Mechanistic Insights Into DREAM and RB Control of the Cell Cycle

Abstract

Two distinct waves of cell cycle regulated genes control progression through the cell cycle: an early wave occurring during G1/S that consists of genes required for entry into S phase driven by the activator E2F transcription factors; and a late wave occurring during G2/M that consists of genes required for mitosis driven by the B-Myb-MuvB-FOXM1 complex. The Retinoblastoma protein (RB) and DREAM complex (DP, RB-like p130, repressor E2F4/5, and MuvB) are transcriptional repressors that inhibit the expression of both the early G1/S and late G2/M cell cycle genes during G0 or the quiescent phase of the cell cycle. Although both RB and DREAM bind and repress an overlapping set of early G1/S cell cycle, E2F-dependent, gene promoters, it remained 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. We observed that DREAM and RB cooperate to repress cell cycle genes during G0 and early G1. We determined that Cyclin D-CDK4 phosphorylation of p130 contributes to disruption of the DREAM complex during cell cycle entry from G0. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as palbociclib-sensitive cancer cell lines. When activated by DNA damage, the tumor suppressor p53 protein contributes to reducing the levels of all cell cycle dependent genes. However, it was unclear whether RB and the DREAM complex were sensitive to this effect of p53. By integrating publicly available datasets with newly generated RNA-sequencing data from primary HFFs, we observed that the early G1/S and late G2/M genes were differentially regulated after p53 activation. DREAM and RB cooperatively repressed G1/S cell cycle genes after p53 activation. In contrast, p130 and the related p107 protein contributed to repression of the late G2/M cell cycle genes after p53 activation and during a normal cell cycle. These results indicate that DREAM cooperates with RB in repressing E2F-dependent G1/S gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.