A novel nk-2-related transcription factor associated with human fetal liver and hepatocellular carcinoma.
A novel cDNA was partially isolated from a HepG2 cell expression library by screening with the promoter-linked coupling element (PCE), a site from the alpha-fetoprotein (AFP) gene promoter. The remainder of the cDNA was cloned from fetal liver RNA using random amplification of cDNA ends. The cDNA encodes a 239-amino acid peptide with domains closely related to the Drosophila factor nk-2. The new factor is the eighth vertebrate factor related to nk-2, hence nkx-2.8. Northern blot and reverse transcriptase polymerase chain reaction analysis demonstrated mRNA in HepG2, two other AFP-expressing human cell lines, and human fetal liver. Transcripts were not detected in adult liver. Cell-free translation produced DNA binding activity that gel shifted a PCE oligonucleotide. Cotransfection of nkx-2.8 expression and PCE reporter plasmids into HeLa cells demonstrated transcriptional activation; NH2-terminal deletion eliminated this activity. Cotransfection into AFP-producing hepatocytic cells repressed AFP reporter expression, suggesting that endogenous activity was already present in these cells. In contrast, cotransfection into an AFP-negative hepatocytic line produced moderate activation of the AFP gene. The cardiac developmental factor nkx-2.5 could substitute for nkx-2.8 in all transfection assays, whereas another related factor, thyroid transcription factor 1, showed a more limited range of substitution. Although the studies have yet to establish definitively that nkx-2.8 is the AFP gene regulator PCF, the two factors share a common DNA binding site, gel shift behavior, migration on SDS-acrylamide gels, and cellular distribution. Moreover, the nk-2-related genes are developmental regulators, and nkx-2.8 is the first such factor associated with liver development.
Apergis
G A
GA
Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Crawford
N
N
Ghosh
D
D
Steppan
C M
CM
Vorachek
W R
WR
Wen
P
P
Locker
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018236:Carcinoma, Embryonal; D006528:Carcinoma, Hepatocellular; D003001:Cloning, Molecular; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008099:Liver / Q000254:growth & development; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000096:biosynthesis* / Q000235:genetics
D009363:Neoplasm Proteins; D014157:Transcription Factors; D000509:alpha-Fetoproteins