AFFINITY ENRICHMENT AND FUNCTIONAL-CHARACTERIZATION OF TRAX1, A NOVELTRANSCRIPTION ACTIVATOR AND X1-SEQUENCE-BINDING PROTEIN OF HLA-DRA

The promoters of all class II major histocompatibility (MHC) genes con tain a positive regulatory motif, the X element. The DNA-binding prote ins specific for this element are presumed to play a critical role in gene expression, although there is a paucity of functional studies sup porting this role. In this study, the X-box-binding proteins of HLA-DR A were affinity purified from HeLa nuclear extracts. Fractions 46 to 4 8 contained an X-box-binding activity and were determined by electroph oretic mobility shift assays to be specific for the X1 element. This X 1 sequence-binding-protein, transcriptional activator X1 (TRAX1), was shown to be a specific transcriptional activator of the HLA-DRA promot er in an in vitro transcription assay. By UV cross-linking analysis, t he approximate molecular mass of TRAX1 including the bound DNA was det ermined to be 40 kDa. When the TRAX1 complex was incubated with antibo dies against a known recombinant X-box-binding protein, RFX1, and test ed in electrophoretic mobility shift assays, TRAX1 was neither shifted nor blocked by the antibody. Further analysis with methylation interf erence showed that TRAX1 bound to the 5' end of the X1 sequence at -10 9 and -108 and created hypersensitive sites at -114, -113, and -97. Th is methylation interference pattern is distinct from those of the know n 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.