H3K9me-Enhanced DNA Hypermethylation of thep16INK4aGene: An Epigenetic Signature for Spontaneous Transformation of Rat Mesenchymal Stem Cells

Abstract

To explore the mechanisms underlying spontaneous transformation of mesenchymal stem cells (MSCs), changes in senescence-associated molecules, particularly the epigenetic modification of the p16^INK4a gene, including histone H3 lysine 27/9 methylation (H3K27/9me) and DNA methylation, were investigated in cultured adult rat bone marrow MSCs at different stages during the transformation process. It was shown that the MSCs underwent replicative senescence after 24 to 25 population doublings, characterized by positive staining for senescence-associated β-galactosidase, increased expression of p16^INK4a and p21, and downregulated phosphorylation of Rb. The upregulation of p16^INK4a was associated with decreased expression of enhancer of the zeste homolog 2 (Ezh2), and reduced levels of H3K27me and DNA methylation in the p16^INK4a gene. At week 4 of senescence, reproliferating cells emerged among the senescent MSCs. These senescence-escaped MSCs lost their senescence-related markers (including p16^INK4a) and became highly proliferative. In addition to H3K27me, another H3 modification pattern, H3K9me, appeared in the p16^INK4a gene, accompanied by an enhanced DNA methylation. With continued culture, the senescence-escaped MSCs did not show any sign of growth arrest and gained the capacity for anchorage-independent growth. These immortalized (transformed) MSCs showed further enhanced DNA methylation of the p16^INK4a gene by increased H3K9me. Ezh2 knockdown with shRNA eliminated H3K27me-mediated DNA methylation of the p16^INK4a gene in presenescent MSCs, but had no effect on H3K9me-enhanced DNA hypermethylation in the cells after senescence escape. These findings identify an Ezh2- and H3K27me-independent, but H3K9me-enhanced, DNA hypermethylation of the p16^INK4a gene, which might be an epigenetic signature for MSC spontaneous transformation.