Analysis of DREAM and E2F1 Competition for Cell Cycle Promoters during G1.
Mentor: James A DeCaprio, MD (Dana Farber Cancer Institute)
The DREAM complex maintains G0 by binding to the sequence specific E2F and cell cycle gene homology region (CHR) promoter elements to repress cell cycle gene expression. The DREAM complex is composed of DP1, retinoblastoma-like p130 or p107, E2F4 or E2F5, and MuvB, which is comprised of LIN37, LIN52, LIN9, LIN54, and RBBP4. The retinoblastoma tumor suppressor protein, RB, cooperates with DREAM to repress cell cycle gene expression by binding and repressing transactivation of gene expression by activator E2F1. RB and DREAM respond similarly to growth factors and oncogenic stimuli, but they are phosphorylated differently. RB loses its binding to E2F1 after it is hypo-phosphorylated by Cyclin D-CDK4/6 in early G1 and hyperphosphorylated by Cyclin E-CDK2 at the late G1 restriction point, allowing for E2F1-driven expression of early cell cycle genes. In contrast, Cyclin D-CDK4/6 phosphorylation of p130 alone results in DREAM complex disruption. However, the mechanism of loss of E2F4 from cell cycle promoters is not known. Temporally, E2F4 is present on cell cycle promoters during early/mid G1 while E2F1-RB is present later during mid-late G1. We hypothesize that E2F4 and E2F1 compete with each other for the E2F promoter elements, with E2F1 having a higher binding affinity. Additionally, E2F1 is removed from cell cycle promoters during G1 by Cyclin A-CDK2 phosphorylation. Because these sites are conserved in E2F4, we hypothesize that Cyclin E-CDK2 phosphorylates E2F4, resulting in loss of E2F4 binding on cell cycle promoters. Cyclin E-CDK2 is active when E2F4 loses binding to cell cycle promoters, so we hypothesize that E2F4 is phosphorylated by this cyclin. The focus of my research is to determine if E2F1 competes with E2F4 for E2F promoter elements, and if E2F1 a higher affinity for these elements. We will clone TFDP1 and E2F4 into a bacterial protein expression vector called pGEX, which has an N-terminal GST tag. We will include TFDP1 (protein name DP1) because heterodimerization of E2F1-DP1 and E2F4-DP1 is required for DNA binding. We will express the proteins in E.coli BL21*(DE3) E.coli. BL21 STAR (DE3) is the best strain of E.coli for our purposes since it has higher mRNA stability and lower protease activity. We will purify the bacterially expressed, N-terminally GST-tagged proteins using immobilized glutathione beads. To test if in the presence of E2F1 DNA-bound E2F4 levels will drop or stay the same, we will perform a DNA-binding affinity assay. We will do this by conjugating biotin to dsDNA sequence with the MCM5 gene promoter that contains E2F and CHR elements. We will pull down DNA using streptavidin beads and qualitatively measure bound protein by immunoblot. To test if Cyclin E-CDK2 affects E2F4 binding affinity, we will incubate E2F4 with Cyclin E-CDK2 and then measure DNA binding affinity. These experiments will give us insight into how DREAM and RB coordinate to repress G1/S phase genes.
In this image, I am immunoblotting with p130, E2F4, and E2F1 antibodies. I incubated the primary antibodies overnight, and washed each nitrocellulose membrane with TBST the following morning. I visualized the antibody binding by using secondary antibody conjugated to Horse Radish Peroxidase and measured chemiluminescence. This experiment can tell me relative amounts of p130, E2F4, and E2F1 in different experimental conditions. Source: