IDENTIFICATION OF 5'-FLANKING SEQUENCE OF RH50 GENE AND THE CORE REGION FOR ERYTHROID-SPECIFIC EXPRESSION

The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes, RHCE and RHD. Rh50 glycoprotein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression of RH50 gene i s restricted to cells with erythroid features. HEL and K562 cells show ed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 fo r Rh. The nucleotide sequence of 5' flanking region of RH50 gene and f unctional promoter assays also supported the erythroid-specific regula tion of the gene, whereas the sequence had lower homology with the pro moter sequence of RH genes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5' flanking sequence. The core promoter of RH50 gene was located within 68bp lengt h from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Kruppel-like facto r were lacking. The inverse GATA motif was the target sequence of GATA -1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-speci fic expression of Rh antigens, but suggest distinct regulatory mechani sms for RH vs RH50 genes. (C) 1998 Academic Press.