pRB Takes an EZ Path to a Repetitive Task

Molecular Cell

Previews The idea that phage infection should be stimulated by high density of host cultures comes mostly from mathematical modeling and may not be true for all phages. In fact, many phages infect cells from high-density cultures poorly, entering into a stable non-replicating pseudolysogenic carrier state (qo s and We˛grzyn, 2012). The increased CRISPRCas activity in this case may be counteracted by phage-encoded mechanisms. Using QS to limit phage infections and HGT appears to be too good a strategy to be used by just one kind of bacterial defense mechanism. The restrictionmodification systems, which have taken biotechnology by storm 35 years before CRISPR-Cas, can be regarded as bacterial innate immunity. In marked difference to the CRISPR-Cas defense, cells carrying restriction-modification systems, when overridden by the phage, produce epigenetically modified viruses that are

fully proficient in infection of protected cells and there is no additional adaptive mechanism like primed adaptation to mend the situation. One can predict that most or even all mechanistically diverse R-M systems of different types should be also responsive to QS to help avoid population crashes caused by modified viral progeny. ACKNOWLEDGMENTS

Datsenko, K.A., Pougach, K., Tikhonov, A., Wanner, B.L., Severinov, K., and Semenova, E. (2012). Nat. Commun. 3, 945. Høyland-Kroghsbo, N.M., Paczkowski, J., Mukherjee, S., Broniewski, J., Westra, E., Bondy-Denomy, J., and Bassler, B.L. (2016). Proc. Natl. Acad. Sci. USA. Published online November 14, 2016. http://dx.doi.org/10.1073/pnas.1617415113. Li, R., Fang, L., Tan, S., Yu, M., Li, X., He, S., Wei, Y., Li, G., Jiang, J., and Wu, M. (2016). Cell Res. http://dx.doi.org/10.1038/cr.2016.135. qos, M., and We˛grzyn, G. (2012). Adv. Virus Res. 82, 339–349.

The CRISPR work in K.S. labs is supported by NIH R01 grant GM10407 and the Russian Science Foundation grant 14-14-00988.

Mulepati, S., and Bailey, S. (2013). J. Biol. Chem. 288, 22184–22192.

REFERENCES

Papenfort, K., and Bassler, B.L. (2016). Nat. Rev. Microbiol. 14, 576–588.

Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). Science 315, 1709–1712.

Patterson, A.G., Jackson, S.A., Taylor, C., Evans, G.B., Salmond, G.P., Przybilski, R., Staals, R.H.J., and Fineran, P.C. (2016). Mol. Cell 64, this issue, 1102–1108.

Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., and van der Oost, J. (2008). Science 321, 960–964.

Tamulaitis, G., Venclovas, C., and Siksnys, V. (2016). Trends Microbiol. Published online October 20, 2016. http://dx.doi.org/10.1016/j.tim.2016.09.012.

pRB Takes an EZ Path to a Repetitive Task Ioannis Sanidas1 and Nicholas J. Dyson1,* 1Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Harvard Medical School, Charlestown, MA 02129, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.11.035

Repetitive DNA elements are essential for genome function; in this issue of Molecular Cell, Ishak et al. (2016) describe a novel mechanism of epigenetic repression at these elements that requires pRB-dependent recruitment of EZH2. The best-known molecular activity of the retinoblastoma tumor suppressor protein (pRB) is an interaction with the family of E2F transcription factors that enable it to repress the transcription of cell cycle related genes. Most studies of pRB’s connection to chromatin have focused on pRB-binding to promoter elements, but surprisingly little attention has been paid to the genome-wide distribution of the protein. In this issue of Molecular Cell, Ishak et al. (2016) provide a fascinating new perspective on pRB function. They demonstrate that a large fraction of pRB associates with intronic and intergenic regions. Here pRB cooperates

with the enhancer of zeste homolog 2 (EZH2), promotes H3K27 methylation, and helps to suppress transcription of repetitive DNA sequences. Changes seen in mice specifically lacking this regulation suggest that this role of pRB is important and that it helps to maintain genome stability and suppress tumorigenesis. Genome sequencing has confirmed that repetitive sequences represent a large portion of most eukaryotic genomes and comprise approximately 50% of the human genome. These regions contain tandem repeat sequences, such as satellite DNA, and interspersed repeats of transposable elements (TEs). TEs can be

non-autonomous (inactive due to mutations eliminating their ability to duplicate or transpose) or autonomous (active, but silenced by epigenetic regulation) (Slotkin and Martienssen, 2007). Chromatin modifications that suppress TEs include DNA methylation and histone tail modifications. Withdrawal of the epigenetic control leads to transcriptional activation of the repetitive genomic elements and the subsequent appearance of genomic changes, such as genomic instability and the cis-activation of proximal protooncogenes, which can promote tumorigenesis (Iskow et al., 2010; Lee et al., 2012).

Molecular Cell 64, December 15, 2016 ª 2016 Elsevier Inc. 1015

Molecular Cell

Previews

When Ishak et al. (2016) HDAC2. Indeed, the high pRB performed pRB chromatin abundance of H3K9Ac in immunoprecipitation seqRB1S/S cells suggests that pRB may influence repetitive uencing (ChIP-seq), they pRB EZH2 elements in additional ways. analyzed their reads using E2Fs E2F1 Nevertheless, pRB/E2F1an alignment approach that H3K27me3 mediated recruitment of prohibits mismatches and aleuchromatin heterochromatin EZH2 is clearly an important lows random distribution of part of the epigenetic regulareads aligning to multiple tion of these loci. locations in the genome. E2F target genes Repetitive elements Transposable elements are This analysis, which facilioften activated in cancer getates the detection of binding nomes (Lee et al., 2012). The sites in repetitive sequences, discovery of this novel pRB gave to a striking result; in function raises the question addition to the expected Cell Cycle arrest Transposon silencing of whether this role contribpRB binding sites near E2FInhibition of cell proliferation utes to pRB’s tumor suppresregulated gene, they deGenome stability Genome stability sor activity. It is established tected pRB binding at many that inactivation of RB1 rerepetitive genomic regions. Figure 1. pRB Acts in Both Euchromatin and Heterochromatin duces the fidelity of chromoIn both mouse and human fipRB’s interaction with E2F transcription factors suppresses the transcription some segregation in both broblasts, pRB associates of cell cycle-regulated genes. In this way, pRB promotes cell cycle arrest, limits normal and cancer cells with a remarkably diverse cell proliferation, and helps to maintain the fidelity of mitosis (left). A specific type of pRB/E2F1 complex recruits EZH2 to heterochromatic regions and by uncoupling the mitotic assortment of repetitive sesilences repetitive DNA sequences by promoting H3K27me3 (right). The pRBcheckpoint control from cell quences, including satellite E2F1-EZH2 complex represses the expression of transposons and promotes cycle progression and by DNA repeats and both autongenome stability. altering centromere structure omous and non-autonomous (Hernando et al., 2004; Manntransposons. While the abundance of pRB at these loci is highest in regulation of these sequences. Ishak ing et al., 