Introduction Histone deacetylase inhibitors (HDIs) like butyrates increase fetal hemoglobin (Hb F) in b-thalassemia major and sickle cell disease patients through a mechanism that is not well understood. Since HDIs act globally and may activate silenced oncogenes, it is necessary to determine how these compounds increase Hb F in order to design targeted therapeutics to increase Hb F and at the same time minimize potentially harmful side effects.
Purpose To examine the developmental window during which sodium butyrate (NaBut) maximally increases Hb F in adult-derived hematopoietic progenitor cells (HPCs), and to determine the effect of NaBut on histone acetylation patterns across the b-like globin gene cluster.
Hypotheses There is a particular window of opportunity during which NaBut optimally up-regulates Hb F and increases g-globin expression. In addition, the NaBut-mediated increase in Hb F involves a change in occupancy of acetylated histones (acH) at specific sites of the b-like globin cluster.
Methods HPCs isolated from adult peripheral blood are treated with 0.5 mM NaBut on day 1, 2, or 7 of in vitro culture and harvested for protein and total RNA on day 10. Control cells are cultured without NaBut. Cell morphology is determined by Giemsa staining, and ELISA and spectrophotometric measurements are performed to determine percent Hb F. Transcription of g-globin is assessed by RT-PCR. Occupancy of acH across the b-like globin cluster is examined by chromatin immunoprecipitation (ChIP) with anti-acH4 antibody, followed by hybridization of ChIP products to custom-made arrays (chip) containing tiled PCR products spanning the 70 kb b-like globin gene cluster.
Results NaBut treatment of HPCs on day 1, 2, or 7 of culture resulted in Hb F levels of 14% (7-fold increase compared to control cells), 11% (5.5-fold), and 5% (2.5-fold), respectively. Results of g-globin mRNA analyses showed a similar trend, with fold induction decreasing with later exposures to NaBut. Preliminary results of ChIP-chip in control cells showed areas of enrichment and depletion of acH4 across the b-like globin cluster during differentiation.
Conclusion The earlier stages of adult erythroid maturation are more amenable to NaBut-mediated increase in Hb F protein and g-mRNA compared to later stages. Further studies will compare and contrast the histone acetylation pattern seen in adult-derived HPCs with that of NaBut-treated adult cells in order to determine and further examine candidate regions and signal pathways involved in drug-mediated increase in Hb F.
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