Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair.

Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Nuclear F-actin and myosins drive relocalization of heterochromatic breaks.

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody.

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.

Nuclear Dynamics of Heterochromatin Repair.

Repairing double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. Recent studies in Drosophila cells revealed that faithful homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of DSBs to the nuclear periphery before Rad51 recruitment. We summarize here the exciting progress in understanding this pathway, including conserved responses in mammalian cells and surprising similarities with mechanisms in yeast that deal with DSBs in distinct sites that are difficult to repair, including other repeated sequences. We will also point out some of the most important open questions in the field and emerging evidence suggesting that deregulating these pathways might have dramatic consequences for human health.

HDACs link the DNA damage response, processing of double-strand breaks and autophagy.

Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.

Nup153 is not required for anchoring heterochromatic DSBs to the nuclear periphery.

Pericentromeric heterochromatin mostly comprises repeated DNA sequences prone to ectopic recombination. In Drosophila cells, 'safe' homologous recombination repair requires relocalization of heterochromatic repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. DSBs are anchored to the nuclear periphery through the Nup107/160 nucleoporin complex. Previous studies suggested that the nuclear pore 'basket' protein Nup153 could also mediate anchoring, but Nup153 RNAi depletion also affects Nup107 association with the pores, preventing a direct assessment of Nup153 role. Using a separation of function mutant, here we show that Nup153 is not required for anchoring heterochromatic DSBs to the nuclear periphery.

Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancers can also be purely initiated by epigenetic alterations, without driver mutations. Specifically, a 24-hours transient down-regulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to drive epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and are characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 down-regulation is performed for extended periods of time remains unclear. Here we show that prolonged depletion of a PRC1 component, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad mis-regulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs, and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects amplify the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin.

Repair of double strand breaks (DSBs) is essential for cell survival and genome integrity. While much is known about the molecular mechanisms involved in DSB repair and checkpoint activation, the roles of nuclear dynamics of radiation-induced foci (RIF) in DNA repair are just beginning to emerge. Here, we summarize results from recent studies that point to distinct features of these dynamics in two different chromatin environments: heterochromatin and euchromatin. We also discuss how nuclear architecture and chromatin components might control these dynamics, and the need of novel quantification methods for a better description and interpretation of these phenomena. These studies are expected to provide new biomarkers for radiation risk and new strategies for cancer detection and treatment.

Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin.

Pericentromeric heterochromatin is highly enriched for repetitive sequences prone to aberrant recombination. Previous studies showed that homologous recombination (HR) repair is uniquely regulated in this domain to enable 'safe' repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. Here, we induce DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR- white reporter and I-SceI expression in spermatogonia of flies. Using this approach, we detect higher frequency of HR repair in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.

Replication fork plasticity upon replication stress requires rapid nuclear actin polymerization.

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase, rapidly extending in number and thickness upon genotoxic treatments, and taking frequent contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork plasticity is linked to reduced recruitment of RAD51 and SMARCAL1 to nascent DNA. Conversely, PRIMPOL gains access to replicating chromatin, promoting unrestrained and discontinuous DNA synthesis, which is associated with increased chromosomal instability and decreased cellular resistance to replication stress. Hence, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

Nuclear actin polymerization rapidly mediates replication fork remodeling upon stress by limiting PrimPol activity.

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase and observed their rapid extension in number and length upon genotoxic treatments, frequently taking contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork remodeling is linked to deregulated chromatin loading of PrimPol, which promotes unrestrained and discontinuous DNA synthesis and limits the recruitment of RAD51 and SMARCAL1 to nascent DNA. Moreover, defective nuclear actin polymerization upon mild replication interference induces chromosomal instability in a PRIMPOL-dependent manner. Hence, by limiting PrimPol activity, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

An Expanding Toolkit for Heterochromatin Repair Studies.

