Schiavoni, F.
Zuazua-Villar, P.
Roumeliotis, T.I.
Benstead-Hume, G.
Pardo, M.
Pearl, F.M.
Choudhary, J.S.
Downs, J.A.
(2022). Aneuploidy tolerance caused by BRG1 loss allows chromosome gains and recovery of fitness. ,
Vol.13
(1),
p. 1731.
show abstract
Aneuploidy results in decreased cellular fitness in many species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is frequently lost in cancer. Here, we use a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. Notably, we find increased tolerance to aneuploidy immediately upon loss of BRG1, and the fitness recovery over time correlates with chromosome gain. These data show that BRG1 loss creates an environment where karyotype changes can be explored without a fitness penalty. At least in some genetic backgrounds, therefore, BRG1 loss can affect the progression of tumourigenesis through tolerance of aneuploidy..
Feng, H.
Lane, K.A.
Roumeliotis, T.I.
Jeggo, P.A.
Somaiah, N.
Choudhary, J.S.
Downs, J.A.
(2022). PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance. ,
.
show abstract
The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer..
Chabanon, R.M.
Morel, D.
Eychenne, T.
Colmet-Daage, L.
Bajrami, I.
Dorvault, N.
Garrido, M.
Meisenberg, C.
Lamb, A.
Ngo, C.
Hopkins, S.R.
Roumeliotis, T.I.
Jouny, S.
Hénon, C.
Kawai-Kawachi, A.
Astier, C.
Konde, A.
Del Nery, E.
Massard, C.
Pettitt, S.J.
Margueron, R.
Choudhary, J.S.
Almouzni, G.
Soria, J.-.
Deutsch, E.
Downs, J.A.
Lord, C.J.
Postel-Vinay, S.
(2021). PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer. ,
Vol.81
(11),
pp. 2888-2902.
show abstract
Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2888/F1.large.jpg..
Lindenburg, L.H.
Pantelejevs, T.
Gielen, F.
Zuazua-Villar, P.
Butz, M.
Rees, E.
Kaminski, C.F.
Downs, J.A.
Hyvönen, M.
Hollfelder, F.
(2021). Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats. ,
Vol.118
(46),
p. e2017708118.
show abstract
Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation..
Flaus, A.
Downs, J.A.
Owen-Hughes, T.
(2020). Histone isoforms and the oncohistone code. Current opinion in genetics & development,
Vol.67,
pp. 61-66.
show abstract
Recent studies have highlighted the potential for missense mutations in histones to act as oncogenic drivers, leading to the term 'oncohistones'. While histone proteins are highly conserved, they are encoded by multigene families. There is heterogeneity among these genes at the level of the underlying sequence, the amino acid composition of the encoded histone isoform, and the expression levels. One question that arises, therefore, is whether all histone-encoding genes function equally as oncohistones. In this review, we consider this question and explore what this means in terms of the mechanisms by which oncohistones can exert their effects in chromatin..
Harrod, A.
Lane, K.A.
Downs, J.A.
(2020). The role of the SWI/SNF chromatin remodelling complex in the response to DNA double strand breaks. Dna repair (amst),
Vol.93,
pp. 102919-102919.
show abstract
Mammalian cells possess multiple closely related SWI/SNF chromatin remodelling complexes. These complexes have been implicated in the cellular response to DNA double strand breaks (DSBs). Evidence suggests that SWI/SNF complexes contribute to successful repair via both the homologous recombination and non-homologous end joining pathways. In addition, repressing transcription near DSBs is dependent on SWI/SNF activity. Understanding these roles is important because SWI/SNF complexes are frequently dysregulated in cancer, and DNA DSB repair defects have the potential to be therapeutically exploited. In this graphical review, we summarise what is known about SWI/SNF contribution to DNA DSB responses in mammalian cells and provide an overview of the SWI/SNF-encoding gene alteration spectrum in human cancers..
Rother, M.B.
Pellegrino, S.
Smith, R.
Gatti, M.
Meisenberg, C.
Wiegant, W.W.
Luijsterburg, M.S.
Imhof, R.
Downs, J.A.
Vertegaal, A.C.
Huet, S.
Altmeyer, M.
van Attikum, H.
(2020). CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks. Nature communications,
Vol.11
(1),
pp. 5775-?.
show abstract
Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled 'chromatin breathing' upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails..
Meisenberg, C.
Pinder, S.I.
Hopkins, S.R.
Wooller, S.K.
Benstead-Hume, G.
Pearl, F.M.
Jeggo, P.A.
Downs, J.A.
(2019). Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability. Molecular cell,
Vol.73
(2),
pp. 212-223.e7.
show abstract
Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer..
Benstead-Hume, G.
Wooller, S.K.
Downs, J.A.
Pearl, F.M.
(2019). Defining Signatures of Arm-Wise Copy Number Change and Their Associated Drivers in Kidney Cancers. ,
Vol.20
(22),
p. E5762.
show abstract
Using pan-cancer data from The Cancer Genome Atlas (TCGA), we investigated how patterns in copy number alterations in cancer cells vary both by tissue type and as a function of genetic alteration. We find that patterns in both chromosomal ploidy and individual arm copy number are dependent on tumour type. We highlight for example, the significant losses in chromosome arm 3p and the gain of ploidy in 5q in kidney clear cell renal cell carcinoma tissue samples. We find that specific gene mutations are associated with genome-wide copy number changes. Using signatures derived from non-negative factorisation, we also find gene mutations that are associated with particular patterns of ploidy change. Finally, utilising a set of machine learning classifiers, we successfully predicted the presence of mutated genes in a sample using arm-wise copy number patterns as features. This demonstrates that mutations in specific genes are correlated and may lead to specific patterns of ploidy loss and gain across chromosome arms. Using these same classifiers, we highlight which arms are most predictive of commonly mutated genes in kidney renal clear cell carcinoma (KIRC)..
Benstead-Hume, G.
Chen, X.
Hopkins, S.R.
Lane, K.A.
Downs, J.A.
Pearl, F.M.
(2019). Predicting synthetic lethal interactions using conserved patterns in protein interaction networks. Plos computational biology,
Vol.15
(4),
pp. e1006888-?.
show abstract
In response to a need for improved treatments, a number of promising novel targeted cancer therapies are being developed that exploit human synthetic lethal interactions. This is facilitating personalised medicine strategies in cancers where specific tumour suppressors have become inactivated. Mainly due to the constraints of the experimental procedures, relatively few human synthetic lethal interactions have been identified. Here we describe SLant (Synthetic Lethal analysis via Network topology), a computational systems approach to predicting human synthetic lethal interactions that works by identifying and exploiting conserved patterns in protein interaction network topology both within and across species. SLant out-performs previous attempts to classify human SSL interactions and experimental validation of the models predictions suggests it may provide useful guidance for future SSL screenings and ultimately aid targeted cancer therapy development..
Jeggo, P.A.
Downs, J.A.
Gasser, S.M.
(2017). Chromatin modifiers and remodellers in DNA repair and signalling. Philosophical transactions of the royal society of london. series b, biological sciences,
Vol.372
(1731).
Hopkins, S.R.
McGregor, G.A.
Murray, J.M.
Downs, J.A.
Savic, V.
(2016). Novel synthetic lethality screening method identifies TIP60-dependent radiation sensitivity in the absence of BAF180. ,
Vol.46,
pp. 47-54.
show abstract
In recent years, research into synthetic lethality and how it can be exploited in cancer treatments has emerged as major focus in cancer research. However, the lack of a simple to use, sensitive and standardised assay to test for synthetic interactions has been slowing the efforts. Here we present a novel approach to synthetic lethality screening based on co-culturing two syngeneic cell lines containing individual fluorescent tags. By associating shRNAs for a target gene or control to individual fluorescence labels, we can easily follow individual cell fates upon siRNA treatment and high content imaging. We have demonstrated that the system can recapitulate the functional defects of the target gene depletion and is capable of discovering novel synthetic interactors and phenotypes. In a trial screen, we show that TIP60 exhibits synthetic lethality interaction with BAF180, and that in the absence of TIP60, there is an increase micronuclei dependent on the level of BAF180 loss, significantly above levels seen with BAF180 present. Moreover, the severity of the interactions correlates with proxy measurements of BAF180 knockdown efficacy, which may expand its usefulness to addressing synthetic interactions through titratable hypomorphic gene expression..
Myrianthopoulos, V.
Gaboriaud-Kolar, N.
Tallant, C.
Hall, M.-.
Grigoriou, S.
Brownlee, P.M.
Fedorov, O.
Rogers, C.
Heidenreich, D.
Wanior, M.
Drosos, N.
Mexia, N.
Savitsky, P.
Bagratuni, T.
Kastritis, E.
Terpos, E.
Filippakopoulos, P.
Müller, S.
Skaltsounis, A.-.
Downs, J.A.
Knapp, S.
Mikros, E.
(2016). Discovery and Optimization of a Selective Ligand for the Switch/Sucrose Nonfermenting-Related Bromodomains of Polybromo Protein-1 by the Use of Virtual Screening and Hydration Analysis. ,
Vol.59
(19),
pp. 8787-8803.
show abstract
Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein-ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5)..
Meisenberg, C.
Ashour, M.E.
El-Shafie, L.
Liao, C.
Hodgson, A.
Pilborough, A.
Khurram, S.A.
Downs, J.A.
Ward, S.E.
El-Khamisy, S.F.
(2016). Epigenetic changes in histone acetylation underpin resistance to the topoisomerase I inhibitor irinotecan. ,
,
pp. gkw1026-gkw1026.
show abstract
The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance..
Kakarougkas, A.
Downs, J.A.
Jeggo, P.A.
(2015). The PBAF chromatin remodeling complex represses transcription and promotes rapid repair at DNA double-strand breaks. Molecular & cellular oncology,
Vol.2
(1),
pp. e970072-e970072.
Alatwi, H.E.
Downs, J.A.
(2015). Removal of H2A Z by INO80 promotes homologous recombination. Embo reports,
Vol.16
(8),
pp. 986-994.
show abstract
The mammalian INO80 remodelling complex facilitates homologous recombination (HR), but the mechanism by which it does this is unclear. Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. H2A.Z is actively incorporated at sites of damage in mammalian cells, raising the possibility that H2A.Z may need to be subsequently removed for resolution of repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. We also report that the histone chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly promotes HR and appears to work on the same pathway as INO80 in these assays. Importantly, we demonstrate that the HR defect in cells depleted of INO80 or ANP32E can be rescued by H2A.Z co-depletion, suggesting that H2A.Z removal from chromatin is the primary function of INO80 and ANP32E in promoting homologous recombination..
Brownlee, P.M.
Meisenberg, C.
Downs, J.A.
(2015). The SWI/SNF chromatin remodelling complex: Its role in maintaining genome stability and preventing tumourigenesis. Dna repair,
Vol.32,
pp. 127-133.
Niimi, A.
Hopkins, S.R.
Downs, J.A.
Masutani, C.
(2015). The BAH domain of BAF180 is required for PCNA ubiquitination. Mutation research/fundamental and molecular mechanisms of mutagenesis,
Vol.779,
pp. 16-23.
Schalbetter, S.A.
Mansoubi, S.
Chambers, A.L.
Downs, J.A.
Baxter, J.
(2015). Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability. Proceedings of the national academy of sciences,
Vol.112
(33).
show abstract
Significance
Genome inheritance requires the complete resolution of all intertwines within parental DNA. This is facilitated by fork rotation and precatenation of the newly replicated DNA. However, the general importance and frequency of fork rotation in vivo are poorly understood. We find that the evolutionarily conserved Timeless and Tipin proteins actively inhibit fork rotation in budding yeast. In their presence, fork rotation appears restricted to hard-to-replicate fragile sites. In their absence, excessive fork rotation leads to damage accumulating in the replicated sister chromatids, especially at known yeast fragile sites. Therefore, fork rotation appears to be restricted to contexts where it is absolutely required for unwinding, and this restriction is required to prevent precatenation inducing excessive chromosomal fragility..
Brownlee, P.M.
Chambers, A.L.
Cloney, R.
Bianchi, A.
Downs, J.A.
(2014). BAF180 promotes cohesion and prevents genome instability and aneuploidy. Cell reports,
Vol.6
(6),
pp. 973-981.
show abstract
BAF180, a subunit of the PBAF chromatin remodeling complex, is frequently mutated in cancer. Although PBAF regulates transcription, it remains unclear whether this is what drives tumorigenesis in cells lacking BAF180. Based on data from yeast, we hypothesized that BAF180 may prevent tumorigenesis by promoting cohesion. Here, we show BAF180 is required for centromeric cohesion in mouse and human cells. Mutations identified in tumor samples are unable to support this activity, and also compromise cohesion-dependent functions in yeast. We provide evidence of genome instability in line with loss of cohesion, and importantly, we find dynamic chromosome instability following DNA damage in cells lacking BAF180. These data demonstrate a function for BAF180 in promoting genome stability that is distinct from its well-characterized role in transcriptional regulation, uncovering a potent mechanism for its tumor-suppressor activity..
Pal, M.
Morgan, M.
Phelps, S.E.
Roe, S.M.
Parry-Morris, S.
Downs, J.A.
Polier, S.
Pearl, L.H.
Prodromou, C.
(2014). Structural Basis for Phosphorylation-Dependent Recruitment of Tel2 to Hsp90 by Pih1. Structure,
Vol.22
(6),
pp. 805-818.
López-Perrote, A.
Alatwi, H.E.
Torreira, E.
Ismail, A.
Ayora, S.
Downs, J.A.
Llorca, O.
(2014). Structure of Yin Yang 1 Oligomers That Cooperate with RuvBL1-RuvBL2 ATPases. Journal of biological chemistry,
Vol.289
(33),
pp. 22614-22629.
Kakarougkas, A.
Ismail, A.
Chambers, A.L.
Riballo, E.
Herbert, A.D.
Künzel, J.
Löbrich, M.
Jeggo, P.A.
Downs, J.A.
(2014). Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin. Molecular cell,
Vol.55
(5),
pp. 723-732.
show abstract
Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity..
Jeggo, P.A.
Downs, J.A.
(2014). Roles of chromatin remodellers in DNA double strand break repair. Experimental cell research,
Vol.329
(1),
pp. 69-77.
Chambers, A.L.
Pearl, L.H.
Oliver, A.W.
Downs, J.A.
(2013). The BAH domain of Rsc2 is a histone H3 binding domain. Nucleic acids research,
Vol.41
(19),
pp. 9168-9182.
Niimi, A.
Chambers, A.L.
Downs, J.A.
Lehmann, A.R.
(2012). A role for chromatin remodellers in replication of damaged DNA. Nucleic acids research,
Vol.40
(15),
pp. 7393-7403.
Brownlee, P.M.
Chambers, A.L.
Oliver, A.W.
Downs, J.A.
(2012). Cancer and the bromodomains of BAF180. Biochemical society transactions,
Vol.40
(2),
pp. 364-369.
show abstract
Chromatin remodelling complexes alter the structure of chromatin and have central roles in all DNA-templated activities, including regulation of gene expression and DNA repair. Mutations in subunits of the PBAF (polybromo/Brg1-associated factor) or SWI/SNF-B remodelling complex, including BAF180, are frequently associated with cancer. There are six potential acetyl-lysine-binding BDs (bromodomains) in BAF180, which may function to target the PBAF complex to promoters or sites of DNA repair. In the present review, we discuss what is currently known about the BDs of BAF180 and their potential significance in cancer..
Chambers, A.L.
Ormerod, G.
Durley, S.C.
Sing, T.L.
Brown, G.W.
Kent, N.A.
Downs, J.A.
(2012). The INO80 chromatin remodeling complex prevents polyploidy and maintains normal chromatin structure at centromeres. Genes & development,
Vol.26
(23),
pp. 2590-2603.
show abstract
The INO80 chromatin remodeling complex functions in transcriptional regulation, DNA repair, and replication. Here we uncover a novel role for INO80 in regulating chromosome segregation. First, we show that the conserved Ies6 subunit is critical for INO80 function in vivo. Strikingly, we found that loss of either Ies6 or the Ino80 catalytic subunit results in rapid increase in ploidy. One route to polyploidy is through chromosome missegregation due to aberrant centromere structure, and we found that loss of either Ies6 or Ino80 leads to defective chromosome segregation. Importantly, we show that chromatin structure flanking centromeres is altered in cells lacking these subunits and that these alterations occur not in the Cse4-containing centromeric nucleosome, but in pericentric chromatin. We provide evidence that these effects are mediated through misincorporation of H2A.Z, and these findings indicate that H2A.Z-containing pericentric chromatin, as in higher eukaryotes with regional centromeres, is important for centromere function in budding yeast. These data reveal an important additional mechanism by which INO80 maintains genome stability..
