Publications by authors named "Susan M Gasser"

161 Publications

Nadeshiko revisited: The situation of women in Japanese research and the measures taken to increase their representation.

EMBO Rep 2021 Mar 26;22(3):e52528. Epub 2021 Feb 26.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

The Japanese government has enacted measures to increase the representation of women in research; the situation is improving but there is still much to do.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.202152528DOI Listing
March 2021

Underappreciated Roles of DNA Polymerase δ in Replication Stress Survival.

Trends Genet 2021 Feb 16. Epub 2021 Feb 16.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; Faculty of Sciences, University of Basel, Klingelbergstrasse 90, CH-4056 Basel, Switzerland. Electronic address:

Recent structural analysis of Fe-S centers in replication proteins and insights into the structure and function of DNA polymerase δ (DNA Pol δ) subunits have shed light on the key role played by this polymerase at replication forks under stress. The sequencing of cancer genomes reveals multiple point mutations that compromise the activity of POLD1, the DNA Pol δ catalytic subunit, whereas the loci encoding the accessory subunits POLD2 and POLD3 are amplified in a very high proportion of human tumors. Consistently, DNA Pol δ is key for the survival of replication stress and is involved in multiple long-patch repair pathways. Synthetic lethality arises from compromising the function and availability of the noncatalytic subunits of DNA Pol δ under conditions of replication stress, opening the door to novel therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tig.2020.12.003DOI Listing
February 2021

Damage-induced chromatome dynamics link Ubiquitin ligase and proteasome recruitment to histone loss and efficient DNA repair.

Mol Cell 2021 02 1;81(4):811-829.e6. Epub 2021 Feb 1.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; Faculty of Natural Sciences, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Electronic address:

Eukaryotic cells package their genomes around histone octamers. In response to DNA damage, checkpoint activation in yeast induces core histone degradation resulting in 20%-40% reduction in nucleosome occupancy. To gain insight into this process, we developed a new approach to analyze the chromatin-associated proteome comprehensively before and after damage. This revealed extensive changes in protein composition after Zeocin-induced damage. First, core histones and the H1 homolog Hho1 were partially lost from chromatin along with replication, transcription, and chromatin remodeling machineries, while ubiquitin ligases and the proteasome were recruited. We found that the checkpoint- and INO80C-dependent recruitment of five ubiquitin-conjugating factors (Rad6, Bre1, Pep5, Ufd4, and Rsp5) contributes to core and linker histone depletion, reducing chromatin compaction and enhancing DNA locus mobility. Importantly, loss of Rad6/Bre1, Ufd4/TRIP12, and Pep5/VPS11 compromise DNA strand invasion kinetics during homology-driven repair. Thus we provide a comprehensive overview of a functionally relevant genome-wide chromatin response to DNA damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.12.021DOI Listing
February 2021

Argonaute NRDE-3 and MBT domain protein LIN-61 redundantly recruit an H3K9me3 HMT to prevent embryonic lethality and transposon expression.

Genes Dev 2021 Jan 10;35(1-2):82-101. Epub 2020 Dec 10.

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

The establishment and maintenance of chromatin domains shape the epigenetic memory of a cell, with the methylation of histone H3 lysine 9 (H3K9me) defining transcriptionally silent heterochromatin. We show here that the SET-25 (SUV39/G9a) histone methyltransferase (HMT), which catalyzes H3K9me1, me2 and me3, can establish repressed chromatin domains de novo unlike the SETDB1 homolog MET-2. Thus, SET-25 is needed to silence novel insertions of RNA or DNA transposons, and repress tissue-specific genes de novo during development. We identify two partially redundant pathways that recruit SET-25 to its targets. One pathway requires LIN-61 (L3MBTL2), which uses its four MBT domains to bind the H3K9me2 deposited by MET-2. The second pathway functions independently of MET-2 and involves the somatic Argonaute NRDE-3 and small RNAs. This pathway targets primarily highly conserved RNA and DNA transposons. These redundant SET-25 targeting pathways (MET-2-LIN-61-SET-25 and NRDE-3-SET-25) ensure repression of intact transposons and de novo insertions, while MET-2 can act alone to repress simple and satellite repeats. Removal of both pathways in the double mutant leads to the loss of somatic H3K9me2 and me3 and the synergistic derepression of transposons in embryos, strongly elevating embryonic lethality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.344234.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778263PMC
January 2021

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mol Cell 2021 01 4;81(1):183-197.e6. Epub 2020 Dec 4.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labéllisée Ligue contre le Cancer, 34396 Montpellier, France; University of Basel and Friedrich Miescher Institute for Biomedical Research, Faculty of Natural Sciences, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address:

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.11.010DOI Listing
January 2021

DNA Damage-Induced Nucleosome Depletion Enhances Homology Search Independently of Local Break Movement.

Mol Cell 2020 10 23;80(2):311-326.e4. Epub 2020 Sep 23.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Faculty of Natural Sciences, 4056 Basel, Switzerland. Electronic address:

To determine whether double-strand break (DSB) mobility enhances the physical search for an ectopic template during homology-directed repair (HDR), we tested the effects of factors that control chromatin dynamics, including cohesin loading and kinetochore anchoring. The former but not the latter is altered in response to DSBs. Loss of the nonhistone high-mobility group protein Nhp6 reduces histone occupancy and increases chromatin movement, decompaction, and ectopic HDR. The loss of nucleosome remodeler INO80-C did the opposite. To see whether enhanced HDR depends on DSB mobility or the global chromatin response, we tested the ubiquitin ligase mutant uls1Δ, which selectively impairs local but not global movement in response to a DSB. Strand invasion occurs in uls1Δ cells with wild-type kinetics, arguing that global histone depletion rather than DSB movement is rate limiting for HDR. Impaired break movement in uls1Δ correlates with elevated MRX and cohesin loading, despite normal resection and checkpoint activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.09.002DOI Listing
October 2020

LifeTime and improving European healthcare through cell-based interceptive medicine.

Nature 2020 11 7;587(7834):377-386. Epub 2020 Sep 7.

Department of Human Genetics, KU Leuven, Leuven, Belgium.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2715-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7656507PMC
November 2020

Disease-associated DNA2 nuclease-helicase protects cells from lethal chromosome under-replication.

Nucleic Acids Res 2020 07;48(13):7265-7278

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5'-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5'-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2's role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1's ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2's role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367196PMC
July 2020

A Nuclear RNA Degradation Pathway Helps Silence Polycomb/H3K27me3-Marked Loci in .

Cold Spring Harb Symp Quant Biol 2019 29;84:141-153. Epub 2020 Apr 29.

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

In fission yeast and plants, RNA-processing pathways contribute to heterochromatin silencing, complementing well-characterized pathways of transcriptional repression. However, it was unclear whether this additional level of regulation occurs in metazoans. In a genetic screen, we uncovered a pathway of silencing in somatic cells, whereby the highly conserved, RNA-binding complex LSM2-8 contributes to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark H3K27me3. Importantly, the LSM2-8 complex works cooperatively with a 5'-3' exoribonuclease, XRN-2, and disruption of the pathway leads to selective mRNA stabilization. LSM2-8 complex-mediated RNA degradation does not target nor depend on H3K9me2/me3, unlike previously described pathways of heterochromatic RNA degradation. Up-regulation of -sensitive loci coincides with a localized drop in H3K27me3 levels in the mutant. Put into the context of epigenetic control of gene expression, it appears that targeted RNA degradation helps repress a subset of H3K27me3-marked genes, revealing an unappreciated layer of regulation for facultative heterochromatin in animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/sqb.2019.84.040238DOI Listing
April 2020

LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay.

Nat Cell Biol 2020 05 6;22(5):579-590. Epub 2020 Apr 6.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-020-0504-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212045PMC
May 2020

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Genes Dev 2020 04 5;34(7-8):560-579. Epub 2020 Mar 5.

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.332213.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111258PMC
April 2020

The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin.

EMBO J 2019 10 12;38(20):e101744. Epub 2019 Sep 12.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

In Saccharomyces cerevisiae, the silent information regulator (SIR) proteins Sir2/3/4 form a complex that suppresses transcription in subtelomeric regions and at the homothallic mating-type (HM) loci. Here, we identify a non-canonical BRCA1 C-terminal domain (H-BRCT) in Sir4, which is responsible for tethering telomeres to the nuclear periphery. We show that Sir4 H-BRCT and the closely related Dbf4 H-BRCT serve as selective phospho-epitope recognition domains that bind to a variety of phosphorylated target peptides. We present detailed structural information about the binding mode of established Sir4 interactors (Esc1, Ty5, Ubp10) and identify several novel interactors of Sir4 H-BRCT, including the E3 ubiquitin ligase Tom1. Based on these findings, we propose a phospho-peptide consensus motif for interaction with Sir4 H-BRCT and Dbf4 H-BRCT. Ablation of the Sir4 H-BRCT phospho-peptide interaction disrupts SIR-mediated repression and perinuclear localization. In conclusion, the Sir4 H-BRCT domain serves as a hub for recruitment of phosphorylated target proteins to heterochromatin to properly regulate silencing and nuclear order.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019101744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792019PMC
October 2019

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis.

Chromosoma 2019 09 6;128(3):453-471. Epub 2019 Jun 6.

Institute for Protein Research, Graduate School of Science, Osaka University, Suita, Osaka, Japan.

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00412-019-00709-5DOI Listing
September 2019

Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei.

Nature 2019 05 22;569(7758):734-739. Epub 2019 May 22.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery. The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity. The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane. In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me, whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factor that is necessary for this second chromatin anchor in intestinal cells. However, MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1243-yDOI Listing
May 2019

The study of protein recruitment to laser-induced DNA lesions can be distorted by photoconversion of the DNA binding dye Hoechst.

F1000Res 2019 25;8:104. Epub 2019 Jan 25.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel, CH-4058, Switzerland.

A commonly used approach for assessing DNA repair factor recruitment in mammalian cells is to induce DNA damage with a laser in the UV or near UV range and follow the local increase of GFP-tagged proteins at the site of damage. Often these measurements are performed in the presence of the blue DNA dye Hoechst, which is used as a photosensitizer. However, a light-induced switch of Hoechst from a blue-light to a green-light emitter will give a false positive signal at the site of damage.  Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.17865.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392149PMC
June 2020

Nuclear Actin and Actin-Binding Proteins in DNA Repair.

Trends Cell Biol 2019 06 4;29(6):462-476. Epub 2019 Apr 4.

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland; University of Basel, Faculty of Natural Sciences, CH-4056 Basel, Switzerland. Electronic address:

Nuclear actin has been implicated in a variety of DNA-related processes including chromatin remodeling, transcription, replication, and DNA repair. However, the mechanistic understanding of actin in these processes has been limited, largely due to a lack of research tools that address the roles of nuclear actin specifically, that is, distinct from its cytoplasmic functions. Recent findings support a model for homology-directed DNA double-strand break (DSB) repair in which a complex of ARP2 and ARP3 (actin-binding proteins 2 and 3) binds at the break and works with actin to promote DSB clustering and homology-directed repair. Further, it has been reported that relocalization of heterochromatic DSBs to the nuclear periphery in Drosophila is ARP2/3 dependent and actin-myosin driven. Here we provide an overview of the role of nuclear actin and actin-binding proteins in DNA repair, critically evaluating the experimental tools used and potential indirect effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tcb.2019.02.010DOI Listing
June 2019

Synergistic lethality between BRCA1 and H3K9me2 loss reflects satellite derepression.

Genes Dev 2019 04 25;33(7-8):436-451. Epub 2019 Feb 25.

