DNA METHYLATION, CHROMATIN AND GLOBIN GENE SILENCING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Increased level of fetal hemoglobin (HbF) is clinically beneficial ill patients with sickle cell anemia. Experiments performed in the baboon model demonstrated that HbF levels could be elevated using pharmacologic agents such as 5-aza-2'-deoxycytidine (decitabine), butyrates, and hydroxyurea. The usefulness of these drugs in patients with sickle cell disease was confirmed in a number of clinical trials. The MSH study demonstrated that hydroxyurea therapy reduced the number of pain crises, incidence of acute chest syndrome, and transfusion requirements in patients. A significant number (10-40%) are refractory to treatment as evidenced by minimal changes in HbF levels. Furthermore, because the increased HbF is distributed heterogeneously among red cells, a large percentage of erythrocytes remain unprotected from intracellular polymerization of deoxy-HbS molecules. New and improved agents and therapies must therefore be developed which increase HbF to higher levels in a greater proportion of patients and maximize the number of F cells produced. It is our goal to develop a better therapeutic regimen for patients with sickle cell disease based upon the use of the demethylating drug decitabine, histone deacetylase inhibitors, and growth factors. We intend to investigate the mechanism of action of these agents by determining the role of DNA methylation and histone acetylation in both the development regulation of globin gene expression and the reactivation of HbF expression in the adult. Analysis of the methylation and histone acetylation status of genes in small numbers of highly purified hematopoietic progenitor cells is now possible using FACS, bisulfite sequencing and immunoprecipitation of formaldehyde-fixed chromatin fragments (CHIP) in combination with PCR. We propose to follow changes in gamma-globin gene expression, DNA methylation, and histone acetylation during fetal development and normal erythroid differentiation, and following augmentation of HbF production induced by administration of decitabine and histone deactylase inhibitors. We will use an in vitro culture system and an in vivo baboon model system that we have used for the past 20 years to study these mechanisms. These studies will define the mechanisms of gamma-globin gene silencing, and will aid in the development of new procedures to augment HbF production in patients with sickle cell disease.