Possible Epigenetic Regulation of Functionally Opposing Groups of Genes Involved in Ex Vivo Expansion of Cord Blood Stem Cells

Recently, we have demonstrated that modifications of chromatin involving DNA methylation and histone acetylation events can change the fate of hematopoietic stem cells (HSC), likely as a result of altered gene expression patterns (Araki H et al. Blood 2007). In our current studies we have examined the epigenetic regulation of genes induced during the expansion of HSC by chromatin modifying agents [5aza-2-deoxycytidine (5azaD), trichostatin A (TSA)]. The expression of the polycomb repressing complex gene, enhancer of zeste homolog 2 (Ezh2) has been implicated in histone methylation and deacetylation. We observed a significant increase in the transcript levels of Ezh2 in addition to self-renewal genes HoxB4, Bmi1 and GATA-2 in 5azaD/TSA treated expanded human cord blood (CB) cells. Ezh2 overexpression has been shown to prevent HSC exhaustion in a murine model by chromatin stabilization. CB CD34+ cells expanded in 5azaD/TSA resulted in retention of relatively higher transcript levels of DNA methyltransferase (DNMT), DNMT1 and lower levels of DNMT3a and DNMT3b in CD34+CD90+ cells, the most primitive subpopulation, in contrast to the relatively mature CD34−CD90− cells. The retention of higher DNMT1, the maintenance methyltransferase, in the most primitive CD34+CD90+ cells may favor the chromosomal stability of expanded grafts. Cytogenetic analysis and DNA ploidy of expanded grafts were normal and the telomere length of expanded CB grafts did not reveal any significant alteration of their lengths in comparison to unmanipulated primary CB grafts. In order to explore the roles of epigenetic modifications on the differentiation of HSC during ex vivo expansion, we have also examined the transcript levels of genes associated with differentiation in 5azaD/TSA expanded cells in comparison to cells expanded in cytokines alone. Interestingly, the transcript levels of differentiation associated genes Pu.1 and GATA-1 decreased following 5azaD/TSA treatment. The lower transcript levels of differentiation inducing genes (Pu.1, GATA-1) and higher levels of self-renewing genes (Ezh2, HoxB4) in 5azaD/TSA expanded cells suggest a possible interaction among these functionally opposing groups of genes which are likely regulated by epigenetic mechanisms. By using a chromatin immunoprecipitation (ChIP) assay we observed significantly increased levels of histone H4 acetylation on the promoter sites of Hox-B4, Bmi-1, and GATA-2 and decreased levels of acetylation on the promoter sites of genes involved in differentiation (Pu.1, GATA-1) in 5azaD/TSA treated CB cells. The degree of histone H4 acetylation of these promoters sites directly correlated with the transcript levels of these genes as evidenced by real time quantitative PCR. Futhermore, silencing of HoxB4 by using short hairpin RNA (shRNA) during 5azaD/TSA mediated expansion resulted in about a 50% reduction in the expansion of CB cells in contrast to cells containing non-silencing control shRNA, indicating HoxB4 is required for HSC expansion. Understanding how the possible interactions between functionally opposing groups of genes regulating the expansion and differentiation of HSC is mediated by epigenetic mechanisms may further facilitate the optimization of ex vivo expansion strategies of CB grafts for therapeutic use.