCAT elements in these promoters.