Characterization and localization of metal-responsive-element-binding transcription factors from tilapia.
Two isoforms of MTF-1, MTF-1L (long form) and MTF-1S (short form), were cloned in tilapia (Ti) and characterized in a tilapia liver cell line, Hepa-T1. The cloned tiMTF-1L has the characteristics of all of the tiMTF-1S identified so far with the zinc finger domain having six fingers, the acidic-rich, proline-rich, and serine/threonine-rich domains; however, the short form encodes for the zinc finger domain with five zinc fingers only and no other domains. The transient transfection of tiMTF-1L into human HepG2 cells showed both constitutive and zinc-induced metal-responsive-element (MRE)-driven reporter gene expression. However, the transfection of tiMTF-1S (which lacks all three transactivation domains) into a human cell line showed reduced transcriptional activities compared with an endogenous control in both basal- and Zn(2+)-induced conditions. The tiMTF-1 isoforms were tagged with GFP and transfected into Hepa-T1 cells (tilapia hepatocytes). The nuclear translocation of tiMTF-1L was observed when the cells were exposed to a sufficient concentration of metals for 6h. However, tiMTF-1S, was localized in the nucleus with or without metal treatment. Electrophoretic mobility shift assay (EMSA) confirmed that both of the isoforms were able to bind to the MRE specifically in vitro. Tissue distribution studies showed that tiMTF-1L was more abundant than tiMTF-1S in all of the tissues tested.
Cheung
Andrew Pok-Lap
AP
Department of Biochemistry, The Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong, China.
Au
Candy Yee-Man
CY
Chan
William Wai-Lun
WW
Chan
King Ming
KM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017210:Tilapia / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology; D000097809:Transcription Factor MTF-1
D004268:DNA-Binding Proteins; D008668:Metallothionein; D020033:Protein Isoforms; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1