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Abstract: We encountered a homozygous −16C>G mutation in cis at identical positions in the promoters of both human γ-globin genes in a subject who was also homozygous for Hemoglobin C (HbC). Subsequent analysis of normal control individuals of African American ancestry revealed that both mutations were always present in cis with an allelic frequency of 3%. Furthermore, 10 out of 11 HbC subjects carried the −16C>G sequence variations, suggesting an association with HbC. The −16C>G mutation disrupts a putative CACCC box positioned between the TATA box and the transcriptional start site. However, the absence of high levels of HbF in HbC subjects homozygous and heterozygous for the −16C>G sequence variation suggested no effect of this mutation on γ-globin gene expression in the adult stage of development. Further functional characterization by means of transient transfections in human erythroleukemic K562 cells showed that the −16C>G promoter sequence variation did not have an effect on γ-globin expression in the fetal stage of development either. We therefore conclude that the −16C>G γ-globin sequence variations are not beneficial to the clinical phenotype of HbC. The unique concurrent presence of this non-functional sequence variation is likely the result of a gene conversion event, and supports the concept of sequence homogenization between the two human γ-globin genes. [Copyright &y& Elsevier]

Abstract: We have established the functional importance of PKR-RE1, a necessary transcriptional regulatory element in the erythroid-specific promoter of the human pyruvate kinase gene (PKLR). Here, we demonstrate by electrophoretic mobility shift assay (EMSA) that the DNA–protein interaction at PKR-RE1 involves a CTGTC motif. Because the same motif is also present in the erythroid-specific promoter of the hexokinase gene (HK1), we confirmed its functional relevance by in vitro transfection in K562 cells. Moreover, EMSA demonstrated that the CTGTC motif in both the PKLR and HK1 promoters mediates binding of the same protein. Therefore, we postulate a more general role of PKR-RE1 in erythroid-specific gene expression. [Copyright &y& Elsevier]