Over the past several years, the transcriptional regulation of major histocompatibility complex (MHC) class II genes has been shown to result from the interaction of cis-acting regulatory elements in the 5' flanking and intronic regions of the class II genes, and a series of transacting factors which can bind to these cis-acting elements. Five motifs of conserved sequence apparently involved in transcriptional regulation have been delineated within the promoter region of the 5' flanking region of class II genes, and these have been denoted the Z, X, and Y boxes, the octamer and the TATA box. The X and Y boxes appear to be critical for efficient transcription. We have developed a methodology based on the Southwestern technique in which a lambda gt11 library is probed with 32P- end labeled double-stranded oligonucleotides corresponding to the regulatory motifs. We have already isolated and sequenced a cDNA encoding a Y box binding protein termed YB-1, and have shown the specificity of YB- 1 for the Y box. In this application, we first propose to characterize YB- 1 with respect to binding and functional parameters. Physical conditions of binding will be assayed by our Southwestern assay, and nucleotides critical to binding will be assayed by the Southwestern, DNAase protection, and methylation interference assays. The inducibility by gamma-interferon of YB-1 mRNA and protein will be assessed respectively by Northern analysis, and by immunoprecipitation of YB-1 with specific antibodies. YB- 1 cDNA will be transfected into inducible U937 cells to test the ability of YB-1 by itself, and later with other binding factors to induce class II expression. The YB-1 genomic gene will be cloned and sequenced, with particular attention to 5' flanking regions potentially involved in the transcriptional regulation of YB-1 itself. cDNA's encoding proteins binding to the other regulatory motifs (the X box, Z box,and octamer) will be isolated by the Southwestern technique and sequenced. The binding proteins will be characterized, and the genomic genes cloned. These experiments should elucidate the mechanism by which proteins binding to the regulatory region of class II genes play a role in class II expression.
