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Telomeres--repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes--mediate genome stability and determine cellular lifespan.sup.1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends.sup.2,3; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase.sup.4-6 and homology-directed DNA repair.sup.7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans. Telomeric-repeat-containing RNA is recruited to telomeres by a mechanism that involves the DNA recombinase RAD51 and the formation of DNA-RNA hybrids, or R-loops--a process similar to that involved in homology-directed DNA repair., Author(s): Marianna Feretzaki [sup.1] , Michaela Pospisilova [sup.2] , Rita Valador Fernandes [sup.1] , Thomas Lunardi [sup.1] , Lumir Krejci [sup.2] [sup.3] , Joachim Lingner [sup.1] Author Affiliations: (1) Swiss [...]

The chromatin-remodelling complex SWI/SNF is highly conserved and has critical roles in various cellular processes, including transcription and DNA-damage repair.sup.1,2. It hydrolyses ATP to remodel chromatin structure by sliding and evicting histone octamers.sup.3-8, creating DNA regions that become accessible to other essential factors. However, our mechanistic understanding of the remodelling activity is hindered by the lack of a high-resolution structure of complexes from this family. Here we report the cryo-electron microscopy structure of Saccharomyces cerevisiae SWI/SNF bound to a nucleosome, at near-atomic resolution. In the structure, the actin-related protein (Arp) module is sandwiched between the ATPase and the rest of the complex, with the Snf2 helicase-SANT associated (HSA) domain connecting all modules. The body contains an assembly scaffold composed of conserved subunits Snf12 (also known as SMARCD or BAF60), Snf5 (also known as SMARCB1, BAF47 or INI1) and an asymmetric dimer of Swi3 (also known as SMARCC, BAF155 or BAF170). Another conserved subunit, Swi1 (also known as ARID1 or BAF250), resides in the core of SWI/SNF, acting as a molecular hub. We also observed interactions between Snf5 and the histones at the acidic patch, which could serve as an anchor during active DNA translocation. Our structure enables us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and to propose a model for how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation.sup.9. The cryo-electron microscopy structure of the yeast SWI/SNF complex bound to a nucleosome substrate provides insights into the chromatin-remodelling function of this family of protein complexes and suggests mechanisms by which the mutated proteins may cause cancer., Author(s): Yan Han [sup.1] , Alexis A Reyes [sup.1] [sup.2] , Sara Malik [sup.1] , Yuan He [sup.1] [sup.2] [sup.3] [sup.4] Author Affiliations: (1) Department of Molecular Biosciences, Northwestern University, [...]

Mutations that attenuate homologous recombination (HR)-mediated repair promote tumorigenesis and sensitize cells to chemotherapeutics that cause replication fork collapse, a phenotype known as 'BRCAness'.sup.1. BRCAness tumours arise from loss-of-function mutations in 22 genes.sup.1. Of these genes, all but one (CDK12) function directly in the HR repair pathway.sup.1. CDK12 phosphorylates serine 2 of the RNA polymerase II C-terminal domain heptapeptide repeat.sup.2-7, a modification that regulates transcription elongation, splicing, and cleavage and polyadenylation.sup.8,9. Genome-wide expression studies suggest that depletion of CDK12 abrogates the expression of several HR genes relatively specifically, thereby blunting HR repair.sup.3-7,10,11. This observation suggests that the mutational status of CDK12 may predict sensitivity to targeted treatments against BRCAness, such as PARP1 inhibitors, and that CDK12 inhibitors may induce sensitization of HR-competent tumours to these treatments.sup.6,7,10,11. Despite growing clinical interest, the mechanism by which CDK12 regulates HR genes remains unknown. Here we show that CDK12 globally suppresses intronic polyadenylation events in mouse embryonic stem cells, enabling the production of full-length gene products. Many HR genes harbour more intronic polyadenylation sites than other expressed genes, and these sites are particularly sensitive to loss of CDK12. The cumulative effect of these sites accounts for the enhanced sensitivity of HR gene expression to CDK12 loss, and we find that this mechanism is conserved in human tumours that contain loss-of-function CDK12 mutations. This work clarifies the function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumour biomarker.The kinase CDK12 suppresses usage of intronic polyadenylation sites and thereby promotes the expression of genes involved in homologous recombination DNA repair., Author(s): Sara J. Dubbury [sup.1] [sup.2] , Paul L. Boutz [sup.1] [sup.3] , Phillip A. Sharp [sup.1] [sup.2] Author Affiliations:(1) Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, [...]

Duplicates of antiaging genes make certain types of jellyfish biologically immortal. The standard jellyfish life cycle begins with larvae that develop into polyps and then grow into the adult 'medusa' [...]

