[
International Worm Meeting,
2021]
Formation of the highly conserved 8-subunit DREAM (Dp, Retinoblastoma-like, E2F, and MuvB) complex culminates in direct repression of cell cycle and developmental genes. We are interested in what determinants of DREAM complex formation are essential for transcriptional repression of target genes. Structural studies on mammalian proteins identified key interaction interfaces between the 3 major components of DREAM, the E2F-DP transcription factor heterodimer (EFL-1 and DPL-1), the Retinoblastoma-like pocket protein (LIN-35), and the 5-subunit MuvB subcomplex (LIN-9, LIN-37, LIN-52, LIN-53, and LIN-54). For example, MuvB interacts with LIN-35 via an LxCxE interacting motif on LIN-52. LIN-35 also interacts directly with the EFL-1 transactivation domain via its pocket domain. DNA-binding by the E2F-DP transcription factor heterodimer and the MuvB subunit LIN-54 coordinate DREAM complex localization to chromatin. We hypothesize that MuvB chromatin occupancy, aided and stabilized by its association with E2F-DP and the pocket protein, establishes and maintains target gene repression. Using CRISPR/Cas9-targeted mutagenesis, we disrupted the interaction between MuvB and the C. elegans pocket protein LIN-35. We expected that severing MuvB from the complex would destabilize DREAM component assembly on chromatin and its repression of target genes. Instead, we observed that disrupting LIN-35-MuvB association did not affect DREAM chromatin occupancy, but we did observe upregulation of DREAM target genes. To continue our evaluation of our model of DREAM complex formation, we identified the conserved amino acid residues that likely mediate the protein-protein and protein-DNA associations for C. elegans DREAM. This analysis lays the groundwork for future application of our CRISPR/Cas9 functional genomics pipeline to establish how the molecular events that drive DREAM complex function contribute to target gene repression.