Class II box consensus sequences in the HLA-DR alpha gene: transcriptional function and interaction with nuclear...

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DR alpha gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DR alpha promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. This Y box-binding protein was present in nuclear extracts of all cell types examined, including human B and T cells and HeLa cells. The molecular mass of the protein, as determined by photoactivated protein-DNA cross-linking, was approximately 40 to 50 kilodaltons. Mutagenesis of the Y box that decreased protein binding also decreased promoter activity, implying that protein binding to this DNA sequence is important for DR alpha promoter function.