Purification by DNA affinity chromatography of two polypeptides that contact the NF-AT DNA binding site in the interleukin 2 promoter.
NF-AT is a lymphoid-specific transcription factor that is though to be largely responsible for determining the cell type-specific expression of the interleukin 2 (IL2) gene. Using two different purification schemes, we found that the NF-AT binding site present in the IL2 promoter is the target for two distinct polypeptides with a relative molecular weight of 45,000 and 90,000. Direct extraction of NF-AT binding activity from highly purified nuclei confirms that p45 and p90 are components of the NF-AT complex. UV-induced covalent cross-linking of p45 and p90 to the NF-AT sequence indicates that both polypeptides interact with DNA. We demonstrate using in vitro transcription that antibodies to subunits p45 and p90 strongly reduce activation of an artificial promoter carrying three NF-AT recognition sites. The complex we have purified has the potential to interact with cis-acting elements identified in the promoter of genes expressed early during T cell activation and to the HIV long terminal repeat. We also show that phosphorylation modulates interaction of NF-AT to its cognate binding site. All together, our data suggest that p45 and p90 form the constitutively expressed nuclear factor(s) proposed by Yaseen et al. (Yaseen, N.R., Maizel, A. L., Wang, F., and Sharma, S. (1993) J. Biol. Chem. 268, 14285-14293).
Corthésy
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Institut de Biologie Animale, Université de Lausanne, Switzerland.
Kao
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D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011687:Purines / Q000378:metabolism; D012492:Salts; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011687:Purines; D012492:Salts; D014157:Transcription Factors; D004247:DNA