Affinity enrichment and functional characterization of TRAX1, a novel transcription activator and X1-sequence-binding protein of HLA-DRA.
The promoters of all class II major histocompatibility (MHC) genes contain a positive regulatory motif, the X element. The DNA-binding proteins specific for this element are presumed to play a critical role in gene expression, although there is a paucity of functional studies supporting this role. In this study, the X-box-binding proteins of HLA-DRA were affinity purified from HeLa nuclear extracts. Fractions 46 to 48 contained an X-box-binding activity and were determined by electrophoretic mobility shift assays to be specific for the X1 element. This X1 sequence-binding-protein, transcriptional activator X1 (TRAX1), was shown to be a specific transcriptional activator of the HLA-DRA promoter in an in vitro transcription assay. By UV cross-linking analysis, the approximate molecular mass of TRAX1 including the bound DNA was determined to be 40 kDa. When the TRAX1 complex was incubated with antibodies against a known recombinant X-box-binding protein, RFX1, and tested in electrophoretic mobility shift assays, TRAX1 was neither shifted nor blocked by the antibody. Further analysis with methylation interference showed that TRAX1 bound to the 5' end of the X1 sequence at -109 and -108 and created hypersensitive sites at -114, -113, and -97. This methylation interference pattern is distinct from those of the known X1-binding proteins RFX1, RFX, NF-Xc, and NF-X. Taken together, our results indicate that TRAX1 is a novel X1-sequence-binding protein and transcription activator of HLA-DRA.
Itoh-Lindstrom
Y
Y
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295.
Peterlin
B M
BM
Ting
J P
JP
D001483:Base Sequence; D002460:Cell Line; D002843:Chromatin / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006684:HLA-DR Antigens / Q000378:metabolism*; D059805:HLA-DR alpha-Chains; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000302:isolation & purification* / Q000378:metabolism; D014157:Transcription Factors
D002843:Chromatin; D004268:DNA-Binding Proteins; D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C091105:transcriptional activator X1