Pericentromeric heterochromatin is mostly composed of repetitive DNA sequences prone to aberrant recombination. Cells have developed highly specialized mechanisms to enable 'safe' homologous recombination (HR) repair while preventing aberrant recombination in this domain. Understanding heterochromatin repair responses is essential to understanding the critical mechanisms responsible for genome integrity and tumor suppression. Here, we review the tools, approaches, and methods currently available to investigate double-strand break (DSB) repair in pericentromeric regions, and also suggest how technologies recently developed for euchromatin repair studies can be adapted to characterize responses in heterochromatin. With this ever-growing toolkit, we are witnessing exciting progress in our understanding of how the 'dark matter' of the genome is repaired, greatly improving our understanding of genome stability mechanisms.

Multi-scale dynamics of heterochromatin repair.

Studies across different organisms show that nuclear architecture and dynamics play central roles in different aspects of homologous recombination (HR) repair. Here we review the most recent discoveries in this field, ranging from directed motions mediating relocalization pathways, to global chromatin mobilization, local DNA looping, and changes in repair focus properties associated with clustering and phase separation. We discuss how these dynamics work in different contexts, including molecular mechanisms and regulatory pathways involved. We specifically highlight how they function in pericentromeric heterochromatin, which presents a unique environment for HR repair given the abundance of repeated DNA sequences prone to aberrant recombination, the 'silent' chromatin state, and the phase separation characterizing this domain.

Live Cell Imaging of Nuclear Actin Filaments and Heterochromatic Repair foci in Drosophila and Mouse Cells.

Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that 'safe' homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here, we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.

The human F-Box DNA helicase FBH1 faces Saccharomyces cerevisiae Srs2 and postreplication repair pathway roles.

The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions.

Complex Chromatin Motions for DNA Repair.

A number of studies across different model systems revealed that chromatin undergoes significant changes in dynamics in response to DNA damage. These include local motion changes at damage sites, increased nuclear exploration of both damaged and undamaged loci, and directed motions to new nuclear locations associated with certain repair pathways. These studies also revealed the need for new analytical methods to identify directed motions in a context of mixed trajectories, and the importance of investigating nuclear dynamics over different time scales to identify diffusion regimes. Here we provide an overview of the current understanding of this field, including imaging and analytical methods developed to investigate nuclear dynamics in different contexts. These dynamics are essential for genome integrity. Identifying the molecular mechanisms responsible for these movements is key to understanding how their misregulation contributes to cancer and other genome instability disorders.

Actin' between phase separated domains for heterochromatin repair.

DNA double-strand breaks (DSBs) are particularly challenging to repair in pericentromeric heterochromatin because of the increased risk of aberrant recombination in highly repetitive sequences. Recent studies have identified specialized mechanisms enabling 'safe' homologous recombination (HR) repair in heterochromatin. These include striking nuclear actin filaments (F-actin) and myosins that drive the directed motion of repair sites to the nuclear periphery for 'safe' repair. Here, we summarize our current understanding of the mechanisms involved, and propose how they might operate in the context of a phase-separated environment.

Nuclear actin filaments in DNA repair dynamics.

Recent development of innovative tools for live imaging of actin filaments (F-actin) enabled the detection of surprising nuclear structures responding to various stimuli, challenging previous models that actin is substantially monomeric in the nucleus. We review these discoveries, focusing on double-strand break (DSB) repair responses. These studies revealed a remarkable network of nuclear filaments and regulatory mechanisms coordinating chromatin dynamics with repair progression and led to a paradigm shift by uncovering the directed movement of repair sites.

Dynamic relocalization of replication origins by Fkh1 requires execution of DDK function and Cdc45 loading at origins.

Chromosomal DNA elements are organized into spatial domains within the eukaryotic nucleus. Sites undergoing DNA replication, high-level transcription, and repair of double-strand breaks coalesce into foci, although the significance and mechanisms giving rise to these dynamic structures are poorly understood. In S. cerevisiae, replication origins occupy characteristic subnuclear localizations that anticipate their initiation timing during S phase. Here, we link localization of replication origins in G1 phase with Fkh1 activity, which is required for their early replication timing. Using a Fkh1-dependent origin relocalization assay, we determine that execution of Dbf4-dependent kinase function, including Cdc45 loading, results in dynamic relocalization of a replication origin from the nuclear periphery to the interior in G1 phase. Origin mobility increases substantially with Fkh1-driven relocalization. These findings provide novel molecular insight into the mechanisms that govern dynamics and spatial organization of DNA replication origins and possibly other functional DNA elements.