Polycomb Group Protein-Mediated Bivalent Domains Regulate Pluripotency of Oct4⁺ Very Small Embryonic-Like (VSEL) Stem Cells In Adult Bone Marrow

Abstract 4788

Recently, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult bone marrow (BM; Leukemia 2006;20:857). These Oct4⁺CXCR4⁺SSEA-1⁺Sca-1⁺CD45^-Lin^- VSELs are capable of differentiation in vitro into cells of all three germ lineages and are able to form spheres (VSEL-derived spheres[VSEL-DS]) composed of primitive stem cells in co-cultures with C2C12 myoblasts. Cells isolated from VSEL-DS are also able to differentiate into all three germ layers (cardiomyocytes, neural cells, and insulin-producing cells). Our previous epigenetic study proved that VSELs exhibit open chromatin structures in the promoters of pluripotent stem cell markers, such as Oct4 and Nanog (Leukemia 2009;23:2042). However, it still remains unclear whether the pluripotency of VSELs could be orchestrated by a mechanism similar to that seen in embryonic stem cells (ESCs). To address this issue, we evaluated whether adult BM-derived VSELs, in a similar way as ESCs, display bivalent-domain–marked histone modifications in the promoter region of homeodomain-containing developmental master transcription factors (TFs), such as Dlx-, Irx-, Lhx-, Pou-, Pax-, and Six-family proteins. To overcome the problem that VSELs sorted by FACS are heterogeneous, we constructed a cDNA library from a small number of cells (~25) by employing the protocols used for single-cell analysis (Nat Protoc 2007;2:739). Analysis of this cDNA library indicated that Oct4⁺ VSELs highly express several epigenetic regulators, especially polycomb repressive complex 2 (PRC2) proteins, including Ezh2, Suz12, and Eed. It is known that PRC2, due to its histone methyltransferase activity on lysine27 of histone3 (H3K27), is responsible for transcriptionally repressive trimethylation of H3K27 (HKK27me3) in promoters for several homeobox domain-containing developmental TFs, such as Sox21, Nkx2.2, Dlx1, Zfpm2, Irx2, Lbx1h, Hlxb9, and Pax5. Moreover, we observed that most of these promoters are “bivalent” and marked with the transcriptionally active histone code that includes trimethylated H3K4 together with H3K27me3, similarly to ESCs. Such bivalent epigenetic marks prevent the premature expression of these TFs in freshly isolated quiescent VSELs. Furthermore, we noticed that during formation of VSEL-DS, Ezh2 expression was decreased and most of the bivalent domains in VSELs were transformed into unmodified histone marks, resulting in the expression of some bivalent-targeted genes, including Hlxb9, HoxA3, and Evx1. Moreover, most of the pluripotent SC markers were also reduced during VSEL-DS progression, which was paralleled by inactivation of the Oct4 promoter. In conclusion, our results suggest that the pluripotency of adult tissue-derived VSELs is modulated, in a similar manner as in ESCs, by transient repression of developmental master TFs through bivalent epigenetic marks. Although PRC2 proteins stimulate cellular proliferation and are highly expressed in various tumors, unique epigenetic reprogramming of genomic imprinting maintains VSEL quiescence under steady-state conditions and also protects these cells from teratoma formation (Leukemia 2009;23:2042). Thus, we suggest that attenuation of this protective mechanism in VSELs, for example during chronic tissue injury and ageing, may lead to tumor formation.