Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

Abstract

i Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.C ellular differentiation is generally controlled by transcriptional activation or repression of specific genes. Consequently, a host of different molecular genetic events can shape the properties of cells during development. Recent evidence indicates that an evolutionarily conserved protein complex known as DREAM is capable of regulating diverse gene expression programs, thereby unifying many disparate events in development into a single molecular machine (1, 2).The DREAM complex was isolated, and its composition was determined from a number of different model organisms. Studies of aberrant growth factor signaling in Caenorhabditis elegans lead to the discovery of complementation groups that contribute to a multivulval (Muv) phenotype (3). Mutation of any two of the synthetic multivulval (synMuv) group A, B, or C genes resulted in worms with elevated numbers of vulvae (4). Group B contains a number of genes (the Lin-9, Lin-37, Lin-52, Lin-53/RBBP4, and Lin-54 genes) whose encoded proteins form the MuvB core complex (5-7). In addition, worm homologues of retinoblastoma protein (RB), E2F, and DP are also group B members (8, 9). The MuvB core was also found to interact with MYB in transcriptional control of cell cycle progression in fruit flies (6, 7). Isolation of MYB and RB revealed that they copurify with MuvB proteins, and this has formed the basis of the DREAM complex (Drosophila RB, E2F, and MuvB). In some organisms it also contains epigenetic readers and writers such as histone deacetylases (HDACs) and L3MBT (6,7,10). The model that has emerged is one in which the DREAM complex can confer both positive and negative regulation of transcripti...

Structural mechanisms of DREAM complex assembly and regulation

“…"Delta DREAM" mice that lack p130 and carry a p107 mutant allele incapable of binding MuvB display a phenotype identical to that of p107/p130 doubleknockout mice, suggesting an intimate relationship between the pocket proteins and MuvB proteins during cell cycle arrest (Forristal et al 2014). While there are some reports of MuvB binding Rb (Gagrica et al 2004;Korenjak et al 2004), other evidence indicates that the DREAM complex only assembles with either p107 or p130 (Litovchick et al 2007;Pilkinton et al 2007;Schmit et al 2007;Forristal et al 2014). p130 expression levels are high during quiescence, and p130 is an established biomarker at this stage of the cell cycle in which DREAM is a repressor of gene expression (Smith et al 1996;Henley and Dick 2012).…”

“…The LxCxE cleft binds viral and endogenous proteins containing an LxCxExφ sequence motif (x is any amino acid, and φ is a hydrophobic amino acid) (Jones et al 1990;Lee et al 1998;Singh et al 2005). A p107 cleft mutant fails to assemble DREAM in vivo (Forristal et al 2014) and we therefore hypothesized that LIN52 directly binds to the LxCxE cleft rather than the E2F TD site. In support of this hypothesis, a purified p107 pocket domain cleft mutant (I931A, N935A, and V939A) fails to bind LIN52 (Fig.…”

“…DREAM formation requires the phosphorylation of MuvB protein LIN52 at serine residue 28 (phosS28) by the DYRK1A kinase (Litovchick et al 2011). "Delta DREAM" mice that lack p130 and carry a p107 mutant allele incapable of binding MuvB display a phenotype identical to that of p107/p130 doubleknockout mice, suggesting an intimate relationship between the pocket proteins and MuvB proteins during cell cycle arrest (Forristal et al 2014). While there are some reports of MuvB binding Rb (Gagrica et al 2004;Korenjak et al 2004), other evidence indicates that the DREAM complex only assembles with either p107 or p130 (Litovchick et al 2007;Pilkinton et al 2007;Schmit et al 2007;Forristal et al 2014).…”

“…p130 expression levels are high during quiescence, and p130 is an established biomarker at this stage of the cell cycle in which DREAM is a repressor of gene expression (Smith et al 1996;Henley and Dick 2012). Loss of p130 results in up-regulated expression of p107 and subsequent recruitment of p107 to MuvB (Litovchick et al 2007;Forristal et al 2014). MuvB dissociation from pocket proteins coincides with cyclin-dependent kinase (Cdk) activity (Pilkinton et al 2007), suggesting that Cdk phosphorylation of p107 and p130 may inhibit DREAM formation in cycling cells.…”

“…Genetics experiments in model organisms reveal an essential role for DREAM components in differentiation, cell proliferation, and tumor suppression (Korenjak et al 2004;Lewis et al 2004;Harrison et al 2006;Litovchick et al 2007; van den Heuvel and Dyson 2008;Reichert et al 2010). DREAM-deficient mice die shortly after birth with bone developmental defects that result from aberrant chondrocyte differentiation (Forristal et al 2014). In human cell culture, DREAM is assembled upon serum starvation in an experimental state of quiescence, and disruption of the DREAM complex drives cells back into the cell cycle despite environmental cues to arrest (Pilkinton et al 2007;Schmit et al 2007;Litovchick et al 2011).…”