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The spatiotemporal configuration of genes with distal regulatory elements is believed to be crucial for transcriptional control, but full mechanistic understanding is lacking. We combine simultaneous live tracking of pairs of genomic loci and nascent transcripts with molecular dynamics simulations to assess the Sox2 gene and its enhancer. We find that both loci exhibit more constrained mobility than control sequences due to stalled cohesin at CCCTC-binding factor sites. Strikingly, enhancer mobility becomes constrained on transcriptional firing, homogenizing its dynamics with the gene promoter, suggestive of their cotranscriptional sharing of a nuclear microenvironment. Furthermore, we find transcription and loop extrusion to be antagonistic processes constraining regulatory loci. These findings indicate that modulating chromatin mobility can be an additional, underestimated means for effective gene regulation.

DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements., (© 2024. The Author(s).)

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the spatiotemporal arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find a close link between chromatin mobility and transcriptional status: active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters. These clusters are refractory to DNA repair through homologous recombination (HR). The mechanisms by which pericentromeric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown. Here, we compare the spatial recruitment of HR factors upon double-stranded DNA breaks (DSBs) induced in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters. We show that while DSBs increase the accessibility of human pericentromeric heterochromatin by disrupting HP1α dimerization, mouse pericentromeric heterochromatin repeat clustering imposes a physical barrier that requires many layers of de-compaction to be accessed. Our results support a model in which the 3D organization of heterochromatin dictates the spatial activation of DNA repair pathways and is key to preventing the activation of HR within clustered repeats and the onset of chromosomal translocations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Replication of the eukaryotic genome is a highly regulated process and stringent control is required to maintain genome integrity. In this review, we will discuss the many aspects of the chromatin and nuclear environment that play key roles in the regulation of both unperturbed and stressed replication. Firstly, the higher order organisation of the genome into A and B compartments, topologically associated domains (TADs) and sub-nuclear compartments has major implications in the control of replication timing. In addition, the local chromatin environment defined by non-canonical histone variants, histone post-translational modifications (PTMs) and enrichment of factors such as heterochromatin protein 1 (HP1) plays multiple roles in normal S phase progression and during the repair of replicative damage. Lastly, we will cover how the spatial organisation of stalled replication forks facilitates the resolution of replication stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wootton and Soutoglou.)

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID) 1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins 1-3 . Although uracils arising in DNA are accurately repaired 1-4 , how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown 1-3 . Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2) 5 and is overexpressed in several cancers 5 . We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a -/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a -/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Centromeric integrity is key for proper chromosome segregation during cell division 1 . Centromeres have unique chromatin features that are essential for centromere maintenance 2 . Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements 3 , little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle 4 . Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA-RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of the RAD51-BRCA1-BRCA2 complex 5 and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity., (© 2021. Crown.)

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR/Cas9 technology can be used to investigate how double-strand breaks (DSBs) occurring in constitutive heterochromatin are getting repaired. This technology can be used to induce specific breaks on mouse pericentromeric heterochromatin, by using a guide RNA specific for the major satellite repeats and co-expressing it with Cas9. Those clean DSBs can be visualized later by confocal microscopy. More specifically, immunofluorescence can be used to visualize the main factors of each DSB repair pathway and quantify their percentage and pattern of recruitment at the heterochromatic region.

Genomic instability can be life-threatening. The fine balance between error-free and mutagenic DNA repair pathways is essential for maintaining genome integrity. Recent advances in DNA double-strand break induction and detection techniques have allowed the investigation of DNA damage and repair in the context of the highly complex nuclear structure. These studies have revealed that the 3D genome folding, nuclear compartmentalization and cytoskeletal components control the spatial distribution of DNA lesions within the nuclear space and dictate their mode of repair., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Genome editing by Clustered Regularly Inter Spaced Palindromic Repeat (CRISPR) associated (Cas) systems has revolutionized medical research and holds enormous promise for correcting genetic diseases. Understanding how these Cas nucleases work and induce mutations, as well as identifying factors that affect their efficiency and fidelity is key to developing this technology for therapeutic uses. Here, we discuss recent studies that reveal how DNA sequence and chromatin structure influences the different steps of genome editing. These studies also demonstrate that a deep understanding of the balance between error prone and error free DNA repair pathways is crucial for making genome editing a safe clinical tool, which does not induce further mutations to the genome., (Copyright © 2020 Chechik, Martin and Soutoglou.)

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery., (© 2019 Caron et al.; Published by Cold Spring Harbor Laboratory Press.)

DNA repair is critical to maintaining genome integrity, and its dysfunction can cause accumulation of unresolved damage that leads to genomic instability. The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex and the nuclear pore-associated transcription and export complex 2 (TREX-2) couple transcription with mRNA export. In this study, we identify a novel interplay between human TREX-2 and the deubiquitination module (DUBm) of SAGA required for genome stability. We find that the scaffold subunit of TREX-2, GANP, positively regulates DNA repair through homologous recombination (HR). In contrast, DUBm adaptor subunits ENY2 and ATXNL3 are required to limit unscheduled HR. These opposite roles are achieved through monoubiquitinated histone H2B (H2Bub1). Interestingly, the activity of the DUBm of SAGA on H2Bub1 is dependent on the integrity of the TREX-2 complex. Thus, we describe the existence of a functional interaction between human TREX-2 and SAGA DUBm that is key to maintaining the H2B/HB2ub1 balance needed for efficient repair and HR., (© 2018 Evangelista et al.)

DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair. We have found that the SUMO E3 ligase CBX4 controls resection through the key factor CtIP. Indeed, CBX4 depletion impairs CtIP constitutive sumoylation and DNA end processing. Importantly, mutating lysine 896 in CtIP recapitulates the CBX4-depletion phenotype, blocks homologous recombination and increases genomic instability. Artificial fusion of CtIP and SUMO suppresses the effects of both the non-sumoylatable CtIP mutant and CBX4 depletion. Mechanistically, CtIP sumoylation is essential for its recruitment to damaged DNA. In summary, sumoylation of CtIP at lysine 896 defines a subpopulation of the protein that is involved in DNA resection and recombination.The choice between non-homologous end-joining and homologous recombination to repair a DNA double-strand break depends on activation of the end resection machinery. Here the authors show that SUMO E3 ligase CBX4 sumoylates subpopulation of CtIP to regulate recruitment to breaks and resection.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.

Identifying DNA fragile sites is crucial to reveal hotspots of genomic rearrangements, yet their precise mapping has been a challenge. A new study in this issue of Molecular Cell (Canela et al., 2016) introduces a highly sensitive and accurate method to detect DNA breaks in vivo that can be adapted to various experimental and clinical settings., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The continuous threats on genome integrity by endogenous and exogenous sources have rendered cells competent to overcome these challenges by activating DNA repair pathways. A complex network of proteins and their modifications participate in orchestrated signaling cascades, which are induced in response to DNA damage and may determine the choice of repair pathway. In this review, we summarize recent findings in the field of DNA Double Strand Break repair with regard to the positioning of the break in the highly compartmentalized nucleus. We aim to highlight the importance of chromatin state along with the nuclear position of the DNA lesions on the choice of DNA repair pathway and maintenance of genome integrity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results in the sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)