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

has two histone H3 Lys9 methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/SUV39H1 related). In worms, we found simple repeat sequences primarily marked by H3K9me2, while transposable elements and silent tissue-specific genes bear H3K9me3. RNA sequencing (RNA-seq) in histone methyltransferase (HMT) mutants shows that MET-2-mediated H3K9me2 is necessary for satellite repeat repression, while SET-25 silences a subset of transposable elements and tissue-specific genes through H3K9me3. A genome-wide synthetic lethality screen showed that RNA processing, nuclear RNA degradation, the BRCA1/BARD1 complex, and factors mediating replication stress survival are necessary for germline viability in worms lacking MET-2 but not SET-25. Unlike mutants, -null worms accumulated satellite repeat transcripts, which form RNA:DNA hybrids on repetitive sequences, additively with the loss of BRCA1 or BARD1. BRCA1/BARD1-mediated H2A ubiquitination and MET-2 deposited H3K9me2 on satellite repeats are partially interdependent, suggesting both that the loss of silencing generates BRCA-recruiting DNA damage and that BRCA1 recruitment by damage helps silence repeats. The artificial induction of MSAT1 transcripts can itself trigger damage-induced germline lethality in a wild-type background, arguing that the synthetic sterility upon BRCA1/BARD1 and H3K9me2 loss is directly linked to the DNA damage provoked by unscheduled satellite repeat transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.322495.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446544PMC
April 2019

Heterochromatic foci and transcriptional repression by an unstructured MET-2/SETDB1 co-factor LIN-65.

J Cell Biol 2019 03 8;218(3):820-838. Epub 2019 Feb 8.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

The segregation of the genome into accessible euchromatin and histone H3K9-methylated heterochromatin helps silence repetitive elements and tissue-specific genes. In , MET-2, the homologue of mammalian SETDB1, catalyzes H3K9me1 and me2, yet like SETDB1, its regulation is enigmatic. Contrary to the cytosolic enrichment of overexpressed MET-2, we show that endogenous MET-2 is nuclear throughout development, forming perinuclear foci in a cell cycle-dependent manner. Mass spectrometry identified two cofactors that bind MET-2: LIN-65, a highly unstructured protein, and ARLE-14, a conserved GTPase effector. All three factors colocalize in heterochromatic foci. Ablation of , but not , mislocalizes and destabilizes MET-2, resulting in decreased H3K9 dimethylation, dispersion of heterochromatic foci, and derepression of MET-2 targets. Mutation of or also disrupts the perinuclear anchoring of genomic heterochromatin. Loss of LIN-65, like that of MET-2, compromises temperature stress resistance and germline integrity, which are both linked to promiscuous repeat transcription and gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201811038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400574PMC
March 2019

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Microb Cell 2019 Jan 7;6(1):1-64. Epub 2019 Jan 7.

Department of Biology, University of Iowa, Iowa City, IA, USA.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15698/mic2019.01.664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334234PMC
January 2019

Meiosis-specific prophase-like pathway controls cleavage-independent release of cohesin by Wapl phosphorylation.

PLoS Genet 2019 01 3;15(1):e1007851. Epub 2019 Jan 3.

Institute for Protein Research, Graduate School of Science, Osaka University, Suita, Osaka, Japan.

