The PRC2 complex directly regulates the cell cycle and controls proliferation in skeletal muscle

Abstract

The polycomb repressive complex 2 (PRC2) is an important developmental regulator responsible for the methylation of histone 3 lysine 27 (H3K27). Here, we show that the PRC2 complex regulates the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. Depletions of the catalytic subunit of the PRC2 complex, EZH2, have shown that EZH2 is required for cell viability, suggesting that EZH2 promotes proliferation. We found that EZH2 directly represses both positive and negative cell cycle genes, thus enabling the PRC2 complex to tightly control the cell cycle. We show that modest inhibition or depletion of EZH2 leads to enhanced proliferation and an accumulation of cells in S phase. This effect is mediated by direct repression of cyclin D1 (Ccnd1) and cyclin E1 (Ccne1) by the PRC2 complex. Our results show that PRC2 has pleiotropic effects on proliferation as it serves to restrain cell growth, yet clearly has a function required for cell viability as well. Intriguingly, we also find that the retinoblastoma protein gene (Rb1) is a direct target of the PRC2 complex. However, modest depletion of EZH2 is not sufficient to maintain Rb1 expression, indicating that the PRC2 dependent upregulation of cyclin D1 is sufficient to inhibit Rb1 expression. Taken together, our results show that the PRC2 complex regulates skeletal muscle proliferation in a complex manner that involves the repression of Ccnd1 and Ccne1, thus restraining proliferation, and the repression of Rb1, which is required for mitotic exit and terminal differentiation.

Keywords: EZH2; PRC2; cell cycle; cyclin D1; cyclin E1; retinoblastoma protein (pRB, RB1); skeletal muscle.

Figure 1.

Stable EZH2 depletion results in increased proliferation and DNA synthesis in C2C12 cells.

Figure 2.

Pro-proliferative cell cycle genes are inhibited by EZH2.

Figure 3.

Negative cell cycle regulators are also directly regulated by the PRC2 complex.

Figure 4.

Transient transfection of shEzh2 in C2C12 mildly decreases cell proliferation.

Figure 5.

Transient loss of EZH2 in primary myoblasts decreases cell viability and also has increased proliferative cell population.

Figure 6.

Transient loss of EZH2 in primary myoblasts leads toincreased MRF and negative cell cycle gene expression while positive cell cycle genes are downregulated.

Figure 7.

Catalytic inhibition of EZH2 by GSK126 at low concentrations enhances proliferation in primary myoblasts.

Figure 8.

EZH2 inhibition by low concentration of GSK126 in C2C12 cells recapitulates increased cell proliferation.