Loss of the Caenorhabditis elegans pocket protein LIN-35 reveals MuvB's innate function as the repressor of DREAM target genes

Abstract

The DREAM (Dp/Retinoblastoma(Rb)-like/E2F/MuvB) transcriptional repressor complex acts as a gatekeeper of the mammalian cell cycle by establishing and maintaining cellular quiescence. How DREAM’s three functional components, the E2F-DP heterodimer, the Rb-like pocket protein, and the MuvB subcomplex, form and function at target gene promoters remains unknown. The current model invokes that the pocket protein links E2F-DP and MuvB and is essential for gene repression. We tested this model by assessing how the conserved yet less redundant DREAM system in Caenorhabditis elegans is affected by absence of the sole C. elegans pocket protein LIN-35. Using a LIN-35 protein null mutant, we analyzed the assembly of E2F-DP and MuvB at promoters that are bound by DREAM and the level of expression of those "DREAM target genes" in embryos. We report that LIN-35 indeed mediates the association of E2F-DP and MuvB, a function that stabilizes DREAM subunit occupancy at target genes. In the absence of LIN-35, the occupancy of E2F-DP and MuvB at most DREAM target genes decreases dramatically and many of those genes become upregulated. The retention of E2F-DP and MuvB at some target gene promoters in lin-35 null embryos allowed us to test their contribution to DREAM target gene repression. Depletion of MuvB, but not E2F-DP, in the sensitized lin-35 null background caused further upregulation of DREAM target genes. We conclude that the pocket protein functions primarily to support MuvB-mediated repression of DREAM targets and that transcriptional repression is the innate function of the evolutionarily conserved MuvB complex. Our findings provide important insights into how mammalian DREAM assembly and disassembly may regulate gene expression and the cell cycle.

Wnt signaling activates gene expression in the absence of theC. elegansDREAM repressor complex in somatic cells

“…This dataset includes the interactions of 296 predicted transcription factors (Bass et al 2016) as prey screened to interact with 534 promoter loci bait sequences (Reece-Hoyes et al 2011). We found that 48 bait sequences in the Y1H dataset were known to be bound by the DREAM complex in their promoter region in late embryos (Goetsch et al 2017). We found that 123 of the 296 TFs showed binding to at least one of the 48 DREAM target promoter loci in the Y1H data set.…”

“…In C. elegans, the DREAM complex alters chromatin structure in the nucleus to transcriptionally repress germline genes in somatic cells (Costello and Petrella 2019;Cui et al 2006;Latorre et al 2015;Petrella et al 2011;Rechtsteiner et al 2019;Unhavaithaya et al 2002;Wang et al 2005;Wu et al 2012). The DREAM complex is completely conserved between mammals and C. elegans, and comprised of eight proteins: E2F-DP heterodimer (EFL-1 & DPL-1), a retinoblastoma-like pocket protein , and a 5-subunit MuvB complex (LIN-9, LIN-37, LIN-52, LIN-53, and the DNA binding subunit LIN-54) (Goetsch et al 2017;Harrison et al 2006;Latorre et al 2015;Sadasivam and DeCaprio 2013). In C. elegans, a mutant form of LIN-54 is incapable of binding the majority of DREAM complex target loci due to a change in a single amino acid in the DNA binding motif (Tabuchi et al 2011).…”

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

“…We tested the impact of this severing on the chromatin localization of DREAM components using chromatin immunoprecipitation (ChIP). We chose 4 genes, set-21, mis-12, polh-1, and air-1, as representative DREAM target genes; in lin-35 null embryos, the chromatin occupancy of DREAM components was greatly diminished at each of their gene promoters [17]. Importantly, DREAM component chromatin occupancy was undetectable at the air-1 promoter in the absence of LIN-35 [17].…”

“…Like pRb, p130 and p107 interact with an E2F-DP transcription factor heterodimer [11][12][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][15][16][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.…”

A Long Lost Key Opens an Ancient Lock:DrosophilaMyb Causes a Synthetic Multivulval Phenotype in Nematodes

“…Drosophila Myb also acts in opposition to the DREAM complex to regulate chorion gene amplification in ovarian follicle cells and programmed neuronal cell death 12,42,43 . Interestingly, recent studies in C. elegans have shown that the MuvB complex can effectively repress gene expression in the absence of the LIN-35 RB-family protein that was previously thought to be required for repression by DREAM complexes 44 . Although C. elegans and other nematode species contain two Myb-related genes that encode homologs of the CDC5/CEF1 splicing factor and the SNAPc small nuclear RNA transcription factor, they do not contain an animal-type Myb gene that might relieve repression by DREAM complexes 3 .…”