Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.
The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.
Moreno
C S
CS
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Emery
P
P
West
J E
JE
Durand
B
B
Reith
W
W
Mach
B
B
Boss
J M
JM
D001483:Base Sequence; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D005802:Genes, MHC Class II; D006207:Half-Life; D006493:Heparin; D006801:Humans; D016393:Lymphoma, B-Cell; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010455:Peptides; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D006493:Heparin