Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box.

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.

Identification and Characterization of TF1phox, a DNA-binding Protein That Increases Expression of gp91phox in PLB985 Myeloid Leukemia Cells

“…Y box-binding proteins, a family of highly conserved RNA-binding proteins (55,56), bind to the MHC class II gene promoter (57) and repress transcription (58). Myef-2, identified by screening a cDNA library for proteins binding the myelin basic protein gene promoter, had Two independent binding assays demonstrated that TF1 phox specifically recognized the BID2-binding site sequence.…”

MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition

“…The p90 ribosomal S6 kinase (RSK) (Stratford et al, 2008), and to a lesser extent AKT (Sutherland et al, 2005), can phosphorylate YB-1 on S102, promoting its nuclear translocation. Within the nucleus, YB-1 binds to inverted CCAAT boxes of genes (Didier et al, 1988) including MDR1 (Bargou et al, 1997), EGFR (Wu et al, 2006), PIK3CA , MET ) and CD44 to increase their transcription and thereby promote tumor progression. Elevated P-YB-1 S102 decreases the sensitivity of HER2-positive breast cancer cells to trastuzumab (Dhillon et al, 2010); however, the intracellular mechanisms underlying this YB-1-mediated trastuzumab resistance remain unclear.…”

The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1

“…Both serine/threonine kinases AKT (Sutherland et al, 2005) and the p90 ribosomal S6 kinase (RSK; Stratford et al, 2008) phosphorylate YB-1 on its S102 residue, thereby altering its nuclear trafficking. Within the nucleus, YB-1 binds to the inverted CCAAT elements known as YB-1-responsive elements (YREs) to affect transcription (Didier et al, 1988). Given the importance of YB-1 in breast cancer we sought to understand how it globally regulates gene expression and therefore a genome-wide promoter screen was performed using the NimbleGen chromatin immunoprecipitation (ChIP)-onchip (COC) platform (Finkbeiner et al, 2009).…”