Expression from the tyrosine aminotransferase promoter (nt -350 to +1) is liver-specific and dependent on the binding of both liver-enriched and ubiquitous trans-acting factors.
The rat tyrosine aminotransferase(TAT) gene promoter (nucleotides -350 to +1; TAT0.35) was able to sustain liver-specific expression both ex vivo in transient transfection (TAT-expressing H411EC3 hepatoma cells vs. TAT non-expressing CCL1.2 fibroblasts) and in in vitro transcription (rat liver vs. spleen crude nuclear extracts). In either case, the index of tissue specificity (6.2 and 6.7 in ex vivo and in vitro experiments, respectively) was close to that obtained with 10 Kb of TAT gene 5'-flanking sequences in transient transfection. Using computer-assisted search of homologies, DNase I footprinting, gel retardation and methylation interference assays, we showed that TAT0.35 sequences spanning nt -156 to -175 and nt -268 to -281 interacted with the liver enriched NF-1Liver (a member of the NF1 gene family) and HNF1 respectively, whereas those encompassing nt -57 to -85 and nt -283 to -288 interacted with the ubiquitous NF-Y and with ubiquitous 'CCAAT'-box binding factor(s), respectively. Competition studies in in vitro transcription carried out with wild type and mutated oligonucleotides, demonstrated that NF-Y cis-elements were crucial for basal TAT promoter activity, both in liver and spleen whereas NF1Liver and HNF1 were only efficient in the liver (supported approximately 60% and 30% of basal TAT0.35 activity respectively). Altogether, these results support the conclusion that TAT0.35 was able to sustain at least part of the liver specificity of TAT gene expression.
Schweizer-Groyer
G
G
CNRS, Institut Jacques Monod, Paris, France.
Groyer
A
A
Cadepond
F
F
Grange
T
T
Baulieu
E E
EE
Pictet
R
R
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014444:Tyrosine Transaminase / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA; D014444:Tyrosine Transaminase; D003850:Deoxyribonuclease I