Chambers, A.L.
Downs, J.A.
(2012). The RSC and INO80 Chromatin-Remodeling Complexes in DNA Double-Strand Break Repair. ,
,
pp. 229-261.
Foster, E.R.
Downs, J.A.
(2009). Methylation of H3 K4 and K79 is not strictly dependent on H2B K123 ubiquitylation. Journal of cell biology,
Vol.184
(5),
pp. 631-638.
show abstract
Covalent modifications of histone proteins have profound consequences on chromatin structure and function. Specific modification patterns constitute a code read by effector proteins. Studies from yeast found that H3 trimethylation at K4 and K79 is dependent on ubiquitylation of H2B K123, which is termed a “trans-tail pathway.” In this study, we show that a strain unable to be ubiquitylated on H2B (K123R) is still proficient for H3 trimethylation at both K4 and K79, indicating that H3 methylation status is not solely dependent on H2B ubiquitylation. However, additional mutations in H2B result in loss of H3 methylation when combined with htb1-K123R. Consistent with this, we find that the original strain used to identify the trans-tail pathway has a genomic mutation that, when combined with H2B K123R, results in defective H3 methylation. Finally, we show that strains lacking the ubiquitin ligase Bre1 are defective for H3 methylation, suggesting that there is an additional Bre1 substrate that in combination with H2B K123 facilitates H3 methylation..
Titman, C.M.
Downs, J.A.
Oliver, S.G.
Carmichael, P.L.
Scott, A.D.
Griffin, J.L.
(2009). A metabolomic and multivariate statistical process to assess the effects of genotoxins in Saccharomycescerevisiae. Molecular biosystems,
Vol.5
(12),
pp. 1913-1913.
Downs, J.A.
(2008). Histone H3 K56 acetylation, chromatin assembly, and the DNA damage checkpoint. Dna repair,
Vol.7
(12),
pp. 2020-2024.
Chambers, A.L.
Downs, J.A.
(2007). The contribution of the budding yeast histone H2A C-terminal tail to DNA-damage responses. Biochemical society transactions,
Vol.35
(6),
pp. 1519-1524.
show abstract
The cellular response to DNA damage involves extensive interaction with and manipulation of chromatin. This includes the detection and repair of the DNA lesion, but there are also transcriptional responses to DNA damage, involving the up- or down-regulation of numerous genes. Therefore changes to chromatin structure, including covalent modification of histone proteins, are known to occur during DNA-damage responses. One of the most well characterized DNA-damage-responsive chromatin modification events is the phosphorylation of the SQ motif found in the C-terminal tail of histone H2A or the H2AX variant in higher eukaryotes. In the budding yeast, a number of additional residues in this region of histone H2A that contribute to the cellular response to DNA damage have been identified, providing an insight into the nature and complexity of the DNA-damage histone code..
Kent, N.A.
Chambers, A.L.
Downs, J.A.
(2007). Dual Chromatin Remodeling Roles for RSC during DNA Double Strand Break Induction and Repair at the Yeast MAT Locus. Journal of biological chemistry,
Vol.282
(38),
pp. 27693-27701.
Downs, J.A.
(2007). Chromatin structure and DNA double-strand break responses in cancer progression and therapy. Oncogene,
Vol.26
(56),
pp. 7765-7772.
Bilsland, E.
Hult, M.
Bell, S.D.
Sunnerhagen, P.
Downs, J.A.
(2007). The Bre5/Ubp3 ubiquitin protease complex from budding yeast contributes to the cellular response to DNA damage. Dna repair,
Vol.6
(10),
pp. 1471-1484.
Downs, J.A.
Nussenzweig, M.C.
Nussenzweig, A.
(2007). Chromatin dynamics and the preservation of genetic information. Nature,
Vol.447
(7147),
pp. 951-958.
Bilsland, E.
Downs, J.A.
(2005). Tails of histones in DNA double-strand break repair. Mutagenesis,
Vol.20
(3),
pp. 153-163.
Harvey, A.C.
Jackson, S.P.
Downs, J.A.
