Relationships between DNA methylation and expression in erythrocyte membrane protein (band 3, protein 4.2, and beta-spectrin) genes during human erythroid development and differentiation.

Red cell membrane proteins are sequentially expressed during erythroid development and differentiation. Spectrins have already been synthesized in early erythroid precursors such as pronormoblasts, and band 3 (B3) appears at nearly the same stage. Protein 4.1 appears next, followed by protein 4.2 (P4.2) at the very late erythroblast stage. The methylation states of the promoter 5'-CG-3' sites are known to be linked to the regulation of promoter function by modulating DNA-protein interactions and the structure of chromatin. Hence, the genes for B3, P4.2, and beta-spectrin (beta-SP) appear to be suitable models to study the relationship between methylation of promoter 5'-CG-3' sites and the sequential expression of genes during human erythroid development and differentiation. We have examined methylation profiles in the promoter regions of the genes (ELB42, EPB3, and SPTB) for the human erythroid membrane proteins P4.2, B3, and beta-SP by applying the bisulfite genomic sequencing method. Our results demon strate the following: (1) The promoter regions of EPB3 and ELB42 are extensively methylated in DNA from human peripheral blood mononuclear cells, but the SPTB promoter is totally unmethylated. (2) During erythroid differentiation, DNA methylation patterns change as follows: (a) ELB42 is unmethylated in DNA from erythroid-committed blastic cells, such as the human cell lin UT-7/EPO, but is methylated in erythroblasts from peripheral blood burst-forming unit erythroid (BFU-E) in the second phase of the liquid-culture method. Messenger RNA (mRNA) from ELB42 is first detected in early erythroblasts, and P4.2 is expressed in late erythroblasts. (b) In contrast, EPB3 is consistently methylated in UT-7/EPO cells and in cultured erythroblasts from BFU-E from human peripheral blood. B3 mRNA and protein are already expressed in early erythroblasts. (c) SPTB remains unmethylated in human DNA from UT-7/EPO cells and from cultured erythroblasts. These results document the diversity of the reactions of human promoter sequences to the modulating influence of DNA methylation. Whereas the human SPTB promoter conforms to expectations in that it is unmethylated and fully active throughout erythroid development, high levels of promoter methylation correlate with promoter activity for the EPB3 and ELB42 genes during their sequential activation in erythrocyte differentiation.