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.

Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit.

The scpAB and sspABC operons of Staphylococcus aureus encode Staphopain cysteine proteases ScpA and SspB, and their respective Staphostatins ScpB and SspC, which are thought to protect against premature activation of Staphopain precursors during protein export. However, we found that the proSspB precursor was secreted and activated without detriment to S. aureus in the absence of SspC function. Our data indicate that this is feasible due to a restricted substrate specificity of mature SspB, a stable precursor structure and slow secretion kinetics. In contrast, mature ScpA had a broad substrate specificity, such that it was prone to autolytic degradation, but also was uniquely able to degrade elastin fibres. Modelling of proScpA relative to the proSspB structure identified several differences, which appear to optimize proScpA for autocatalytic activation, whereas proSspB is optimized for stability, and cannot initiate autocatalytic activation. Consequently, recombinant proSspB remained stable and unprocessed when retained in the cytoplasm of Escherichia coli, whereas proScpA initiated rapid autocatalytic activation, leading to capture of an activation intermediate by ScpB. We conclude that the status of sspBC in S. aureus, as paralogues of the ancestral scpAB genes, facilitated a different activation mechanism, a stable proSspB isoform and modified Staphostatin function.

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1^ΔG) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1^ΔG/ΔG mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1^ΔG/ΔG MEFs display a magnitude of E2F target gene derepression similar to that of Rb1^−/− cells, even though Rb1^ΔG/ΔG cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1^ΔG/ΔG MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G₁ to arrest proliferation. Some Rb1^ΔG/ΔG mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.