| Abstract |
Screening of an expression library for regulatory factors interacting with the -492 to -477 region of the human a-globin promoter (site G7), revealed a cDNA designated as human Cold Shock Domain protein A (hCSDA) and a cDNA identical to a previously described as DNA binding protein B (DbpB) (Sakura et al., 1988). hCSDA is highly homologous to the previously reported DNA binding protein A (DbpA) (Sakura et al., 1988) and identical to DbpAv (Coles et al., 1996). These proteins belong to an ancient family characterized by a highly conserved structural motif, the "Cold Shock Domain" (Schindelin et al., 1993; Schnuchel et al., 1993), which has affinity for a variety of DNA sequence motifs as well as RNA. Members of this family have been identified in a number of organisms including bacteria, (Av-Gay et al., 1992; Jones et al., 1992; Willimsky et al., 1992), plants (de Oliveira et al., 1990) and animals (Tafuri et al., 1993; Tafuri and Wolffe, 1990; Wolffe, 1993; Wolffe et al., 1992). Phylogenetic analysis shows that the "Cold Shock Domain" protein family is highly conserved throughout evolution, and contains members which can be subdivided in distinct subfamilies. hCSDA cDNA fragments were used for chromosomal localization of the hCSDA genes by Southern analysis of monochromosomal human/rodent cell hybrid DNA panel. Isolated genomic clones (YAC and PAC) corresponding to hCSDA sequences were used for FISH analysis revealing an intron-less pseudogene mapped to chromosome 16p11.2. Examination of the specific distribution of hCSDA and DbpB mRNAs in human and mouse tissues and cell lines and in mouse embryos, carried out by Northern analysis, revealed that hCSDA and DbpB genes show differential levels of expression in different tissues and cell lines and their levels of expression decline in late stages of mouse embryo development. In addition, their expression levels are downregulated after differentiation of erythroid and myeloid cell lines (K562, MEL and HL-60). There is an inverse correlation of hCSDA and DbpB mRNA levels to the levels of the α-globin mRNA in differentiated erythroid cell lines (K562 and MEL) which is an indication of possible negative regulation of the α-globin gene promoter by these proteins. Moreover, downregulation of hCSDA and DbpB expression in differentiated cell lines may suggest a possible implication of these proteins in mechanisms underlying cell proliferation. To test DNA binding specificity to the G7 site and determine the consensus binding sequence for ds and ssDNA, hCSDA and DbpB cDNAs were cloned and expressed through bacterial expression vectors and the respective recombinant proteins, were used for DNA-protein binding studies. A 10 fold higher binding affinity was estimated for the ss compared to ds oligonucleotide corresponding to the G7 site. However, the sequence recognition specificity to dsDNA was determined to be more stringent compared to ssDNA. The consensus binding site for both ds and ssDNA is a polypyrimidine stretch which could participate in the formation of triple helix H-DNA structure. A possible way of short distance regulation of transcription by these proteins could be through their binding and stabilization on an H-DNA structure which could prevent the binding of neighboring positive regulators such as the Sp1 factor. In order to define the function of these proteins on the transcriptional activity of the human α-globin promoter, the cDNAs were cloned in eukaryotic expression vectors and used in transient cotransfection experiments together with a variety of α-globin promoter - CAT reporter constructs in K562, COS-7 and HeLa cells. This analysis suggested that hCSDA and DbpB affect negatively the transcription activity of the human α-globin promoter through their binding on the G7 site and similar sequences of the promoter. We also examined protein-protein interactions of hCSDA and DbpB proteins with the Sp1 transcription factor, which has an adjacent binding site to the G7 site, by gel shift analysis, and we identified that both proteins can physically interact with the Sp1 factor. Sp1 is the major positive factor of the α-globin promoter and the region of the promoter containing multiple Sp1 binding sites is necessary for its interaction with the enhancer regulatory element HS-40 (Pondel et al., 1995). It has been speculated that it may participate in LCR mediated erythroid specific activation of globin promoters through its ability to interact with the erythroid specific transcription factor GATA-1 (Merika and Orkin, 1995). GATA-1 binding on HS40 is crucial for HS-40 mediated transactivation of the α-globin promoters (Zhang et al., 1995; Rombel et al., 1995). A possible way of long-range action of hCSDA and DbpB could be through prevention of Sp1-GATA-1 interaction and subsequently the interaction of the HS-40 with the α-globin gene promoters.
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