The DREAM complex (DP, Rb-like p130/p107, repressor E2F4/5, and MuvB) establishes quiescence (G0) by binding and repressing cell cycle-regulated promoters. Rb, a protein related to p130, controls E2F-dependent genes at the G1/S boundary by inhibiting activator E2Fs promoter of G1/S early cell cycle gene expression. The distinct contributions of p130 and Rb to regulation of this overlapping set of cell cycle E2F-regulated promoters are not well understood. Cyclin-CDK complexes phosphorylate Rb to relieve repression of activator E2Fs to allow transactivation of E2F cell cycle regulated promoters. Little is known about the mechanism of relief of DREAM repression of cell cycle genes, and whether it is required for activator E2F transactivation of gene expression. Our data indicates that Cyclin D-CDK4 promotes DREAM complex disruption. E2F4 persists on promoters without phosphorylated p130 or MuvB. We seek to determine the mechanism of E2F4 release from chromatin and its relationship to the occupancy of activating E2Fs during G1. The sufficiency of DREAM to regulate E2F promoters during cell cycle reentry remains unknown, but we observed that RB1 knockout HFFs have similar cell cycle gene expression kinetics and retain sensitivity to palbociclib, a Cyclin D-CDK4/6 specific inhibitor. These results suggest that DREAM complex may regulate E2F promoters independently of Rb. We will distinguish the contributions of Rb and DREAM-mediated repression of cell cycle gene expression and specific contribution of Cyclin D-CDK4/6 inactivation of Rb and DREAM to cellular transformation. We propose that DREAM complex disruption by Cyclin D-CDK4/6 promotes cell cycle gene expression and transformation independently of pRB.
In Aim 1, we will determine whether Cyclin D-CDK4/6 phosphorylation of p130 is sufficient to disrupt the DREAM complex by causing release of p130 from E2F4 and cell cycle regulated promoters. We will determine which phosphorylation sites on p130 contribute to its binding to E2F4 and regulate disassociation from early cell cycle gene promoters.
In Aim 2, we will determine the mechanism of loss of E2F4 from cell cycle regulated promoters during G1. We will obtain a global view of chromatin association kinetics of DREAM members and E2F1 during G1. We will determine the mechanism of co-regulation of repressor and activator E2Fs of cell cycle genes during G1.
In Aim 3, we will determine if Cyclin D-CDK4/6 mediated disruption of the DREAM complex is required for cellular transformation. We will distinguish pRB and DREAM-mediated repression of cell cycle gene expression and specific contributions as tumor suppressors. We will determine if DREAM restricts cell cycle gene expression independent of Rb and if disruption of DREAM is required to transform fibroblasts. Distinguishing Rb and DREAM cell cycle control will elucidate the previously unappreciated tumor suppressor role of p130. If DREAM is sufficient to repress cell cycle gene expression in Rb null transformed cells, the therapeutic range of CDK4/6 inhibitors may be expanded to tumors with normal DREAM to promote cell cycle exit.

Public Health Relevance

The DREAM complex represses cell cycle gene expression to maintain the resting, quiescent state. The proposed project seeks to determine the mechanism of relief of DREAM-mediated repression of gene expression to allow cell cycle reentry, and the specific contribution of that relief to cellular transformation. Successful completion of the proposed research will provide novel insights into the unappreciated tumor suppressor role of the DREAM complex.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31CA220800-01
Application #
9392358
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Schmidt, Michael K
Project Start
2017-09-01
Project End
2019-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115