Polymorphic Pvu II Sites in the Stage-Selector Elements of the Baboon (P. anubis) γ-Globin Gene Promoter May Determine HbF Levels by Reducing DNA Methylation Density.
Authors: Lavelle Donald , Singh Mahipal , Vaitkus Kestas , Hankewych Maria , Wilson Lauren , DeSimone Joseph 
Published: October 2019

Abstract
Phylogenetic footprinting studies previously identified conserved CpG residues in the γ-globin promoter in all simian primates that express the γ-globin gene during the fetal period of development that are absent in prosimians that express this gene during the embryonic period. (Tagle et al, J Mol Biol203:439–455, 1988). These CpG residues are methylated in adults but unmethylated in fetal tissues expressing the γ-globin gene. Reactivation of HbF synthesis in adults following treatment with the DNA methyltransferase inhibitor decitabine suggests that methylation of these CpG residues in adults may be instrumental in silencing the γ-globin gene. Sequence analysis of the baboon γ-globin gene promoter isolated from baboon (P. anubis) white blood cell DNA lambda phage libraries identified differences between the baboon and other simian primates in the nucleotide sequence at two of these CpG sites located at −51 and +16 that result in a CpG to CpA change at both positions in the baboon. At both positions these nucleotide sequence differences introduce new Pvu II sites. Three subspecies of baboons, P. anubis (n=12), P. cynocephaus (n=6) and P, hamadryas (n=6) were genotyped for the presence or absence of these Pvu II sites in the γ-globin gene promoter. Pvu II sites were detected in the γ-globin promoters of all baboons and were not present in Macaca mullatta (n=2). Individual baboons differed in the number and arrangement of polymorphic Pvu II sites. Both polymorphic sites are located in regions previously reported to act as stage selector elements (Jane et al, EMBO J11:2961–2969, 1992; Amrolia et al, J Biol Chem270:12892–12898, 1995). A family of methylated DNA binding proteins that bind methylated CpG residues and act as transcriptional repressors has been described. Repression of transcription by MeCP2, one member of this family, depends on the density of DNA methylation of the promoter region. MeCP2 loses the ability to repress transcription when the density of methyl CpG residues is reduced to <1 per 100bp (Nan et al, Cell88:471–481, 1994). Therefore we investigated the effect of these polymorphisms in combination with decitabine treatment on methylation density of the γ-globin gene promoter. Bisulfite sequence analysis of DNA obtained from erythroid precursors purified from bone marrow aspirates obtained pre- and post-treatment was performed. In pre-treatment samples obtained from two phlebotomized baboons, HbF levels were 5.4% and 9.0% and γ-globin promoter DNA methylation densities (mCpG/100bp) were 2.8 and 2.0 respectively. Following decitabine treatment (0.52mg/kg/day; 10–13d sc), HbF levels rose to 51.1% and 76% corresponding to respective DNA methylation densities of 0.85 and 0.95. High-level reactivation of the γ-globin promoter is therefore associated with a reduction in the DNA methylation density of the γ-globin promoter to <1 in purified bone marrow erythroid precursors suggesting that MeCP2 or a similar methylated DNA binding protein may repress γ-globin gene expression. We hypothesize that these polymorphisms contribute to the strong HbF response to hematopoietic stress and pharmacological agents of the baboon, compared to human, by reducing the DNA methylation density of the γ-globin gene promoter.