Cyclin D–CDK4 relieves cooperative repression of proliferation and cell cycle gene expression by DREAM and RB

Abstract

The Retinoblastoma protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related protein p130 forms the DREAM (DP, RB-like, E2F and MuvB) complex and contributes to repression of cell cycle dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F dependent gene promoters, it remains unclear if they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and p130 knockout cells in primary human fibroblasts. Knockout of both p130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB played a dominant role in E2F dependent gene repression during mid to late G1 while DREAM activity was more prominant during G0 and early G1. Cyclin D - Cyclin Dependent Kinase 4 (CDK4) dependent phosphorylation of p130 occurred during early G1 and led to the release of p130 and MuvB from E2F4 and decreased p130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and p130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation

“…In a recently published report that investigated serum-starved fibroblasts, no evidence was found for cell-cycle genes that are exclusively regulated by RB (56). In contrast, our data reveal that a substantial set of genes is solely repressed by RB and not by DREAM after p53 activation (Figure 5).…”

RB, p130 and p107 differentially repress G1/S and G2/M genes after p53 activation

“…In addition to RB, the RB-like protein p130 (encoded by the RBL2 gene) binds the repressor E2F4 and the MuvB ( m ulti v ulva class B ) core consisting of LIN9, LIN37, LIN52, LIN54 and RBBP4 to form the DREAM complex ( D P, R B-like, E 2F a nd M uvB) (10–12). The repressor E2F4 component of DREAM binds to the E2F sites in the G1/S gene promoters (11,13,14). The DREAM complex and RB cooperate to repress G1/S gene expression during G0 (13,15).…”

“…The repressor E2F4 component of DREAM binds to the E2F sites in the G1/S gene promoters (11,13,14). The DREAM complex and RB cooperate to repress G1/S gene expression during G0 (13,15).…”

“…The late or G2/M wave of cell cycle genes encodes factors required for mitosis(3,5). During G0 and G1, the DREAM complex binds and represses G2/M genes by MuvB specific binding to CHR elements present in their promoters (11,13–15). During S/G2, the MuvB core sequentially recruits BMYB ( MYBL2 ) and then FOXM1 to CHR elements in G2/M gene promoters (16–19).…”

“…Relief of repression by RB and DREAM of G1/S gene expression during cell cycle entry is dependent, at least in part, on Cyclin-CDK phosphorylation of p130 and RB. During G1, RB is phosphorylated by Cyclin D-CDK4/6 and Cyclin E-CDK2 (13,22,23). Cyclin D-CDK4/6 phosphorylation of p130 during mid-G1 results in DREAM complex disruption in a two step process when phosphorylated p130 and MuvB are released together from chromatin followed by separation of p130 from MuvB and E2F4 release from chromatin (13).…”