SUMMARY Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

fThe 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 ⌬G ) 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 ⌬G/⌬G mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1⌬G/⌬G MEFs display a magnitude of E2F target gene derepression similar to that of Rb1 ؊/؊ cells, even though Rb1 ⌬G/⌬G cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1 ⌬G/⌬G MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G 1 to arrest proliferation. Some Rb1 ⌬G/⌬G 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.T he retinoblastoma tumor suppressor protein (pRB) has a central role in the regulation of the G 1 -to-S-phase transition. Inactivation of its control over cell cycle progression is one of the most common events in cancer (1). The RB protein is thought to regulate entry into S phase through its ability to repress E2F-dependent transcription (2). In the G 1 phase of the cell cycle, a direct interaction between the large pocket domain of pRB (RBLP) and the transactivation domain of E2Fs blocks transcription and recruits chromatin regulators that maintain the cell in G 1 (3). Activation of cyclin-dependent kinases (CDKs) results in the phosphorylation of pRB and the release of E2F transcription factors (4). Free E2Fs then activate a transcriptional program that drives the cell into S phase (3). This model of pRB regulation of E2F dominates our understanding of G 1 -to-S-phase control. Much of our knowledge of this model was derived from studies using viral oncoproteins encoded by small DNA tumor viruses (5, 6). Of particular note, the human papillomavirus E7 protein has been shown to compete for pRB-E2F interactions to deregulate proliferation (7, 8). However, E7 must also target pRB for degradation in order to induce proliferation (8). Thus, the experimental system that gave rise to the pRB-E2F regulatory axis in cell cycle control also suggests that pRB may engage other growth-suppressing activities beyond E2F regulation. By comparison with the pRB-E2F pathway, we know very little about pRB's non-E2F-dependent growth control mechanisms and their relative contribution to cell cycle regulation and tumor suppressor activities.The minimal growth-suppressive region of pRB...

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...

SummaryThe 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.

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. FIG 1 CRISPR/Cas9-induced mutations in RB1 cause DNA damage. (A) Ethidium bromide-stained agarose gel showing examples of wild-type, heterozygous, and homozygous mutant RB1 genotypes that are detected by PCR amplification of exon 22 sequences. MW, molecular weight. (B, top) Representative Western blot showing RB expression in control, heterozygous, and homozygous mutant cells. (Bottom) Sp1 loading control.(C) Immunofluorescence microscopy was used to detect RB expression (green) in cultures of control, heterozygous, or homozygous mutants. Cells were counterstained with DAPI to visualize nuclei (blue). (D) Representative confocal microscopy images of ␥H2AX foci (red) in control, heterozygous, and homozygous RB1 mutant cells. Cells were counterstained with DAPI to visualize nuclei (blue). (E) Counts of ␥H2AX foci for each of the U2OS RB1 genotypes. The average proportions of cells with discrete numbers of foci are shown as histograms, while the cumulative frequency of foci for each genotype is shown in the inset. The average distributions of foci for RB1 wild-type (4 different clones), heterozygous (3 different clones), and knockout (4 different clones) cells were compared using the Kolmogorov-Smirnov test (*, P Ͻ 0.05). (F) U2OS cells were transfected with CRISPR/Cas9 constructs targeting either a safe-harbor site in the genome or exon 2 of the RB1 gene. Three clones were selected under both control and knockout conditions, and ␥H2AX foci were quantified by fluorescence microscopy. The average proportions of ␥H2AX foci for both RB1 wild-type and knockout genotypes are shown as histograms, while the cumulative relative frequency of foci is shown in the inset. Focus distributions were again compared by a Kolmogorov-Smirnov test. (G) H460 lung cancer cells were stained for ␥H2AX foci and analyzed as described above for panel F. (H) H1792 non-small cell lung cancer cells were analyzed as described above for panel F. All error bars are ϩ1 standard error of the mean (SEM). *, P Ͻ 0.05. RB1Deletion Causes ...

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Bovine spongiform encephalopathy (BSE) is a transmissible fatal neurodegenerative disorder, presenting a characteristic spongiform degeneration of cattle brain due to the accumulation of a pathogenic and protease-resistant infectious protein (prion). Two deletion/insertion polymorphisms of the prion protein gene (23 bp at the promoter region and 12 bp at intron 1) were analyzed in three beef cattle herds (Aberdeen Angus, Charolais, and Franqueiro) to verify allele frequencies for possible use in selection of resistant animals. High frequencies of susceptibility alleles (23 and 12 bp deletion) and haplotype (23 del/12 del) were observed in the Aberdeen Angus and Charolais herds, but Franqueiro presented one of the highest frequencies of resistant alleles so far described. These data indicate the need for selection in Aberdeen Angus and Charolais breeds to increase the frequency of resistant animals in order to reduce the probabilities of BSE outbreaks in these populations.

ABSTRACT. The melanocortin 1 receptor (MC1R) gene has been described as responsible for the black color in some breeds of sheep, but little is known about its function in many colored breeds, particularly those with a wide range of pigmentation phenotypes. The Brazilian Creole is a local breed of sheep from southern Brazil that has a wide variety of wool colors. We examined the MC1R gene (Extension locus) to search for the e allele and determine its role in controlling wool color variation in this breed. One hundred and twenty-five animals, covering the most common Creole sheep phenotypes (black, brown, dark gray, light gray, and white), were sequenced to detect the mutations p.M73K and p.D121N. Besides these two mutations, three other synonymous sites (429, 600, and 725) were found. The dominant allele (E