Epigenetic Reprogramming and Inheritance of the Cellular Differentiation Status Following Transient Expression of a Nonfunctional Dominant-Negative Retinoblastoma Mutant in Murine Mesenchymal Stem Cells

The retinoblastoma gene product (Rb1), a master regulator of the cell cycle, plays a prominent role in cell differentiation. Previously, by analyzing the differentiation of cells transiently overexpressing the ΔS/N DN Rb1 mutant, we demonstrated that these cells fail to differentiate into mature adipocytes and that they constitutively silence <i>Pparγ2</i> through CpG methylation. Here, we demonstrate that the consequences of the transient expression of ΔS/N DN Rb1 are accompanied by the retention of <i>Cebpa</i> promoter methylation near the TSS under adipogenic differentiation, thereby preventing its expression. The CGIs of the promoters of the <i>Rb1</i>, <i>Ezh2</i>, <i>Mll4</i>, <i>Utx</i>, and <i>Tet2</i> genes, which are essential for adipogenic differentiation, have an unmethylated status regardless of the cell differentiation state. Moreover, Dnmt3a, a de novo DNA methyltransferase, is overexpressed in undifferentiated ΔS/N cells compared with wild-type cells and, in addition to Dnmt1, Dnmt3a is significantly upregulated by adipogenic stimuli in both wild-type and ΔS/N cells. Notably, the chromatin modifier Ezh2, which is also involved in epigenetic reprogramming, is highly induced in ΔS/N cells. Overall, we demonstrate that two major genes, <i>Pparγ2</i> and <i>Cebpa</i>, which are responsible for terminal adipocyte differentiation, are selectively epigenetically reprogrammed to constitutively silent states. We hypothesize that the activation of Dnmt3a, Rb1, and Ezh2 observed in ΔS/N cells may be a consequence of a stress response caused by the accumulation and malfunctioning of Rb1-interacting complexes for the epigenetic reprogramming of <i>Pparγ2/Cebpa</i> and prevention of adipogenesis in an inappropriate cellular context. The failure of ΔS/N cells to differentiate and express <i>Pparγ2</i> and <i>Cebpa</i> in culture following the expression of the DN Rb1 mutant may indicate the creation of epigenetic memory for new reprogrammed epigenetic states of genes.