Epigenetic Changes in the γ-Globin Gene Promoter in Hematopoietic Progenitor Cells at Different Stages of Differentiation.
Authors: Singh Mahipal , Vaitkus Kestas , Hankewych Maria , Lavelle Donald , Mahmud Nadim , DeSimone Joseph 
Published: October 2019

Abstract
The role of epigenetic modifications, which represent a second layer of genetic regulation, appear to play an important role during hematopoiesis. How the chromatin structure changes from a preprogrammed hematopoietic stem cell to progressively differentiated cells and how the cell’s inner and surrounding environment affects orchestrating the epigenetic modifications is not clearly understood. This investigation was initiated to determine the pattern of DNA methylation in the γ-globin gene promoter region in the cells isolated from different stages of erythroid development in baboon (P. anubis). Baboon fetal liver hematopoietic stem progenitor cells (HSPC) were purified by passage through a Miltenyi magnetic column (to deplete mature erythroblasts) followed by flow cytometric cell sorting of erythroblast depleted cells into various sub populations depending upon the expression of CD36 antigen. Three types of cell populations i.e. CD34+CD36−, CD34+CD36+ and CD34−CD36+ were collected. Clonal analysis was performed to verify the degree of differentiation of CD34+ cells based on their co-expression of CD36. CFC assays of CD34+CD36−, CD34+CD36+ and CD34−CD36+ cells revealed a cloning efficiency of 89%, 38.5% and 12% respectively, after 12 days of culture using methylcellulose media supplemented with a cocktail of growth factors and serum. Only CD34−CD36+ cells produced detectable CFU-E colonies while CD34+CD36− and CD34+CD36+ generated mostly BFU-Es. CD34+CD36− cells produced about 2 fold more BFU-Es as compared to CD34+CD36+ cell population. Moreover, the colony size also decreased as the cells progressed towards maturation. Our data suggest that CD34+CD36−cells are most primitive erythroid progenitors while CD34−CD36+ cells are the most committed mature erythroid progenitors and the CD34+CD36+ cells represent the intermediate stage. The methylation pattern of 5 CpG sites in the γ-globin promoter region in the above purified cell populations along with erythroblast fraction was assayed using bisulfite sequencing. Two independent fetuses, 56 and 58 days post conception (dpc) were analysed. Bisulfite treated genomic DNA was used to amplify the γ-globin promoter region and the PCR products were subcloned into pCR-TOPO vector. Sequences of fifteen independent clones per sample were analysed. Our results indicated almost complete methylation of the γ-globin promoter region in earlier stages of differentiation and a progressive decrease in methylation as the cells progress towards maturation. The methylation pattern observed was 98.3%, 63.3%, 28.3% and 7.4 % in 58d fetus and 92.6%, 69.2%, 26.3% and 0.0% in 56d fetus in CD34+CD36−, CD34+CD36+, CD34−CD36+ and erythroblast cell populations respectively. Only the −54 CpG site exhibited hypomethylation in the most primitive CD34+CD36− cell population. In conclusion, our results show a progressive decrease in methylation as the HSPC mature into erythroblasts and progressively accumulate more hemoglobin indicating a direct role of DNA methylation in regulation of hemoglobin production. Status of histone methylation and acetylation along with DNA methylation pattern of ε- and β-globin gene promoters in hematopoietic progenitors at various differentiated stages should enhance our understanding of γ-globin gene regulation in a non-human primate model which closely mimics human beings.