Identification of three NFAT binding motifs in the 5'-upstream region of the human CD3gamma gene that differentially bind NFATc1, NFATc2, and NF-kappa B p50.
Human immunodeficiency virus, type 1 (HIV-1) infection of CD4(+) T cells progressively abrogates T cell receptor (TCR).CD3 function and surface expression by specifically interfering with CD3gamma gene transcription. Our data show that the loss of CD3gamma transcripts begins very early after infection and accumulates to a >90% deficiency before a significant effect on surface receptor density is apparent. Blocking TCR.CD3-directed NFAT activation with cyclosporin A provokes a partial re-expression of CD3gamma gene transcripts and surface complexes in a time- and dose-dependent manner. We have identified three NFAT consensus sequences (5'-GGAAA-3') in the 5'-upstream region of the human CD3gamma gene at: -124 to -120 (NFAT(gamma1)), -384 to -380 (NFAT(gamma2)), and +450 to +454 (NFAT(gamma3)) from the first transcription initiation site. Using electrophoretic mobility shift and supershift assays, we show that NFATc2 alone binds to the NFAT(gamma2) motif; however, complexes containing either NFATc2 or NFATc1 plus NF-kappaB p50 bind to the NFAT(gamma1) and NFAT(gamma3) sites. We further demonstrate that NFATc1 and NF-kappaB p50 bind in the same protein.DNA complex and that a fourth Ala added to the core sequence (5'-GGAAAA-3') in NFAT(gamma1), and NFAT(gamma3) is critical for their binding. Finally, we have shown that an increase in the binding of nuclear NFATc2, NFATc1, and NF-kappaB p50 to these three motifs is correlated with a progressive loss of CD3gamma transcripts after HIV-1 infection.
Badran
Bassam M
BM
Laboratory of Experimental Hematology, Faculty of Medicine, University of Brussels, 121 Blvd. de Waterloo, Brussels B1000, Belgium.
Wolinsky
Steven M
SM
Burny
Arsène
A
Willard-Gallo
Karen E
KE
D020121:5' Untranslated Regions; D020816:Amino Acid Motifs; D001483:Base Sequence; D001667:Binding, Competitive; D017252:CD3 Complex / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004996:Ethidium / Q000494:pharmacology; D005434:Flow Cytometry; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000494:pharmacology; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D020121:5' Untranslated Regions; D017252:CD3 Complex; C452842:CD3 antigen, gamma chain; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; C494006:NFATC2 protein, human; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016572:Cyclosporine; D004247:DNA; D004996:Ethidium