Zfhep-1 and Zfhep-2 and their alternative promoters are active in myoblast cell line and repress myoglobin gene transcription

MANAVELLA PA, DOS SANTOS R, NUNEZ MT, DARLING DS, CABANILLAS AM

Carlos Paz, Córdoba, Argentina.

Congreso; X Congreso de la Sociedad Latinoamericana de Tiroides (SLAT).; 2003

T3 induces myoglobin (Mb) gene expression in cardiac and skeletal muscle cells. EMSA studies also shown that T3 increased the binding of an E-box-associated protein to Mb gene. Zfhep (Zinc Finger Homeodomain Enhancer Protein) is an E-box binding protein which is highly expressed in rat central nervous system and heart as two isoforms, termed Zfhep-1 and Zfhep-2. The Zfhep-2 isoform lacks the first exon of the gene, and the protein is predicted to lack half of one zinc finger domain and therefore it may have different DNA-binding activity. We have shown that Zfhep -1 but not Zfhep -2, inhibits T3-mediated activation of the growth hormone gene. Zfhep isoforms are regulated by two different promoters but none of them were characterized yet. The goals of this work were: 1) characterize both promoters in different cell lines to know whether their activities were expressed differentially in muscle and other cell types; and 2) determine the ability of this E-box binder, Zfhep-1 and -2, to regulate transcription of the T3-dependent gene, myoglobin, in myoblasts. We subcloned PCR fragments of Zfhep promoters into a luciferase reporter vector pGL3-basic (Z-1pLuc and Z-2pLuc).Transfection (Tfx) assays were performed by Ca-phosphate precipitation method in several cell lines: P19 (embryonal carcinoma), C2C12 (myoblast), COS-7, CHO-K1 (chinese hamster ovary), and JEG-3 (choriocarcinoma). CMVb (to normalize Tfx efficiency) and Z-1pLuc or Z-2pLuc or pGL3-basic plasmids were incorporated to the cells grown in DMEM/10% fetal bovine serum. b-galactosidase and luciferase activities were assayed 48hs after Tfx by standard methods. Z-1pLuc activity was 15-20 (COS-7), 12 (JEG-3), 50-70 (C2C12, CHO-K1), and 10-20 (P19) times higher than the respective controls. Z-2pLuc activity was 20-22 fold over control in C2C12 and CHO-K1 and absent in COS-7 and JEG-3. For the second goal, pGL3-basic or Mb promoter (MbLuc) and expression vectors of Zfhep-1 or Zfhep-2 or the empty vector and TRa1 were transfected to C2C12 cells in the presence or not of 100nM T3. Both, Zfhep-1 and Zfhep-2 repressed Mb promoter activity in the presence of T3. Zfhep-1 repression was higher than that of Zfhep-2 (10 fold vs. 3 fold) which is in accordance with the activity of Zfhep promoters in C2C12 cells. These results show that both Zfhep-1 and Zfhep-2 promoters and their respective protein isoforms can be active in muscle cells and that the myoglobin gene could be one of the targets of the transcriptional effect of this transcription factor. Zfhep effect on myoglobin gene would not be dependent on N-terminus of Zfhep. b (to normalize Tfx efficiency) and Z-1pLuc or Z-2pLuc or pGL3-basic plasmids were incorporated to the cells grown in DMEM/10% fetal bovine serum. b-galactosidase and luciferase activities were assayed 48hs after Tfx by standard methods. Z-1pLuc activity was 15-20 (COS-7), 12 (JEG-3), 50-70 (C2C12, CHO-K1), and 10-20 (P19) times higher than the respective controls. Z-2pLuc activity was 20-22 fold over control in C2C12 and CHO-K1 and absent in COS-7 and JEG-3. For the second goal, pGL3-basic or Mb promoter (MbLuc) and expression vectors of Zfhep-1 or Zfhep-2 or the empty vector and TRa1 were transfected to C2C12 cells in the presence or not of 100nM T3. Both, Zfhep-1 and Zfhep-2 repressed Mb promoter activity in the presence of T3. Zfhep-1 repression was higher than that of Zfhep-2 (10 fold vs. 3 fold) which is in accordance with the activity of Zfhep promoters in C2C12 cells. These results show that both Zfhep-1 and Zfhep-2 promoters and their respective protein isoforms can be active in muscle cells and that the myoglobin gene could be one of the targets of the transcriptional effect of this transcription factor. Zfhep effect on myoglobin gene would not be dependent on N-terminus of Zfhep.