Distinct Epigenetic Modifications of Hematopoietic Cells Define Undifferentiated Status and Susceptibility to Epigenetic Reprogramming.

Epigenetic modification is a key regulatory element in cell fate decision and development, but their role in hematopoietic regulation remains unclear. To characterize the epigenetic status of undifferentiated hematopoietic cell populations, we compared genome-wide DNA methylation pattern of human umbilical cord blood-derived CD34+ and CD34- cells. The methylated CpG-enriched DNA fractions were immunoprecipitated from CD34+ andCD34- cells using antibody against methylated CpG dinucleotide, and analyzed through microarray-based platform (244K Human CpG Island Microarray, Agilent). Compared to CD34- cells, CD34+ cells exhibited a characteristic hypomethylation pattern over the regions around 200 bp of the transcription start site (TSS). The hypomethylation around TSS was observed for both CpG island (CpGI) positive and negative loci. However, TSS of CpGI negative loci were flanked by hypermethylation in the regions up to 1 kb of the TSS, indicating a bivalent configuration of DNA methylation in the CD34+ cells. Interestingly, targets of pluropotent genes including those of NOS (Nanog, Oct-4 and Sox-2) exhibited similar bivalent DNA methylation in CD34+ hematopoietic cells. We next compared the histone modification of CD34+ and CD34- cells. CD34+ cells exhibited higher levels of histone acetylation (H4-Ac) and active form histone modifications (H3K4-methyl), whereas CD34- cells were enriched with repressive histone modification (H3K9-methyl).Importantly, pulse-chase labeling assay showed that CD34+ cells are under higher turn-overrate of histone acetylation than CD34- cells, indicating a dynamic maintenance of open chromatin structure in undifferentiated hematopoietic cells. Next, to investigate the regulatory roles of epigenetic modification in hematopoietic cells, we examined the effects of epigenetic blockers, 5-Azacytidine (AZA) and trichostatin (TSA) on hematopoietic cells at different stage of differentiation. For effects on in-vivo repopulating cells, murinebone marrow cells were transplanted into irradiated recipient mice and recipients were i.p. injected with TSA or AZA for 2 weeks. CRU assays performed 18 weeks after the transplantation showed that TSA, rather than AZA increased HSCs during reconstitution(22, 738, and 248 CRUs for control, TSA, and AZA group, respectively). When examined for in-vitro culture, primitive Lin-Sca-1+c-kit+ (LSK) cells treated with TSA and AZA for 5days retained significantly higher levels of undifferentiated cells than control (45% vs. 12%of LSK cells for treated and control group, respectively, p<0.05), indicating their effects for maintenance of undifferentiated states. Next, more differentiated cells were tested for reprogramming to undifferentiated cells. When Lin-Sca-1-c-kit- cells were treated with AZA/TSA, extensive apoptosis was seen in 24 hrs (82%), but 10% of the surviving cells acquired LSK phenotype. Similarly, terminal differentiated cells (Sca-1-Mac-1/Gr-1+, orSca-1-B220+) exhibited 95% of cell death by AZA/TSA treatment underwent, but 30-50% of the surviving cells acquired lineage-negative phenotypes, indicating a phenotypicde-differentiation. However, transplantation of these de-differentiated cells did not show multi-lineage repopulation in the recipient mice, indicating a limitation in their de-differentiation to the HSC level. Taken together, un-differentiated state of hematopoietic cell is facilitated by a characteristic epigenetic modification with dynamic maintenance of open chromatin structures, whereas differentiated state is maintained by stable, condensed chromatin structures with resistance to the alteration in epigenetic modifications or cell fate changes for de-differentiation.