Cloning And Function Analysis Of Novel Human Heart Development Candidate Gene Hnulp1

The basic helix-loop-helix (bHLH) protein is a distinct eukaryotic transcription factor involved in many important physiological processes, which include neurogenesis, myogenesis, tumorgenesis, hemagenesis and development process. In the present study, we report the identification and characterization of a novel basic helix-loop-helix protein, HNULP1, from a human embryonic heart cDNA library. The complete sequence of the hnulpl cDNA is 2270-bp in length and contains a putative open reading frame of 2031 nucleotides, which encodes a putative protein of 676 amino acids. This protein contains a bHLH domain and a DUF654 domain in N-terminal and C-terminal, respectively. HNULP1 is highly conserved from yeast, zebrafish, chicken, A. thaliana, C, elegans, Drosophilae to mouse, which suggests that it plays an important role in evolution. Northern blotting analysis shows that a 2.3-kb transcript expressed broadly in both early human embryonic and adult tissues, especially with a higher level in adult heart. The analysis of the GAL4 luciferase reporter gene indicated that HNULP1 function as a transcriptional suppressor and can inhibit the transcriptional activities of SRF. Furthermore, the DUF654 motif represents the basal transcriptional repressive activity. These results suggest that during the late development of heart hnu1p1 may be involved in the regulation of transcriptional activities of SRF through DUF654 domain.Moreover, we identified that the heart specific expression gene FHL2 is a candidate interacting protein of HNULP1 by using the yeast two-hybridization system. Next, we confirmed the interaction of these two proteins in the cells by using immmunoprecipitation analysis. Subcellular co-localization of these two proteins indicated that HNU1P1 and FHL2 co-localized in cytoplasma and nuclear. GAL4 luciferase reporter gene analysis shows that FHL2 inhibits the transcriptional activity of hNU1P1. Taken together, these results therefore lays solid basis for the study of the functional interaction of FHL2 and HNULP1.