γ-Globin Gene (HBG) Promoter Methylation and Gene Expression Is Affected by the Micro-Environment In Adult but Not Fetal Erythroid Cells

During adult erythroid differentiation, there is a period of transient γ-globin (HBG) gene expression that coincides with transient DNA hypomethylation of the HBG gene promoter. This precedes the final pattern of high β-globin (HBB) gene expression in terminally differentiated adult erythroid cells (the ‘maturational switch’). Possibly, the micro-environment can modulate the period of transient HBG promoter hypomethylation and gene expression, which could have implications for efforts to sustain HBG expression for therapeutic objectives. To evaluate the responsiveness of the maturational switch to external factors, CD34+ hematopoietic progenitor cells from adult baboon bone marrow were cultured with and without AFT024 murine fetal liver stromal cell line feeder layers, as the micro-environmental variable.

All cultures contained Iscove's media with 30% fetal bovine serum, 200ng/ml stem cell factor, 2U/ml erythropoietin, and 1μM dexamethasone. Globin chain synthesis was measured by biosynthetic radiolabelling of globin chains on d11 and d14 followed by HPLC separation, HBG transcript levels were determined by real time PCR, and F cells were analyzed by flow cytometry. Results: γ-globin chain synthesis was significantly lower in the presence of AFT024 feeder layers (0.07±0.05 γ/γ+β, n=4 versus 0.34±0.09 γ/γ+β, n=4, mean±SD, p<0.01). HBG mRNA was also significantly lower in the presence of AFT024 feeder layers (0.09±0.05 γ/total β-like globin mRNA, n=3, versus 0.32±0.10 γ/total β-like globin mRNA, n=3, p<0.025). Accordingly, F cell numbers were substantially decreased in the presence of feeder layers (38 versus 64%). Cell growth was similar in the presence and absence of feeder layers (115±48 versus 86±8.5 fold expansion, n=3). To test whether decreased γ-globin expression in the presence of ATF024 feeder cells is secondary to preferential expansion of more differentiated progenitors less capable of HBG expression, BM cells were fractionated into CD34+ CD36+ and CD34+ CD36- subpopulations prior to culture with and without feeder layers. Expression of HBG in both subpopulations was similarly reduced by the feeder layers, suggesting that decreased HBG expression was due to a direct effect of the feeder layer rather than to selective expansion of a more differentiated subpopulation. Bisulfite sequence analysis was performed to determine if differences in HBG expression were associated with differences in DNA methylation. DNA methylation of 5 CpG residues in the HBG promoter in purified erythroid cells from two independent cultures was 97.6 and 97.7% in the presence of feeder layers and 68.6 and 69.7% without feeder layers. Addition of the DNA methyltransferase inhibitor decitabine (0.5 × 10^-6 M), to cells cultured in the presence of feeder layers increased γ-globin synthesis nearly tenfold (0.60 γ/γ+β) compared to untreated controls (0.063 γ/γ+β), consistent with mechanistic role for DNA methylation in HBG repression associated with feeder layer culture. In contrast to the results with adult-derived CD34+ BM cells, cord blood derived CD34+ cells sustained high levels of HBG expression (0.84 γ/γ+β)in the presence of feeder layers. Conclusion: The switch from HBG to HBB gene expression that occurs during the differentiation of adult erythroid cells is responsive to the micro-environment. Furthermore, this switch depends on DNA methylation, and depleting DNMT1 counteracts the micro-environmentally induced switch to sustain γ-globin expression in adult erythroid cells. In contrast to adult erythroid cells, fetal erythroid cells were resistant to micro-environmental induction of a HBG to HBB switch, consistent with previous studies showing that intrinsic differences between adult and fetal erythroid cells are a critical component in developmental stage-specific differences in globin gene expression.

No relevant conflicts of interest to declare.