The Rb-E2F pathway is a critical signaling axis that controls cell cycle transitions. The E2F family of transcription factors comes in two varieties: activators (E2F1-3) and repressors (E2F4-8). The RB tumor suppressor can repress E2F target gene expression through physical interaction with both E2F1-3 activators by inhibiting their transactivation function and E2F4-6 repressors by forming protein complexes that inhibit gene expression. The non-canonical E2F7 and 8 repress gene expression independent of RB. It is generally accepted that modulation of gene expression is a direct result of promoter binding by a transcription factor. As evidence, site-specific transcription factors, such as E2F, appear to require a consensus DNA binding sequence to assert their function. However, it is unclear how a transcription factor family with such varied mechanisms of action can regulate the same genes purportedly through the same DNA binding site. Thus, the purpose of this study is to test the limits of the assumption that all E2Fs require the presence of an intact DNA binding site to regulate target gene expression in a periodic fashion during the cell cycle and development.
We have taken an alternative approach to investigate the requirement of E2F-binding for transcriptional regulation of genes through the cell cycle in both mouse embryo fibroblasts (MEFs) and intact mouse tissues. To this end, we have generated a novel knock-in mouse of a critical cell cycle gene, cyclin A2 (Ccna2) by introducing a promoter mutation disrupting the established E2F binding site (STOCK-Ccna2tm2Gle). In cycling cells, Ccna2 expression is dynamic, increasing in late G1, peaks in early G2 and is shut off before the end of mitosis. In the developing liver, Ccna2 expression is also dynamic, showing high expression during embryogenesis and early postnatal development, but is nearly undetectable by sexual maturity. Using real time PCR and western blot, we demonstrate that in knock-in MEFs and mouse liver, the expression of Ccna2 RNA and protein is static over the cell cycle and developmental time, respectively. Additionally, we have shown using immunohistochemistry that mutant animals have higher liver macrophage infiltration than their wildtype counterparts, suggesting disruption to E2F binding affects tissue homeostasis in addition to ectopic expression of E2F target genes. We conclude that the E2F binding site in the Ccna2 promoter is required for cell cycle- and developmentally-dependent oscillatory expression of Ccna2, yielding higher levels of inflammation in developing livers.