Linking CRISPR-Cas9 double-strand break profiles to gene editing precision with BreakTag.

Cas9 can cleave DNA in both blunt and staggered configurations, resulting in distinct editing outcomes, but what dictates the type of Cas9 incisions is largely unknown. In this study, we developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. Overall, we assessed cleavage by SpCas9 at more than 150,000 endogenous on-target and off-target sites targeted by approximately 3,500 single guide RNAs. We found that approximately 35% of SpCas9 DSBs are staggered, and the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants. A machine learning model shows that Cas9 incision is dependent on the protospacer sequence and that human genetic variation impacts the configuration of Cas9 cuts and the DSB repair outcome. Matched datasets of Cas9 and engineered variant incisions with repair outcomes show that Cas9-mediated staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, demonstrating that a scission-based gRNA design can be used to correct clinically relevant pathogenic single-nucleotide deletions.

High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue.

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.

53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling.

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double-strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.

RECON syndrome is a genome instability disorder caused by mutations in the DNA helicase RECQL1.

Despite being the first homolog of the bacterial RecQ helicase to be identified in humans, the function of RECQL1 remains poorly characterized. Furthermore, unlike other members of the human RECQ family of helicases, mutations in RECQL1 have not been associated with a genetic disease. Here, we identify 2 families with a genome instability disorder that we have named RECON (RECql ONe) syndrome, caused by biallelic mutations in the RECQL gene. The affected individuals had short stature, progeroid facial features, a hypoplastic nose, xeroderma, and skin photosensitivity and were homozygous for the same missense mutation in RECQL1 (p.Ala459Ser), located within its zinc binding domain. Biochemical analysis of the mutant RECQL1 protein revealed that the p.A459S missense mutation compromised its ATPase, helicase, and fork restoration activity, while its capacity to promote single-strand DNA annealing was largely unaffected. At the cellular level, this mutation in RECQL1 gave rise to a defect in the ability to repair DNA damage induced by exposure to topoisomerase poisons and a failure of DNA replication to progress efficiently in the presence of abortive topoisomerase lesions. Taken together, RECQL1 is the fourth member of the RecQ family of helicases to be associated with a human genome instability disorder.

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability.

Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis.

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces marked chromosome condensation, but following reentry into the G1 phase of the cell cycle, chromosomes reestablish their interphase organization. Here, we tested the role of RNA polymerase II (RNAPII) in this transition using a cell line that allows its auxin-mediated degradation. In situ Hi-C showed that RNAPII is required for both compartment and loop establishment following mitosis. RNAPs often counteract loop extrusion, and in their absence, longer and more prominent loops arose. Evidence from chromatin binding, super-resolution imaging, and in silico modeling allude to these effects being a result of RNAPII-mediated cohesin loading upon G1 reentry. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

Genome-wide detection of DNA double-strand breaks by in-suspension BLISS.

sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4+ T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.

53BP1-DNA repair enters a new liquid phase.

In response to DNA damage, transient repair compartments in the nucleus concentrate repair proteins and activate downstream signaling factors. In this issue of The EMBO Journal, Kilic et al show that DNA repair focal assemblies marked by accumulation of 53BP1 are phase separated liquid compartments. This liquid droplet-like behavior of 53BP1 compartments might help to coordinate local lesion recognition with global gene activation in response to DNA damage.

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.

Dynamics of Double-Strand Breaks: Implications for the Formation of Chromosome Translocations.

Illegitimate joining of chromosome breaks can lead to the formation of chromosome translocations, a catastrophic type of genome rearrangements that often plays key roles in tumorigenesis. Emerging evidence suggests that the mobility of broken DNA loci can be an important determinant in partner search and clustering of individual breaks, events that can influence translocation frequency. We summarize here the recent literature on the mechanisms that regulate chromatin movement, focusing on studies exploring the motion properties of double-strand breaks in the context of chromatin, the functional consequences for DNA repair, and the formation of chromosome fusions.

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types.

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression.

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1-/-) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1-/- mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1-/- animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1-/-/MGMT-/- double knockout (DKO) mice developed more, but much smaller tumors than MGMT-/- animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1-/- cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

Studies of the DNA Damage Response by Using the Lac Operator/Repressor (LacO/LacR) Tethering System.

Maintaining the integrity of genetic information is essential for the survival of cells. Recent advances in cell biological and microscopy methodologies have complemented traditional genetic and biochemical approaches, and they now permit the observation of spatiotemporal aspects of damaged chromosomal loci. In one of these approaches, integrated LacO/TetO operator sequences can be used as binding sites to physically tether onto chromatin any protein of interest when genetically fused to the respective repressors (LacR/TetR). This methodology has been the basis of several models to probe the spatial dynamics of DNA repair in the eukaryotic nucleus and to visualize genomic loci in yeast, fly, nematodes, and in mammalian cells. Further applications are the induction of localized DNA damage by immobilizing endonucleases at different genome sites in vivo, the assessment of the hierarchy of protein interactions within repair complexes, and the activation of the DNA damage response (DDR) by the physical tethering of DSB-repair factors on chromatin in the absence of damage. We outline here a protocol for the quantification of DDR activation upon the prolonged immobilization of single repair factors on chromatin or upon tethering of the endonuclease FokI. The outlined protocol requires basic cell culture and microscopy skills and allows the tethering of any protein of interest within 2-3 days.