An enhancer LEF-1/TCF-1 site is essential for insertion site-independent transgene expression in thymus.
Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein-protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.
Haynes
T L
TL
Department of Pediatrics, University of Cincinnati, OH 45229, USA.
Thomas
M B
MB
Dusing
M R
MR
Valerius
M T
MT
Potter
S S
SS
Wiginton
D A
DA
D000619:Aminohydrolases / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D051538:Hepatocyte Nuclear Factor 1-alpha; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000235:genetics; D051822:T Cell Transcription Factor 1; D013950:Thymus Gland / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes
D004268:DNA-Binding Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C497573:LEF1 protein, human; C497574:Lef1 protein, mouse; D051821:Lymphoid Enhancer-Binding Factor 1; D012333:RNA, Messenger; D051822:T Cell Transcription Factor 1; C497576:TCF7 protein, human; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; D000619:Aminohydrolases; C018774:adenine deaminase