Complex developmental patterns of histone modifications associated with the human β-globin switch in primary cells
Received 23 January 2009; received in revised form 2 April 2009; accepted 20 April 2009. published online 29 April 2009.
Objective
The regulation of the β-globin switch remains undetermined, and understanding this mechanism has important benefits for clinical and basic science. Histone modifications regulate gene expression and this study determines the presence of three important histone modifications across the β-globin locus in erythroblasts with different β-like globin-expression profiles. Understanding the chromatin associated with weak γ gene expression in bone marrow cells is an important objective, with the goal of ultimately inducing postnatal expression of weak γ-globin to cure β-hemoglobinopathies.
Materials and Methods
These studies use uncultured primary fetal and bone marrow erythroblasts and human embryonic stem cell−derived primitive-like erythroblasts. Chromatin immunoprecipitation with antibodies against modified histones reveals DNA associated with such histones. Precipitated DNA is quantitated by real-time polymerase chain reaction for 40 sites across the locus.
Results
Distribution of histone modifications differs at each developmental stage. The most highly expressed genes at each stage are embedded within large domains of modifications associated with expression (acetylated histone H3 [H3ac] and dimethyl lysine 4 of histone H3 [H3K4me2]). Moderately expressed genes have H3ac and H3K4me2 in the immediate area around the gene. Dimethyl lysine 9 of histone H3 (H3K9me2), a mark associated with gene suppression, is present at the ε and γ genes in bone marrow cells, suggesting active suppression of these genes.
Conclusion
This study reveals complex patterns of histone modifications associated with highly expressed, moderately expressed, and unexpressed genes. Activation of γ postnatally will likely require extensive modification of the histones in a large domain around the γ genes.
aDepartment of Microbiology and Immunology, and Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH., USA
bDepartment of Medicine, Department of Pharmacology and Toxicology, and Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH., USA
cEinstein Center for Human Embryonic Stem Cell Research, Department of Medicine, Hematology and Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY., USA
Offprint requests to: Steven Fiering, Ph.D., Department of Microbiology and Immunology, Dartmouth Medical School, 622 Rubin Building, 1 Medical Center Drive, Lebanon, NH 03756
ABSTRACT