2010). It is tempting to imagine arrested cells, pRB association with re- et al. (2016) observe a specific and sig- that activation of TEs may also contribute petitive sequences is also detected in nificant reduction in the abundance of to the genomic instability in RB1 null tuH3K27me3 in repetitive DNA regions and mor cells, and this undoubtedly will be a proliferating cells. pRB’s ability to bind to DNA is thought a concomitant increase of H3K9Ac topic for future investigation. Both gainto be largely dependent on its association enrichment in RB1S/S cells. However, of-function and loss-of-function Ezh2 muwith E2F. Earlier studies had shown that other epigenetic modifications that have tations have been identified in cancer pRB can interact with E2F1 in a way that been implicated in the silencing of TEs, (Kim and Roberts, 2016), and it will be differs from its interactions with other such as H4K20me3, H3K9me3, and DNA interesting to discover if either of these E2Fs (Dick and Dyson, 2003). Using an methylation, were not altered by the activities is linked to its interaction with RB1S allele, which contains an F832A RB1S allele. When the authors examined pRB. It is conceivable that some oncosubstitution and is defective for this the recruitment of EZH2, the methyl- genic signals may target the interaction E2F1-specific binding site, Ishak et al. transferase catalyzing the H3K27me3, between pRB and EZH2 to undermine its (2016) found that pRB’s association with they found that EZH2 enrichment at the tumor suppressor activity. repetitive genomic regions is dependent repetitive sequences was pRB depenThe analysis of RB1S/S mice provides dent. ChIP-seq analysis confirmed that some insights into the biological signifion this selective interaction with E2F1. RB1S/S MEFs display increased expres- EZH2 co-localizes with pRB at both TEs cance of the pRB/E2F1/EZH2 interaction. sion levels of TEs, satellites, and simple and major satellite repeats and that this Although H3K27me3 is reduced at repetDNA repeats. Strikingly, the control of co-localization is strictly dependent on itive DNA sequences in chromatin isothe repetitive sequences expression is pRB’s interaction with E2F1. These find- lated from RB1S/S splenocytes, the limited to this specific pRB/E2F1 interac- ings suggest an appealing new model expression of these regions varies betion. Other mutations in RB1 that affect that brings the expression of repetitive tween animals. Ishak et al. (2016) suggest the regulation of canonical E2F targets, genomic elements under pRB/E2F1/ that cells expressing repetitive sequences such as mutations that abrogate a binding EZH2 control (Figure 1). pRB/E2F1 is not stimulate an immune response, an idea site that interacts with the transactivation needed for EZH2 to be recruited to all of supported by the activation of an interdomain of multiple E2F proteins or the its binding sites, and it seems unlikely feron response in RB1S/S splenocytes, LXCXE-binding cleft of pRB, retain the that EZH2 will be the only activity re- and this may limit the accumulation of ability to silence endogenous retroviral cruited by pRB to repetitive sequences, such cells in young adult mice. Histone since pRB also interacts with several tail modifications play important roles in sequences. pRB association with repetitive ele- other chromatin modifiers, including the silencing of transposons when DNA ments contributes to the epigenetic the histone de-acetylases HDAC1 and methylation is lost in embryonic stem cells 1016 Molecular Cell 64, December 15, 2016

