Disruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice.
Previous work in transfected cell lines and in nuclear extracts has led to the identification of two cis-acting elements important for transcription of the human alpha-1-antitrypsin (A1AT) gene, which bind to two liver specific trans-acting factors, LF-A1 and LF-B1. Mutations EM3 and PM1, which abolish the binding of LF-A1 and LF-B1 respectively, drastically reduce transcription activity of the A1AT gene in vitro and in cell culture. The same mutants have now been introduced in a larger DNA context and their effect has been tested in transgenic mice. A stretch of DNA was constructed which carries two transcriptional units: 18 kb of the human retinol binding protein (RBP) gene, driving the expression of the bacterial chloramphenicol acetyl transferase, linked to 17.5 kb containing the entire A1AT coding sequence with additional 5' and 3' flanking sequences. Transcription from the RBP promoter was shown to predominate in liver, and could be used as an internal marker of 'active copy number'. Mutations in the A1AT gene promoter were introduced by homologous recombination in bacterial cells. The results show that base pair substitutions in the binding site for LF-A1 and LF-B1 drastically reduce transcription in non-hepatic adult tissues, yolk sac, and fetal liver, whereas only LF-B1 binding site mutations have a marked, albeit variable, effect in adult liver.
Tripodi
M
M
Dipartimento di Biopatologia umana, Università la Sapienza, Policlinico Umbert I, Roma, Italy.
Abbott
C
C
Vivian
N
N
Cortese
R
R
Lovell-Badge
R
R
D000375:Aging / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003360:Cosmids; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009687:Nuclear Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis; D012177:Retinol-Binding Proteins / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D000515:alpha 1-Antitrypsin / Q000235:genetics*
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; C071258:LF-A1 protein, human; D009687:Nuclear Proteins; D012177:Retinol-Binding Proteins; D014157:Transcription Factors; D000515:alpha 1-Antitrypsin; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase