DREAM Interrupted: Severing MuvB from DREAM’s pocket protein impairs gene repression but not DREAM assembly on chromatin

Abstract

The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in mammalian systems, p107 and p130 are the ancestral pocket proteins. The Rb-like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an E2F-DP transcription factor heterodimer, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex. DREAM complex assembly on chromatin culminates in repression of target genes mediated by the MuvB subcomplex. Here, we examined how the Rb-like pocket protein contributes to DREAM formation and function by disrupting the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. A triple alanine substitution of LIN-52s LxCxE motif 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. However, RNA-seq revealed limited upregulation of DREAM target genes when LIN-35-MuvB association was severed, as compared to gene upregulation following LIN-35 loss. Based on chromatin immunoprecipitation, disrupting LIN-35-MuvB association did not affect the chromatin localization of E2F-DP, LIN-35, or MuvB components. In a previous study we showed that in worms lacking LIN-35, E2F-DP and MuvB chromatin occupancy was reduced genome-wide. With LIN-35 present but unable to associate with MuvB, our present study suggests that the E2F-DP-LIN-35 interaction promotes E2F-DPs chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket proteins association with MuvB supports DREAM function but is not required for DREAMs chromatin occupancy.