B-cell factor 1 is required for optimal expression of the DRA promoter in B cells.

The X box in the DRA promoter of the human histocompatibility complex is required for expression of the DRA gene in B cells. We show that a B-cell factor binds to a sequence that is clearly distinguishable from binding sites for the previously described X box binding nuclear proteins RF-X, NF-X, NF-Xc, NF-S, hXBP, and AP-1. Mutations in the DRA X box that disrupt the binding of this factor result in a lower level of gene expression, as does the presence of Id (a trans-dominant regulatory protein that negatively regulates helix-loop-helix proteins). Furthermore, this factor is recognized by antibodies directed against the helix-loop-helix protein A1, a mouse homolog of the immunoglobulin enhancer binding proteins E12/E47, and it binds to sequences in other genes that were previously shown to bind these proteins. By these criteria, this factor is BCF-1.     

Mutational analysis of the DRA promoter: cis-acting sequences and trans-acting factors.

Class II major histocompatibility genes are expressed at high levels in B lymphocytes and are gamma interferon (IFN-gamma) inducible in many other cells. Previously, we observed that DRA promoter sequences from positions -150 to +31 determine the tissue specificity of this class II gene. Moreover, Z and X boxes located between positions -145 and -87 conferred B-cell specificity and IFN-gamma inducibility upon a heterologous promoter. In this study, sequences from positions -145 to -35 in the DRA promoter were systematically mutated by using oligonucleotide cassettes. Z (-131 to -125), pyrimidine (-116 to -109), X (-108 to -95), Y (-73 to -61), and octamer (-52 to -45) boxes were required for B-cell specificity and, with the exception of the octamer box, for IFN-gamma inducibility. Z box and sequences flanking Z and X boxes helped to determine low levels of expression in T and uninduced cells. In phenotypically distinct cells, shared and distinct proteins bound to these conserved upstream sequences. However, few correlations between expression and DNA-binding proteins could be made. Similar proteins bound to Z and X boxes, and the Z box most likely represents a duplication of the X box.     

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

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.     

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.