Sister chromatid cohesion on chromosome arms is essential for the segregation of homologous chromosomes during meiosis I while it is dispensable for sister chromatid separation during mitosis. It was assumed that, unlike the situation in mitosis, chromosome arms retain cohesion prior to onset of anaphase-I. Paradoxically, reduced immunostaining signals of meiosis-specific cohesin, including the kleisin Rec8, were observed on chromosomes during late prophase-I of budding yeast. This decrease is seen in the absence of Rec8 cleavage and depends on condensin-mediated recruitment of Polo-like kinase (PLK/Cdc5). In this study, we confirmed that this release indeed accompanies the dissociation of acetylated Smc3 as well as Rec8 from meiotic chromosomes during late prophase-I. This release requires, in addition to PLK, the cohesin regulator, Wapl (Rad61/Wpl1 in yeast), and Dbf4-dependent Cdc7 kinase (DDK). Meiosis-specific phosphorylation of Rad61/Wpl1 and Rec8 by PLK and DDK collaboratively promote this release. This process is similar to the vertebrate "prophase" pathway for cohesin release during G2 phase and pro-metaphase. In yeast, meiotic cohesin release coincides with PLK-dependent compaction of chromosomes in late meiotic prophase-I. We suggest that yeast uses this highly regulated cleavage-independent pathway to remove cohesin during late prophase-I to facilitate morphogenesis of condensed metaphase-I chromosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1007851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317811PMC
January 2019

Challenges and guidelines toward 4D nucleome data and model standards.

Nat Genet 2018 10 27;50(10):1352-1358. Epub 2018 Sep 27.

Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum Munchen, München, Germany.

Due to recent advances in experimental and theoretical approaches, the dynamic three-dimensional organization (3D) of the nucleus has become a very active area of research in life sciences. We now understand that the linear genome is folded in ways that may modulate how genes are expressed during the basic functioning of cells. Importantly, it is now possible to build 3D models of how the genome folds within the nucleus and changes over time (4D). Because genome folding influences its function, this opens exciting new possibilities to broaden our understanding of the mechanisms that determine cell fate. However, the rapid evolution of methods and the increasing complexity of data can result in ambiguity and reproducibility challenges, which may hamper the progress of this field. Here, we describe such challenges ahead and provide guidelines to think about strategies for shared standardized validation of experimental 4D nucleome data sets and models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-018-0236-3DOI Listing
October 2018

Chromosome Dynamics in Response to DNA Damage.

Annu Rev Genet 2018 11 12;52:295-319. Epub 2018 Sep 12.

Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland; email:

Recent advances in both the technologies used to measure chromatin movement and the biophysical analysis used to model them have yielded a fuller understanding of chromatin dynamics and the polymer structure that underlies it. Changes in nucleosome packing, checkpoint kinase activation, the cell cycle, chromosomal tethers, and external forces acting on nuclei in response to external and internal stimuli can alter the basal mobility of DNA in interphase nuclei of yeast or mammalian cells. Although chromatin movement is assumed to be necessary for many DNA-based processes, including gene activation by distal enhancer-promoter interaction or sequence-based homology searches during double-strand break repair, experimental evidence supporting an essential role in these activities is sparse. Nonetheless, high-resolution tracking of chromatin dynamics has led to instructive models of the higher-order folding and flexibility of the chromatin polymer. Key regulators of chromatin motion in physiological conditions or after damage induction are reviewed here.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-genet-120417-031334DOI Listing
November 2018

Asymmetric Processing of DNA Ends at a Double-Strand Break Leads to Unconstrained Dynamics and Ectopic Translocation.

Cell Rep 2018 09;24(10):2614-2628.e4

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Faculty of Natural Sciences, 4056 Basel, Switzerland. Electronic address:

Multiple pathways regulate the repair of double-strand breaks (DSBs) to suppress potentially dangerous ectopic recombination. Both sequence and chromatin context are thought to influence pathway choice between non-homologous end-joining (NHEJ) and homology-driven recombination. To test the effect of repetitive sequences on break processing, we have inserted TG-rich repeats on one side of an inducible DSB at the budding yeast MAT locus on chromosome III. Five clustered Rap1 sites within a break-proximal TG repeat are sufficient to block Mre11-Rad50-Xrs2 recruitment, impair resection, and favor elongation by telomerase. The two sides of the break lose end-to-end tethering and show enhanced, uncoordinated movement. Only the TG-free side is resected and shifts to the nuclear periphery. In contrast to persistent DSBs without TG repeats that are repaired by imprecise NHEJ, nearly all survivors of repeat-proximal DSBs repair the break by a homology-driven, non-reciprocal translocation from ChrIII-R to ChrVII-L. This suppression of imprecise NHEJ at TG-repeat-flanked DSBs requires the Uls1 translocase activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.07.102DOI Listing
September 2018

Repressive Chromatin in : Establishment, Composition, and Function.

