DREAM Interrupted: Severing LIN-35-MuvB association in Caenorhabditis elegans impairs DREAM function but not its chromatin localization

The highly conserved DREAM transcriptional repressor complex contains an RB-like pocket protein, an E2F-DP transcription factor heterodimer, and the 5-subunit MuvB complex. Using CRISPR/Cas9 targeted mutagenesis, we disrupted the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52. A triple alanine substitution of LIN-52's LxCxE motif (3A) severed LIN-35-MuvB association and caused classical DREAM mutant phenotypes, including synthetic multiple vulvae, high-temperature arrest, and ectopic expression of germline genes in the soma. We performed RNA-seq in lin-52(3A) mutant late embryos (4 replicates) compared to lin-52(WT) wild-type late embryos (4 replicates) to assess the genome-wide effects on gene expression that result from severing LIN-35-MuvB association. We examined 4 biological replicates each of RNA samples (8 total samples) from lin-52(bn139[lin-52::GFP::3xFLAG]) (WT) and lin-52(bn151(lin-52[L42A,C44A,E46A]::GFP::3xFLAG)) (3A) late embryos. Gene expression was compared between 3A and WT to identify misexpressed genes following loss of LIN-35-MuvB association.