HNF3beta and GATA-4 transactivate the liver-enriched homeobox gene, Hex.
The orphan homeobox gene, Hex, has a limited domain of expression which includes the developing and adult mouse liver. Hex is expressed in the developing liver coincident with the forkhead/winged helix transcription factor, Hepatocyte Nuclear Factor 3beta (HNF3beta). Although preliminary characterization of the mouse Hex promoter has recently been reported, the identity of the molecular regulators that drive liver expression is not known. We hypothesized that putative HNF3beta and GATA-4 elements within the Hex promoter would confer liver-enriched expression. A series of Hex promoter-driven luciferase reporter constructs were transfected in liver-derived HepG2 and fibroblast-like Cos cells+/-HNF3beta or GATA expression plasmids. The Hex promoter region from nt -235/+22 conferred basal activity in both HepG2 and Cos cells, with the region from -103/+22 conferring liver-enriched activity. HNF3beta and GATA-4 transactivated the promoter via response elements located within nt -103/+22, whereas Sp1 activated the -235/+22 construct. Mutation of the HNF3 element significantly reduced promoter activity in HepG2 cells, whereas this element in isolation conferred HNF3beta responsiveness to a heterologous promoter. Electrophoretic mobility shift assays were performed to confirm transcription factor:DNA binding. We conclude that HNF3beta and GATA-4 contribute to liver-enriched expression of Hex.
Denson
L A
LA
The Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. lee.denson@yale.edu
McClure
M H
MH
Bogue
C W
CW
Karpen
S J
SJ
Jacobs
H C
HC
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050991:GATA4 Transcription Factor; D005801:Genes, Homeobox / Q000235:genetics; D051542:Hepatocyte Nuclear Factor 3-beta; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496503:Foxa2 protein, mouse; C496504:Foxa2 protein, rat; D050991:GATA4 Transcription Factor; C078492:HHEX protein, human; C513080:Hhex protein, mouse; C513079:Hhex protein, rat; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D051705:Sp3 Transcription Factor; D004247:DNA; D008156:Luciferases