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

The mammalian Retinoblastoma (Rb) protein family, collectively called pocket proteins, regulate entry into and exit from the cell cycle [1-5]. Although pRb plays a dominant role, the Rb-like homologs p130 and p107 represent the ancestral proteins [6, 7] and functionally overlap with pRb to repress cell cycle gene expression during cellular quiescence (G0) [8-10]. Like pRb, p130 and p107 interact with an E2F-DP transcription factor heterodimer [11-13]. Unlike pRb, they also interact with the highly conserved 5-subunit MuvB complex, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex, which mediates transcriptional repression through MuvB [8, 14-17]. To address how the Rb-like pocket protein contributes to MuvB-mediated gene repression, we disrupted the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. Disrupting the LIN-35-MuvB association did not affect DREAM chromatin occupancy but did cause a highly penetrant synthetic multivulval (SynMuv) phenotype, indicating that blocking DREAM assembly impairs MuvB function. Some DREAM target genes became derepressed, indicating that for those genes MuvB chromatin binding alone is not sufficient for gene repression and that direct LIN-35-MuvB association potentiates MuvBs innate repressive activity. Our previous study [17] showed that in worms lacking LIN-35, E2F-DP and MuvB chromatin occupancy is reduced genome-wide. With LIN-35 present, this study demonstrates 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 protein family may recruit regulatory factors like MuvB to chromatin through E2F-DP to facilitate their transcriptional activity.nnGraphical AbstractnnO_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/671024v1_ufig1.gif" ALT="Figure 1">nView larger version (33K):norg.highwire.dtl.DTLVardef@71a25org.highwire.dtl.DTLVardef@45afacorg.highwire.dtl.DTLVardef@1106c52org.highwire.dtl.DTLVardef@137b682_HPS_FORMAT_FIGEXP M_FIG C_FIG