The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance.

DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.

Life or Death after a Break: What Determines the Choice?

DNA double-strand breaks (DSBs) pose a constant threat to genomic integrity. Such DSBs need to be repaired to preserve homeostasis at both the cellular and organismal levels. Hence, the DNA damage response (DDR) has evolved to repair these lesions and limit their toxicity. The initiation of DNA repair depends on the activation of the DDR, and we know that the strength of DDR signaling may differentially affect cellular viability. However, we do not fully understand what determines the cytotoxicity of a DSB. Recent work has identified genomic location, (in)correct DNA repair pathway usage, and cell-cycle position as contributors to DSB-induced cytotoxicity. In this review, we discuss how these determinants affect cytotoxicity, highlight recent discoveries, and identify open questions that could help to improve our understanding about cell fate decisions after a DNA DSB.

Distinct Roles for Condensin's Two ATPase Sites in Chromosome Condensation.

Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.

Reduction of chromosomal instability and inflammation is a common aspect of adaptation to aneuploidy.

Aneuploidy, while detrimental to untransformed cells, is notably prevalent in cancer. Aneuploidy is found as an early event during tumorigenesis which indicates that cancer cells have the ability to surmount the initial stress responses associated with aneuploidy, enabling rapid proliferation despite aberrant karyotypes. To generate more insight into key cellular processes and requirements underlying adaptation to aneuploidy, we generated a panel of aneuploid clones in p53-deficient RPE-1 cells and studied their behavior over time. As expected, de novo-generated aneuploid clones initially display reduced fitness, enhanced levels of chromosomal instability (CIN), and an upregulated inflammatory response. Intriguingly, after prolonged culturing, aneuploid clones exhibit increased proliferation rates while maintaining aberrant karyotypes, indicative of an adaptive response to the aneuploid state. Interestingly, all adapted clones display reduced CIN and reduced inflammatory signaling, suggesting that these are common aspects of adaptation to aneuploidy. Collectively, our data suggests that CIN and concomitant inflammation are key processes that require correction to allow for fast proliferation in vitro. Finally, we provide evidence that amplification of oncogenic KRAS can promote adaptation.

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.

Centrosomes: Please keep your social distance!

Accurate control of centrosome number is essential for proper chromosome segregation, and it is well established that centrosome abnormalities can trigger a p53-dependent cell cycle arrest. Two new studies published in The EMBO Journal demonstrate how PIDD1 is recruited to centrosomes and that the localization of PIDD1 to distal appendages of centrosomes is required for PIDDosome activation at clustered supernumerary centrosomes.

Unexpected gene activation following CRISPR-Cas9-mediated genome editing.

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSBs) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiently generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are not yet well understood. Here, we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of Taxol-resistant colonies. We show that these colonies upregulate ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 on-target effect that researchers need to be aware of when using lentiviral vectors for genome editing.

Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening.

Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites.

Double-strand break toxicity is chromatin context independent.

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.

Life of double minutes: generation, maintenance, and elimination.

Advances in genome sequencing have revealed a type of extrachromosomal DNA, historically named double minutes (also referred to as ecDNA), to be common in a wide range of cancer types, but not in healthy tissues. These cancer-associated circular DNA molecules contain one or a few genes that are amplified when double minutes accumulate. Double minutes harbor oncogenes or drug resistance genes that contribute to tumor aggressiveness through copy number amplification in combination with favorable epigenetic properties. Unequal distribution of double minutes over daughter cells contributes to intratumoral heterogeneity, thereby increasing tumor adaptability. In this review, we discuss various models delineating the mechanism of generation of double minutes. Furthermore, we highlight how double minutes are maintained, how they evolve, and discuss possible mechanisms driving their elimination.

Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment.

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Targeting the cyclin-dependent kinase 5 in metastatic melanoma.

The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.

Consequences of Genomic Diversification Induced by Segregation Errors.

Chromosome segregation errors are an important source of genomic diversification that promote tumor heterogeneity and evolution. However, the aneuploidy induced by chromosome missegregations causes cellular stress at many levels, raising the question of how segregation errors can be tolerated in cancer. Additionally, we now know that chromosome segregation errors can lead to activation of the innate immune system, producing yet another challenge for chromosomally unstable cells. These observations imply that several liabilities are encountered during tumor evolution, which could potentially be exploited for cancer therapies. Here, we provide an overview of the different causes of segregation errors, their impact on cellular and genomic homeostasis, and discuss recent studies that help to understand how tolerance towards imbalanced karyotypes can be obtained.

PHF6 promotes non-homologous end joining and G2 checkpoint recovery.

The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Börjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint.

Transient activation of p53 in G2 phase is sufficient to induce senescence.

DNA damage can result in a transient cell-cycle arrest or lead to permanent cell-cycle withdrawal. Here we show that the decision to irreversibly withdraw from the cell cycle is made within a few hours following damage in G2 cells. This permanent arrest is dependent on induction of p53 and p21, resulting in the nuclear retention of Cyclin B1. This rapid response is followed by the activation of the APC/C(Cdh1) (the anaphase-promoting complex/cyclosome and its coactivator Cdh1) several hours later. Inhibition of APC/C(Cdh1) activity fails to prevent cell-cycle withdrawal, whereas preventing nuclear retention of Cyclin B1 does allow cells to remain in cycle. Importantly, transient induction of p53 in G2 cells is sufficient to induce senescence. Taken together, these results indicate that a rapid and transient pulse of p53 in G2 can drive nuclear retention of Cyclin B1 as the first irreversible step in the onset of senescence.

Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition.

During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy of fluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.

Uncovering SUMOylation dynamics during cell-cycle progression reveals FoxM1 as a key mitotic SUMO target protein.

Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation. SUMOylation of FoxM1 peaks during G2 and M phase, when FoxM1 transcriptional activity is required. We found that a SUMOylation-deficient FoxM1 mutant was less active compared to wild-type FoxM1, implying that SUMOylation of the protein enhances its transcriptional activity. Mechanistically, SUMOylation blocks the dimerization of FoxM1, thereby relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression.

Sustained CHK2 activity, but not ATM activity, is critical to maintain a G1 arrest after DNA damage in untransformed cells.

BACKGROUND: The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized. RESULTS: Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1 h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover. CONCLUSION: Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction.