No abstract

The expression of HLA class H genes is regulated by a series of cis-acting elements and trans-acting factors. Several cis-acting elements have been identified and have been termed the Z box, X box, Y box, octamer, and "TATA" box. The Y box contains an inverted CCAAT box. By probing a phage Agtii library with double-stranded oligonucleotides, we have directly isolated a cDNA encoding a Y box-binding protein designated YB-1. YB-1 binding has an absolute requirement for the CCAAT box and relative specificity for the Y box. It has a M, of 35,414, contains 18% basic residues, and contains putative nuclear localization signals. An inverse correlation of YB-1 and HLA-DR (3 chain mRNA levels suggests that YB-1 is a negative regulatory factor.The transcription of major histocompatibility complex class II genes is regulated by a series of cis-and trans-acting elements (1). For class II genes, the majority of the cis-acting elements has been localized to the 5' flanking and intronic regions (2, 3). The promoter region within the 5' flanking region contains a series of sequence motifs that are highly conserved among all class II genes and the invariant chain gene (1-4). These motifs have been termed the Z box, X box, Y box, octamer, and "TATA" box. The Y box contains an inverted CCAAT box. The functional role of these motifs has been explored by using transfection and transgenic systems. Both the X box and Y box have been shown to be essential for transcription, while y interferon inducibility has been ascribed variably to the X box, Y box, and Z box (1,(5)(6)(7)(8) (12). ds Oligonucleotide Synthesis. ds oligonucleotides were prepared as described (11). Either the complementary strands were first hybridized and then 'y-32P-end-labeled, or each strand was y-32P-end-labeled and then hybridized. The labeled ds oligonucleotides were purified by gel electrophoresis through a 15% polyacrylamide gel.Filter Hybridization. The amplified library was plated, and the plates were overlaid with nitrocellulose filters saturated with 10 mM isopropyl thiogalactoside. Filters were prehybridized with 5% Carnation instant milk in 10 mM Hepes (pH 8.0) for 1 hr at room temperature and then washed twice for 10 min with 10 mM Hepes (pH 8.0) (13). Hybridization was done in 250 mM NaCl/5 mM MgC12/1 mM dithiothreitol/0.1 mM EDTA/10 mM Hepes, pH 8.0, containing 10 .tg of salmon sperm DNA or 10 gg of poly(I-C) per ml. After 10 min, 108 cpm of end-labeled ds oligonucleotide was added, and the incubation was continued for 2 hr at room temperature. Filters were washed twice for 10 min with hybridization buffer at room temperature and were autoradiographed.Molecular Weight Estimation of the Fusion Protein 13-Galactosidase-YB-1. Escherichia coli strain Y1088 or Y1090 (108) was infected with 106 plaque-forming units of the phage Agtll-YB-1 and grown at 370C until partial lysis was achieved.

On the non-autoimmune C57BL/6 (B6) background, the chromosome 7-derived lupus susceptibility loci Sle3 and Sle5 have been shown to mediate an elevated CD4:CD8 ratio with an increase in activated CD4+ T cells, decreased susceptibility to apoptosis, and a break in humoral tolerance. Development of subcongenic strains has subsequently shown that the elevated CD4:CD8 ratio is due to Sle3 but that both loci contribute to the development of autoantibodies. To elucidate the functional expression patterns of these loci, adoptive transfer experiments were conducted. All possible combinations of bone marrow reconstitution, including syngenic, were conducted between the congenic B6 and B6.Sle3/5 strains. It was found that the Sle3/5 locus was functionally expressed by bone marrow-derived cells, but not by host cells, and that the elevated CD4:CD8 phenotype could be reconstituted in radiation chimeras. Using Ly5-marked congenic strains and B6 host mice, additional experiments surprisingly demonstrated that the elevated CD4:CD8 ratio was neither an intrinsic property of the T cells nor of single positive thymocytes. Allotype-marked chimeras indicated that autoantibody production by B cells was also an extrinsic property, as shown by the fact that B cells without the Sle3/5 interval contributed to autoantibody production. These experiments strongly suggest that a gene within the B6.Sle3/5 interval was expressed by a bone marrow-derived, nonlymphocyte population in the thymus and periphery and was affecting T cell selection and/or survival.