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

Abstract

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 Pparγ2 through CpG methylation. Here, we demonstrate that the consequences of the transient expression of ΔS/N DN Rb1 are accompanied by the retention of Cebpa promoter methylation near the TSS under adipogenic differentiation, thereby preventing its expression. The CGIs of the promoters of the Rb1, Ezh2, Mll4, Utx, and Tet2 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, Pparγ2 and Cebpa, 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 Pparγ2/Cebpa and prevention of adipogenesis in an inappropriate cellular context. The failure of ΔS/N cells to differentiate and express Pparγ2 and Cebpa in culture following the expression of the DN Rb1 mutant may indicate the creation of epigenetic memory for new reprogrammed epigenetic states of genes.

Keywords: Cebpa; DNMTs; MSCs; Rb1; adipogenesis; epigenetic reprogramming.

Figure 1

The ΔS/N cells with ILYAS amino acid deletion overexpress Rb1 in the undifferentiated state. (a) Schematic diagram representing the structural domains of the Rb1 protein along with the 27 exons of the gene and showing the location of the 6-amino acid deletion in exon 22. (b) Ribbon diagrams of the 3D atomic structure of the RB tumor suppressor bound to the transactivation domain of E2F. Deletion of the ILQYAS amino acid of the alpha helix of the RB1 B pocket domain at positions 768-763 of human RB1 resulted in 761-766 ILQYAS in mice. Adapted from 1n4m PDBe complex ID: PDB-CPX-139032. (c) Real-time PCR demonstrated that the p130 mRNA expression level was not changed in ΔS/N cells. (d) Real-time qPCR revealed that the mRNA expression level of Rb1 was greater in ΔS/N cells than in wild-type cells (n = 3); n, number of biological replicates. The error bars indicate the SEMs. (e) The ATF-2 binding site in the Rb1 promoter contains a CpG. (f) Partial chromatogram of the Rb1 promoter demonstrating that the ATF-2 binding site is not methylated.

Figure 2

Real-time qPCR revealed that the mRNA expression levels of Dnmt3a and Ezh2 are greater in undifferentiated ΔS/N cells than in wild-type cells (n = 3); n, number of biological replicates. The error bars indicate the SEMs. (a) Comparison of Dnmts expression in differentiated und undifferentiated ΔS/N cells and wild-type cells. (b) Comparison of Ezh2 and Utx expression in differentiated und undifferentiated ΔS/N cells and wild-type cells.

Figure 3

The hypermethylated promoter of Cebpa retained CG methylation near the TSS in differentiated ΔS/N cells. (a) Gel photograph showing amplification of the proximal region of the Cebpa gene and adipogenic-related gene promoters using bisulfite-treated DNA as a template and a pair of bisulfite-specific primers. (b) Bisulfite sequencing analyses of the Cebpa promoter. The distribution of CpG sites in differentiated and undifferentiated 10T1/2 and ΔS/N cells is shown. At the bottom of the illustrations, the methylation status of the CpGs is shown. (c) Real-time qPCR revealed that the mRNA expression level of Cebpa was lower in both undifferentiated and differentiated ΔS/N cells than in wild-type cells (n = 3); n, number of biological replicates. The error bars indicate the SEMs.

Figure 4

The putative Nrf1 binding site preserves the methylation of two CG sites of methylation-sensitive Nrf1. (a) The Cebpa promoter region contains the putative Nrf1 binding site. Unerased CG sites detected via Cebpa promoter methylation analysis upon stimulation of adipogenesis in ΔS/N cells are shown as a pictogram. (b) Analysis of a 35-bp region containing an unerased methylated CpG located downstream of the TSS using the UCSC Genome Browser transcription factor binding search tool in mice (GRCm39/mm39). (c) JASPAR NRF1 profile summary, Matrix ID MA0506.1.

Figure 5

The unerased CpG methylation observed in the Cebpa promoter upstream of the TSS may represent a target for methylation-dependent Cebpg/b transcription factors. (a) Analysis of a 35-bp region containing an unerased methylated CpG located upstream of the TSS using the UCSC Genome Browser transcription factor binding search tool in mice (GRCm39/mm39). (b) JASPAR CEBPG/B profile summary, Matrix ID MA0838.1/MA0466.2.

Figure 6

Bisulfite sequencing analyses of the Pparγ2 promoter. The distribution of CpG sites in differentiated and undifferentiated 10T1/2 and ΔS/N cells is shown. At the bottom of the illustrations, the methylation status of the CpGs is shown.