Molecular Cell

Previews (Walter et al., 2016). It is possible, therefore, that an alternative epigenetic mark may dominantly repress repetitive sequences in adult tissues. However, older RB1S/S mice develop lymphomas and show a significantly reduced tumor-free survival in comparison to wild-type mice. Moreover, the transcriptional activation of repetitive DNA sequences can be detected in these tumors. These observations suggest that the link between pRB, E2F1, and EZH2 is not just interesting molecular biology but that it is also relevant for tumor suppression. In summary, the observations described by Ishak et al. (2016) give us an altered perspective on the genomic loci that are targeted by pRB. The silencing of repetitive genomic elements by a pRB/ E2F1/EZH2 complex takes us into a new direction in studies of pRB’s tumor suppressor activity. Aberrant expression of

DNA repeats may increase the susceptibility of RB1 null tumors to immune therapy, and these changes may suggest new approaches for targeting RB1 mutant tumor cells. It will be interesting to discover whether the activation of TEs in RB1-deficient tumors is indeed mutagenic and whether these changes increase acquired resistance to therapy. ACKNOWLEDGMENTS N.J.D. is the James and Shirley Curvey Massachusetts General Hospital Research Scholar.

Ishak, C.A., Marshall, A.E., Passos, D.T., White, C.R., Kim, S.J., Cecchini, M.J., Ferwati, S., MacDonald, W.A., Howlett, C.J., Welch, I.D., et al. (2016). Mol. Cell 64, this issue, 1074–1087. Iskow, R.C., McCabe, M.T., Mills, R.E., Torene, S., Pittard, W.S., Neuwald, A.F., Van Meir, E.G., Vertino, P.M., and Devine, S.E. (2010). Cell 141, 1253–1261. Kim, K.H., and Roberts, C.W. (2016). Nat. Med. 22, 128–134. Lee, E., Iskow, R., Yang, L., Gokcumen, O., Haseley, P., Luquette, L.J., 3rd, Lohr, J.G., Harris, C.C., Ding, L., Wilson, R.K., et al.; Cancer Genome Atlas Research Network (2012). Science 337, 967–971. Manning, A.L., Longworth, M.S., and Dyson, N.J. (2010). Genes Dev. 24, 1364–1376.

REFERENCES Dick, F.A., and Dyson, N. (2003). Mol. Cell 12, 639–649. Hernando, E., Nahle´, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hemann, M., Michel, L., Mittal, V., Gerald, W., Benezra, R., et al. (2004). Nature 430, 797–802.

Slotkin, R.K., and Martienssen, R. (2007). Nat. Rev. Genet. 8, 272–285. Walter, M., Teissandier, A., Pe´rez-Palacios, R., and Bourc’his, D. (2016). eLife 5, http://dx.doi.org/10. 7554/eLife.11418.

Stressing Out About RAD52 Alberto Ciccia1,* and Lorraine S. Symington2,* 1Department

of Genetics and Development of Microbiology and Immunology Columbia University Irving Medical Center, New York, NY 10032, USA *Correspondence: [email protected] (A.C.), [email protected] (L.S.S.) http://dx.doi.org/10.1016/j.molcel.2016.11.036 2Department

The role of mammalian RAD52 has been mysterious due to the lack of a strong DNA repair phenotype of RAD52-deficient cells. In this issue of Molecular Cell, studies by Bhowmick et al. (2016) and Sotiriou et al. (2016) reveal an unexpected role for RAD52 in promoting DNA synthesis following replication stress. Genome instability, one of the hallmarks of cancer, is driven by replication stress. Replication stress can result from oncogene activation, depletion of nucleotides, DNA damage to the template strands, or physical impediments to the replication machinery, such as tightly bound proteins, R loops, or noncanonical DNA structures (Berti and Vindigni, 2016). Cells have multiple mechanisms to deal with replication stress (Figure 1). In particular, stalled fork structures can be remodeled into four-way structures, known as reversed forks, thereby allowing DNA synthesis to proceed past the block by

using the complementary, nascent DNA strand as a template (Figure 1, IV–VI). Defective protection of stalled forks after replication stress can result in fork collapse, generating single-ended DNA breaks that can be repaired by homology-dependent strand invasion and DNA synthesis (Figure 1, X–XIII). In this issue, Bhowmick et al. (2016) and Sotiriou et al. (2016) identify an unexpected role for RAD52 in promoting DNA synthesis following replication stress. While RAD52 is essential for all homologydependent repair (HDR) in budding and fission yeasts, its role in mammalian cells

has remained mysterious because mice lacking it have a mild HDR phenotype. This observation has been attributed to the functional redundancy between RAD52 and BRCA2 in mediating RAD51dependent HDR in mammalian cells (Liu and Heyer, 2011). Importantly, the current studies illuminate a new function for RAD52 that is independent of RAD51 and BRCA2. The Hickson lab had previously shown that replication stress activates DNA repair synthesis in mitosis (MiDAS) at common fragile site (CFS) loci (Minocherhomji et al., 2015). MiDAS requires the

Molecular Cell 64, December 15, 2016 ª 2016 Elsevier Inc. 1017