Abstract

The β-globin locus contains a number of developmentally regulated genes that undergo switching of expression. In the embryo the ε-globin gene is expressed with a subsequent switch to γ-globin in the fetal liver, followed by a final switch to β-globin in definitive cells. Recently, the importance of chromatin structure in regulating gene expression has been recognized, and it has been proposed that modifications of histones (i.e. acetylation, methylation) have a direct effect on gene regulation. This hypothesis would suggest that changes in the histone code underlie the differences in globin gene expression during development. To address this we studied the pattern of histone acetylation across the β-globin locus at different stages of human development. Two different time points of human fetal erythropoiesis (approximately 55dpc and 98dpc) were studied. At these stages the level of γ mRNA is approximately 98%, there is no detectable ε mRNA, while β mRNA is about 2% of total mRNA. The adult stage of erythropoiesis was represented by erythroblasts derived from cultured adult CD34+ cells where γ mRNA was approx. 25% of total mRNA. Nuclei from the fetal and adult erythroblasts were analyzed by Chromatin Immunoprecipitation (ChiP) using antibodies to histone H3 acetylation and H4 acetylation. Quantification of enrichment was carried out by SYBR green real-time PCR, with PCR primers spanning the globin locus. The LCR region showed high levels of enrichment for acetylation in all stages of development. The ε-globin gene showed minimal levels of enrichment in all stages of development. In the 55dpc human erythroblasts γ-globin showed the highest level of enrichment of all globin genes, with the acetylation spreading across the promoter as well as the whole γ-coding region. In the 98dpc fetal erythroblasts, the acetylation at γ-globin was also enriched.

In the cultured adult erythroblasts a reduced level of γ-globin acetylation was present, which was expected since there was less γ-globin synthesis in these samples. There was a higher level of acetylation at the δ- and β-globin genes in the adult cells, as would be expected for the adult stage of development. These results demonstrate that dynamic changes in histone acetylation take place as the human β-globin gene region undergoes its developmental switches. Whether these changes represent primary events in the course of globin gene switching remains to be determined.

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