Molecular cloning and characterization of acDNA encoding HTF4: A human helix-loop-helix protein

Yi Zhang, Purdue University

Abstract

Previous studies have shown that the simian virus 40 (SV40) AP4 site plays important roles in both SV40 early and late transcription. In order to identify the cellular factors that interact with this region of SV40 DNA, a HeLa cell cDNA library was screened with an oligonucleotide probe containing multiple copies of the AP4 site. The experiment yielded two distinct cDNA clones, both of which encode proteins of the helix-loop-helix family. One protein, HE47, represents one of the products of the human E2A gene. The other, HTF4, is a new human helix-loop-helix protein. Comparative DNA binding analysis demonstrates that both HTF4 and HE47 interact with E-box (CANNTG) motifs which are recognized by the basic helix-loop-helix proteins. Both proteins can form heterodimers with several muscle-specific transcription factors including MyoD, Myogenin and MRF4. The heterodimeric complexes show a high affinity for E-box motifs in the muscle creatine kinase gene enhancer. The initial HTF4 cDNA clone contained only the 320 amino acid carboxyl-terminal portion of a complete protein. A cDNA was reconstructed that contained the coding sequence for a full length protein designated HTF4a. Polyclonal antibody was raised against HTF4 and Western blot analysis showed that in the cell extract the antibody reacts predominantly with a 32-kDa protein as well as with two other polypeptides migrating at about 80-kDa. The HTF4a mRNA is about 5.5 kb and can be detected in many cell lines including HeLa cells; BJAB, a human B-cell lymphoma; 70z/3, a murine pre-B cell line; Jurkat and HSB-2, two human T-cell lines and HepG2, a human liver cell line. Among these cell lines HTF4a is expressed at a much higher level in Jurkat cells suggesting that HTF4 might play a dominant role in T lymphocytes. Northern blot analysis also showed that HTF4-related mRNAs are expressed at relatively high levels in human heart, skeletal muscle and kidney, at medium levels in brain, placenta and lung, and at undetectable levels in liver and pancreas. Two overlapping genomic clones have been isolated and they define a 23 kb segment of the HTF4 gene. This segment includes three exons (76 bp, 80 bp and 200 bp long) corresponding to the HTF4a cDNA. The genomic clones also include another exon which encodes a 24 amino acids long sequence that is not found in HTF4a. However, comparative sequence analysis indicates that this exon is utilized in alternatively spliced mRNA species found in mouse and chicken. Fluorescence hybridization was used to show that the HTF4 gene maps to human chromosome 15, 15q21. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Bina, Purdue University.

Subject Area

Molecular biology

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