Genetics 2018 02;208(2):491-511

Friedrich Miescher Institute for Biomedical Research (FMI), 4058 Basel, Switzerland, and

Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in , focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in , and should inform our understanding of repressed chromatin in other animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.117.300386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788517PMC
February 2018

Chromatin and nucleosome dynamics in DNA damage and repair.

Genes Dev 2017 11;31(22):2204-2221

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

Chromatin is organized into higher-order structures that form subcompartments in interphase nuclei. Different categories of specialized enzymes act on chromatin and regulate its compaction and biophysical characteristics in response to physiological conditions. We present an overview of the function of chromatin structure and its dynamic changes in response to genotoxic stress, focusing on both subnuclear organization and the physical mobility of DNA. We review the requirements and mechanisms that cause chromatin relocation, enhanced mobility, and chromatin unfolding as a consequence of genotoxic lesions. An intriguing link has been established recently between enhanced chromatin dynamics and histone loss.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.307702.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769766PMC
November 2017

The Importance of Satellite Sequence Repression for Genome Stability.

Cold Spring Harb Symp Quant Biol 2017 13;82:15-24. Epub 2017 Nov 13.

Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

Up to two-thirds of eukaryotic genomes consist of repetitive sequences, which include both transposable elements and tandemly arranged simple or satellite repeats. Whereas extensive progress has been made toward understanding the danger of and control over transposon expression, only recently has it been recognized that DNA damage can arise from satellite sequence transcription. Although the structural role of satellite repeats in kinetochore function and end protection has long been appreciated, it has now become clear that it is not only these functions that are compromised by elevated levels of transcription. RNA from simple repeat sequences can compromise replication fork stability and genome integrity, thus compromising germline viability. Here we summarize recent discoveries on how cells control the transcription of repeat sequence and the dangers that arise from their expression. We propose that the link between the DNA damage response and the transcriptional silencing machinery may help a cell or organism recognize foreign DNA insertions into an evolving genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/sqb.2017.82.033662DOI Listing
November 2017

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

Mol Cell 2017 Oct 12;68(2):431-445.e5. Epub 2017 Oct 12.

Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Faculty of Natural Sciences, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address:

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2017.09.019DOI Listing
October 2017

The INO80 remodeller in transcription, replication and repair.

Philos Trans R Soc Lond B Biol Sci 2017 Oct;372(1731)

Institute for Cell and Molecular Biosciences, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

The accessibility of eukaryotic genomes to the action of enzymes involved in transcription, replication and repair is maintained despite the organization of DNA into nucleosomes. This access is often regulated by the action of ATP-dependent nucleosome remodellers. The INO80 class of nucleosome remodellers has unique structural features and it is implicated in a diverse array of functions, including transcriptional regulation, DNA replication and DNA repair. Underlying these diverse functions is the catalytic activity of the main ATPase subunit, which in the context of a multisubunit complex can shift nucleosomes and carry out histone dimer exchange. studies showed that INO80 promotes replication fork progression on a chromatin template, while it was shown to facilitate replication fork restart after stalling and to help evict RNA polymerase II at transcribed genes following the collision of a replication fork with transcription. More recent work in yeast implicates INO80 in the general eviction and degradation of nucleosomes following high doses of oxidative DNA damage. Beyond these replication and repair functions, INO80 was shown to repress inappropriate transcription at promoters in the opposite direction to the coding sequence. Here we discuss the ways in which INO80's diverse functions help maintain genome integrity.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rstb.2016.0290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577468PMC
October 2017

Chromatin modifiers and remodellers in DNA repair and signalling.

Philos Trans R Soc Lond B Biol Sci 2017 10;372(1731)

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rstb.2016.0279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577457PMC
October 2017