Abstract
Understanding the mechanism responsible for the developmental regulation of the β-like globin genes would be important in the design of future pharmacologic therapies to increase fetal hemoglobin (HbF) in patients with sickle cell disease and β-thalassemia. The baboon is a valuable and relevant experimental animal model to study the regulation of globin gene expression because the structure of the β-globin gene complex and developmental pattern of globin gene expression are similar to human, and HbF levels are greatly increased following treatment of baboons with the DNA methyltransferase inhibitor Dacogen (5-aza-2′-deoxycytidine; DAC). To investigate the relationship between DNA methylation, chromatin structure and globin gene expression, the pattern of acetylated histone H3 (ac-H3) and H4 (ac-H4) within the β-globin gene complex was compared in purified erythroblasts from baboon fetal liver (FL; n=2) and bone marrow (ABM; n=2) of adult baboons pre and post DAC treatment. HbF increased to high levels (67.8%, 61.9%) in respective animals and methylation of 18 CpG sites within the ε- and γ globin genes was reduced >50% following DAC treatment. Enrichment of ac-H3 and ac-H4 throughout the β-globin gene complex was measured by chromatin immunoprecipitation (ChIP) followed by real time PCR. In FL, equivalent levels of ac-H3 and ac-H4 were observed near the ε-globin and γ-globin promoters that were 3 fold higher than near the Aγ-enhancer and pseudo-β gene and 5–14 fold higher than near the β-globin promoter. In pretreatment ABM, levels of ac-H3 and ac-H4 near the β-globin promoter were 4–6 fold greater than near the γ-globin promoter, Aγ-enhancer, and pseudo-β gene and 10-15 fold higher than near the ε-globin promoter. The lowest levels of histone acetylation were observed in a 6kb subdomain within the γ-β intergenic region extending from the duplicated Alu sequence to 3′ of the δ-globin gene. Following DAC treatment, histone acetylation of the ε-, γ-, and pseudo-β genes and Aγ-enhancer increased 4-10 fold, while histone acetylation of the β-globin gene remained unchanged. This resulted in equivalent levels of histone acetylation associated with the γ-globin gene, Aγ-enhancer, pseudo-β-, and β-globin genes that were 3 fold greater than with the ε-globin gene. The levels of histone acetylation within the 6 kb subdomain of the γ-β intergenic region remained low. Our results suggest that three subdomains of chromatin are present within the baboon β-globin gene complex. One subdomain that encompasses the ε-, γ-, and pseudo-β genes is characterized by high levels of histone acetylation in FL and low levels in ABM. DAC treatment increases histone acetylation within this region to levels observed near the β-globin gene. A second subdomain near the β-globin gene is characterized by high levels of histone acetylation in ABM and low levels in FL. Histone acetylation of the β-globin gene within this subdomain remains high following DAC. A third subdomain found within the γ-β intergenic region surrounding the duplicated Alu sequences is characterized by a low level of histone acetylation in both FL and ABM. The level of histone acetylation of this region remains low following DAC. We conclude that three chromatin subdomains within the β-globin gene complex are differentially sensitive to DAC-induced changes in histone acetylation.
The upstream area of the chicken α-globin gene domain is transcribed in both directions in the same cells