Suha Malik “The Role of Retinoblastoma and E2Fs in the Cell Cycle”
Mentor: Gustavo Leone, MCW Cancer Center
Poster #89
The Retinoblastoma (RB)-E2F axis, consisting of the RB tumor suppressor protein and the E2F family of transcription factors, has been determined to be perturbed in nearly every human cancer. E2Fs, encoded by eight genes and forming nine distinct protein products, can act as canonical activators (E2F1, E2F2, E2F3), canonical repressors (E2F4, E2F5, E2F6), or atypical repressors (E2F7, E2F8), and their expression levels vary as the cell cycle and differentiation progresses. The universality of this axis in each cell and its vital role in cell cycle regulation, through control of the G1-S restriction point, underlies the importance of investigating the exact mechanistic action of both RB and E2Fs. This project explores how RB and E2Fs individually play a direct role in the control of cell cycle-dependent gene expression. We bred RB knockout, E2F knockout, and fluorescently ubiquitination-based cell-cycle indicator (FUCCI) mice, collected embryos, separated cells based on the cell cycle fluorescent tags, isolated RNA to generate cDNA libraries for next generation sequencing, and analyzed gene expression (including its relative levels) based on cell cycle phase in each cohort of mice. These results will be validated using standard quantitative PCR expression assays and statistically analyzed through methods such as standard student t-tests. Bioinformatic analysis demonstrates the statistically significant number of up- and down-regulated genes, as well as genes common and unique to cell cycle phases. Preliminary results indicate increased E2F target expression in RB knockout mice, in accordance with literature. Further gene expression analysis will be performed to identify specific genes and pathways of interest, and breeding will continue to obtain test animals for repetition of the experiment. Elucidating specific genes and pathways affected when either RB or E2Fs are knocked out is critical to correcting uncontrolled cell proliferation and lack of apoptotic signal recognition seen in cancerous cells.