The histone demethylase PHF8 has been implicated in multiple pathological disorders, including X-linked mental retardation and tumorigenesis. However, it is not clear how the abundance and function of PHF8 are regulated. Here, we report that PHF8 physically associates with the deubiquitinase USP7. Specifically, we demonstrated that USP7 promotes deubiquitination and stabilization of PHF8, leading to the upregulation of a group of genes, including cyclin A2, that are critical for cell growth and proliferation. The USP7-encoding gene was also transcriptionally regulated by PHF8, via positive feedback. USP7 was overexpressed in breast carcinomas, and the level of expression positively correlated with expression of PHF8 and cyclin A2 and with the histological grade of breast cancer. We showed that USP7 promotes breast carcinogenesis by stabilizing PHF8 and upregulating cyclin A2 and that the interaction between USP7 and PHF8 is augmented during DNA damage. Moreover, USP7-promoted PHF8 stabilization conferred cellular resistance to genotoxic insults and was required for the recruitment of BLM and KU70, which are both essential for DNA double-strand break repair. Our study mechanistically links USP7 to epigenetic regulation and DNA repair. Moreover, these data support the pursuit of USP7 and PHF8 as potential targets for breast cancer intervention, especially in combination with chemo- or radiotherapies., Introduction Posttranslational modification of histone proteins, which is accomplished by means of a variety of enzymatic reactions, plays an important role in chromatin structure and function in eukaryotic cells (1). [...]

Somatic mutations are the driving force of cancer genome evolution (1). The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, [...]

Promoters are DNA sequences that have an essential role in controlling gene expression. While recent whole cancer genome analyses have identified numerous hotspots of somatic point mutations within promoters, many [...]

Loss of REV7 is shown to regulate end resection of double-stranded DNA breaks in BRCA1-deficient cells, leading to PARP inhibitor resistance and restoration of homologous recombination; REV7 dictates pathway choice in BRCA1-deficient cells and during immunoglobulin class switching. MAD2L2/REV7 promotes genome integrity DNA polymerase [zeta], composed of REV3, REV7 and an associated factor, REV1, mediates a type of DNA repair involving translesion synthesis, and hence its activity is highly mutagenic. Two studies exploring the DNA damage response have converged on REV7 (also known as MAD2L2) as a factor that, by itself, can promote maintenance of genome integrity. Several protective mechanisms that prevent telomere ends being recognized as a double-strand breaks (DSBs) and triggering an inappropriate DNA damage response were known. Jacqueline Jacobs and colleagues now show that REV7/MAD2L2 suppresses homology-dependent repair at deprotected telomeres and at irradiation-induced DSBs by inhibiting resection of the 5' end. As a consequence, the ends are shunted into the non-homologous end-joining pathway. Sven Rottenberg and colleagues came to a similar conclusion by studying the development of resistance to PARP inhibitors. They found that REV7/MAD2L2 dictates pathway choice in BRCA-deficient cells and during immunoglobulin class switching. Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway.sup.1. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers.sup.2,3. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration.sup.4. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases.sup.5. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance.sup.6. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells., Author(s): Guotai Xu [sup.1] , J. Ross Chapman [sup.2] , Inger Brandsma [sup.3] , Jingsong Yuan [sup.4] , Martin Mistrik [sup.5] , Peter Bouwman [sup.6] , Jirina Bartkova [sup.7] , [...]

MAD2L2 regulates DNA repair at deprotected telomeres and at ionizing-radiation-induced double-stranded DNA breaks by inhibiting resection of the 5' ends; the ends are thus shunted into the non-homologous end-joining pathway. MAD2L2/REV7 promotes genome integrity DNA polymerase [zeta], composed of REV3, REV7 and an associated factor, REV1, mediates a type of DNA repair involving translesion synthesis, and hence its activity is highly mutagenic. Two studies exploring the DNA damage response have converged on REV7 (also known as MAD2L2) as a factor that, by itself, can promote maintenance of genome integrity. Several protective mechanisms that prevent telomere ends being recognized as a double-strand breaks (DSBs) and triggering an inappropriate DNA damage response were known. Jacqueline Jacobs and colleagues now show that REV7/MAD2L2 suppresses homology-dependent repair at deprotected telomeres and at irradiation-induced DSBs by inhibiting resection of the 5' end. As a consequence, the ends are shunted into the non-homologous end-joining pathway. Sven Rottenberg and colleagues came to a similar conclusion by studying the development of resistance to PARP inhibitors. They found that REV7/MAD2L2 dictates pathway choice in BRCA-deficient cells and during immunoglobulin class switching. Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and ageing.sup.1,2. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3' telomeric overhangs, indicating that MAD2L2 inhibits 5' end resection. End resection blocks NHEJ while committing to homology-directed repair, and is under the control of 53BP1, RIF1 and PTIP.sup.3. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5' end resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end resection at irradiation-induced DNA double-strand breaks and promotes end-joining of DNA double-strand breaks in several settings, including during immunoglobulin class switch recombination. These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis.sup.4. Together, our data establish MAD2L2 as a crucial contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5' end resection downstream of RIF1., Author(s): Vera Boersma [sup.1] , Nathalie Moatti [sup.1] , Sandra Segura-Bayona [sup.1] , Marieke H. Peuscher [sup.1] , Jaco van der Torre [sup.1] , Brigitte A. Wevers [sup.1] , Alexandre [...]