Differences in the Pattern of Histone H3 (K4) and (K27) Methylation throughout the β-Globin Gene Locus in Baboon Fetal Liver and Adult Bone Marrow Erythroblasts Pre- and Post-Decitabine Treatment.

Abstract

Understanding the role of chromatin structure in specifying the pattern of β-like globin gene expression during development would be important in the design of future pharmacologic therapies to increase fetal hemoglobin in patients with sickle cell disease and β-thalassemia. The baboon is an important 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 man, and HbF levels are greatly increased in baboons treated with the DNA methyltransferase inhibitor decitabine (5-aza-2′-deoxycytidine). To investigate the relationship between chromatin structure, DNA methylation, and globin gene regulation, the distribution of acetyl histone H3 (ac-H3), acetyl histone H4 (ac-H4), histone H3 (K4) dimethyl and trimethyl, and histone H3 (K27) dimethyl throughout the β-globin gene locus was determined in purified primary erythroblasts from baboon fetal liver (FL), and adult bone marrow (BM) pre- and post-decitabine treatment. Analysis was performed by chromatin immunoprecipitation (ChIP) of formaldehyde-fixed chromatin followed by real time PCR using 18 primer sets spanning the baboon β-globin gene locus from the 5′ region of the ε-globin gene to the β-globin gene. Comparison of the pattern of ac-H3 and ac-H4 suggested the presence of three subdomains of chromatin within the β-globin locus characterized by different levels of histone acetylation that exhibited a differential response to decitabine treatment. Histone H3 (K4) dimethyl was relatively enriched in the region containing the ε- and γ-globin genes and in the γ-β intergenic region 5′ to the duplicated Alu sequence in FL. Levels associated with the ε-, γ-, and γ-globin genes in adult BM were similar and relatively unaffected by decitabine treatment. In contrast, high levels of histone H3 (K4) trimethylation and pol II distribution were associated with the promoters and transcribed regions of active genes. Differences in the levels of H3 (K4) trimethylation and pol II associated with individual genes were well correlated with differences in their relative levels of expression in FL and adult BM pre- and post-decitabine treatment. The level of histone H3 (K4) trimethyl associated with the promoter of the developmentally inactive ε-globin gene was very low and not enriched compared to inactive necdin gene or the γ-β intergenic regon in adult BM suggesting that the ε-globin gene is not maintained in a “poised” transcriptional state by the presence of the histone H3 (K4) trimethyl mark near the ε-globin promoter. The pattern of histone H3 (K27) dimethyl differed in FL and adult BM. Levels of H3 (K27) dimethyl associated with the ε- and γ-globin genes in FL were 2–4 fold less than near the duplicated Alu sequence in the γ-β intergenic region, while levels were 4–10 fold higher near the ε- and γ-globin genes and γ-β intergenic region compared to the promoter and transcribed region of the β-globin gene in adult BM. Reactivation of γ-globin expression following decitabine treatment was associated with a relative decrease in the level of H3 (K27) dimethyl near the γ-globin gene. Increased H3 (K27) methylation in regions surrounding the silenced ε- and γ-globin genes suggests that the polycomb group (PcG) protein EZH2, a histone H3 (K27) methyltransferase, may be involved in globin gene silencing.