MODIFICATION OF HEMATOPOIETIC STEM CELL DIFFERENTIATION USING INHIBITORS OF HISTONE MODIFYING ENZYMES
Gabriela Sanchez, E Camilla Forsberg, Rebekah Sousae, Fernando Ugarte.
University of California, Santa Cruz, Santa Cruz, CA.
Hematopoietic stem cells (HSCs) uniquely possess the ability to self-renew and produce all blood lineages. During differentiation, key changes in chromatin occur as HSCs differentiate toward final mature lineages; their relatively open chromatin converts to increasingly more closed heterochromatin. To identify the role of specific chromatin modifiers during HSC differentiation, inhibitors were used to target histone and DNA methylation. We focused on 4 chromatin-modifying inhibitors: SGI-1027, EPZ-5676, Decitabine, and DZNEP. In addition, we used inhibitor UNC0638 as a comparison for the other inhibitors. In vitro treatment of HSCs with UNC0638 shows an increase in stem/progenitor cells, defined as ckit+lin-Sca1+ (KLS) cells. UNC0638 targets the G9a histone methyl transferase, G9a-mediated methylation of histone H3 promoting heterochromatin formation. Each inhibitor was combined with UNC0638 to investigate whether our observed accumulation of KLS is specific to G9a inhibition. To test whether the accumulated KLS cells were functional hematopoietic stem and progenitor cells, we transplanted these populations into irradiated mice and analyzed their ability to reconstitute mature blood lineages. Cell sample data for the mice with just the UNC0638 drug confirmed that the cells are, in fact, KLS cells. Each of the other 4 inhibitors show similar results but at a much lower KLS percentage and, when treated in sync with UNC0638, the KLS fraction represented the same results as those of the UNC0638 only. These results highlight the importance of G9a in the transition from HSCs to more mature lineages and emphasize the role of chromatin condensation during stem cell differentiation.