Transcriptional regulation of cell cycle exit, also called quiescence, and cell cycle progression plays an essential role in organismal development, tissue homeostasis, and cellular differentiation. The DREAM (Dp, Rb, E2F, and MuvB) transcriptional repressor complex, which includes an E2F-DP transcription factor heterodimer, a Retinoblastoma (RB)-like pocket protein, and the 5-subunit MuvB complex, plays a key role in transcriptional regulation of the cell cycle. In mammalian cells, DREAM functionally overlaps with the Retinoblastoma (RB) protein, but the former represents the ancestral regulator of the cellular quiescent program. The highly-conserved MuvB subcomplex plays a critical role, as it mediates repression of genes during cell cycle quiescence and activation of genes during the late cell cycle. The long-term goal of my lab is to elucidate how the conserved, but less redundant, DREAM complex functions in the Caenorhabditis elegans model system. The objective of this proposal is to establish how the molecular events that drive DREAM complex formation contribute to target gene repression. The rationale of this proposal is that the C. elegans DREAM complex regulatory system represents an integral component of cell cycle transcriptional control whose function is obscured by molecular redundancy present in mammalian systems. The central hypothesis is that MuvB chromatin localization, aided by its association with E2F-DP and the pocket protein, mediates gene repression. We will address the central hypothesis by pursuing the following specific aims: 1) We will investigate the effects that follow disruption of DREAM complex formation. Using CRISPR/Cas9-mediated genome editing to disrupt known associations between E2F-DP, the pocket protein, MuvB, and chromatin, we will test how blocking DREAM assembly affects target gene expression. 2) We will establish the temporal dynamics of DREAM complex repression. Using the Auxin-Induced Degron (AID) system to rapidly deplete DREAM subunits, we will selectively eliminate and restore MuvB chromatin occupancy near target genes and measure the effects on DREAM target gene expression. 3) We will assess whether MuvB acts as a transcriptional activator in the C. elegans germline. Using the AID system, we will establish tissue-specific depletion of MuvB and test how its loss affects germline gene expression and germline function. The innovation of this study is that this investigation exploits advances in genomic editing and genetic tools to dissect and characterize an essential transcriptional process in vivo. The significance of this study is that we will develop a genetic toolkit with the capability to precisely perturb and analyze the function of the DREAM complex, a key regulatory component of the cell cycle. Developing this toolkit is an essential step towards understanding and manipulating the molecular components that govern transcriptional regulation of the cell cycle.
Careful regulation of the balance between cell cycle exit, called cellular quiescence, and cell cycle progression protects organismal health throughout its lifespan, from embryo through maturity. This project will determine the molecular mechanism by which assembly of the highly conserved 8-protein DREAM complex establishes transcriptional repression of cell cycle genes and maintains cellular quiescence. Our studies will provide key insights into how the dynamics of cell cycle gene regulation underlie organismal development.
