Abstract
The maintenance of undifferentiated state and ability for self-renewal constitutes key properties of hematopoietic stem cell (HSC). To explore the possibility that epigenetic modification contributes to regulation of HSC functions, we have studied whether distinctive epigenetic modification can be correlated to different functions of hematopoietic cells.As a first approach, the global DNA methylations in non-coding repetitive elements were examined using mIAP, mEtn, and mc.satellite regions as marker loci. Sequencing of sodium bisulfite-modified CpG islands in these loci showed that most primitive Lin-c-kit+CD34− cells displayed highest level of DNA methylation as compared to the progenitor-enriched Lin-c-kit+CD34+ cells or differentiated counterpart (Lin+ cells) in mc satellite regions (85.7%, 60 %, 65%, respectively) and mEtn regions (73%, 58%, 74%, respectively), but not in mIAP region. Interestingly, whereas most primitive Lin-c-kit+CD34− cells expressed highest level of methyl cytosine binding protein (MeCp2) or DNA methyl transferase 3 (DNMT3a, 3b), the Lin+ cells expressed bare to minimal level of these gene products despite their high maintenance of DNA methylation, suggesting a differential de-novo methylations between these cells.Similarly, the most primitive Lin-c-kit+CD34− cells exhibited highest level of total acetylated histone (Ac-H4) but these cells expressed also high levels of histone deacetylase (HDAC) as well as histone acetyl transferase (HAT). Subsequent pulse-labeling with C14-acetate demonstrated that immature bone marrow cells (lin-), but not mature Lin+ cells, exhibited active acetylation of H4 with higher turn-overs, thus showing active remodeling of chromatin structures in immature hematopoietic cells. Next, to explore whether alterations in epigenetic modification could influence HSC function, the effect of epigenetic blockers (5-Azacytidine or TSA) were examined for their influence on in-vivo self-renewing activity of transplanted HSCs. Thus, recipients that had been lethally irradiated and transplanted with congeneic HSCs were treated with blockers during first two weeks of recovery, and transplanted into secondary recipients 18 weeks later to determine CRU numbers regenerated. The result of this CRU assay revealed that HSCs had underwent 32-folds higher self-renewal with inhibition of HDAC (22 vs. 738 CRUs), and 11-folds higher self-renewal with inhibition of DNA methylation (22 vs. 248 CRUs) showing that self-renewing potential of “stimulated” HSC is regulated by epigenetic modification. Next, to see if stem cell fate can be also influenced by epigenetic reprogramming, the effects of epigenetic blockers during “stationary” phase of hematopoiesis were examined by treating donor mice with epigenetic blockers for 3 weeks before sacrifice. Limiting dilution transplantation of these marrows revealed that the CRU frequencies in the treated (5-Azacytidine + TSA) donor marrows were 7-folds higher compared to the un-treated donor marrow cells (1/22,9000 vs. 1/3000) in the absence of increase in total marrow cell numbers, suggesting de-novo generation of CRUs with epigenetic manipulations.Taken together, these results show that the epigenetic modification should be an important regulatory mechanism for self-renewal and fate decisions for normal HSCs in-vivo.
Published Version
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