(2005). Saccharomyces cerevisiae Histone H2A Ser122 Facilitates DNA Repair. Genetics,
Vol.170
(2),
pp. 543-553.
show abstract
Abstract
DNA repair takes place in the context of chromatin. Recently, it has become apparent that proteins that make up and modulate chromatin structure are involved in the detection and repair of DNA lesions. We previously demonstrated that Ser129 in the carboxyl-terminal tail of yeast histone H2A is important for double-strand-break responses. By undertaking a systematic site-directed mutagenesis approach, we identified another histone H2A serine residue (Ser122) that is important for survival in the presence of DNA-damaging agents. We show that mutation of this residue does not affect DNA damage-dependent Rad53 phosphorylation or G2/M checkpoint responses. Interestingly, we find that yeast lacking H2A S122 are defective in their ability to sporulate. Finally, we demonstrate that H2A S122 provides a function distinct from that of H2A S129. These data demonstrate a role for H2A S122 in facilitating survival in the presence of DNA damage and suggest a potential role in mediating homologous recombination. The distinct roles of H2A S122 and S129 in mediating these responses suggest that chromatin components can provide specialized functions for distinct DNA repair and survival mechanisms and point toward the possibility of a complex DNA damage responsive histone code..
Zaid, O.
Downs, J.A.
(2005). Histones as tumour suppressor genes. Cellular and molecular life sciences,
Vol.62
(15),
pp. 1653-1656.
WARDLEWORTH, B.N.
DOWNS, J.A.
(2005). Spotting new DNA damage-responsive chromatin-binding proteins. Biochemical journal,
Vol.388
(1).
show abstract
In response to DNA damage, cells initiate multiple repair mechanisms that all contribute to the survival of both the cell and the organism. These responses are numerous and variable, and can include cell cycle arrest, transcriptional activation of DNA repair genes and relocalization of repair proteins to sites of DNA damage. If all else fails, in multicellular organisms the initiation of apoptosis is also a potential cellular response to DNA damage. Despite a wealth of information about these events, it is clear that we do not yet have a comprehensive picture of the cellular responses to DNA damage. In this issue of the Biochemical Journal, a proteomics approach was used by Lee et al. to identify proteins that bind to chromatin in a DNA damage-inducible manner. The proteins identified, nucleophosmin, hnRNP C1 (heterogeneous nuclear ribonucleoprotein C1) and hnRNP C2, were proteins that would not necessarily have been predicted to behave this way. These studies have the potential to be extended and contribute to our knowledge of the cellular response to DNA damage..
Downs, J.A.
Cote, J.
(2005). Dynamics of Chromatin during the Repair of DNA Double-Strand Breaks. Cell cycle,
Vol.4
(10),
pp. 1373-1376.
Downs, J.A.
Jackson, S.P.
(2004). A means to a DNA end: the many roles of Ku. Nature reviews molecular cell biology,
Vol.5
(5),
pp. 367-378.
Downs, J.A.
Allard, S.
Jobin-Robitaille, O.
Javaheri, A.
Auger, A.
Bouchard, N.
Kron, S.J.
Jackson, S.P.
Côté, J.
(2004). Binding of Chromatin-Modifying Activities to Phosphorylated Histone H2A at DNA Damage Sites. Molecular cell,
Vol.16
(6),
pp. 979-990.
Downs, J.A.
Kosmidou, E.
Morgan, A.
Jackson, S.P.
(2003). Suppression of Homologous Recombination by the Saccharomyces cerevisiae Linker Histone. Molecular cell,
Vol.11
(6),
pp. 1685-1692.
Buratowski, R.M.
Downs, J.
Buratowski, S.
(2002). Interdependent Interactions between TFIIB, TATA Binding Protein, and DNA. Molecular and cellular biology,
Vol.22
(24),
pp. 8735-8743.
show abstract
ABSTRACT
Temperature-sensitive mutants of TFIIB that are defective for essential interactions were isolated. One mutation (G204D) results in disruption of a protein-protein contact between TFIIB and TATA binding protein (TBP), while the other (K272I) disrupts an interaction between TFIIB and DNA. The TBP gene was mutagenized, and alleles that suppress the slow-growth phenotypes of the TFIIB mutants were isolated. TFIIB with the G204D mutation [TFIIB(G204D)] was suppressed by hydrophobic substitutions at lysine 239 of TBP. These changes led to increased affinity between TBP and TFIIB. TFIIB(K272I) was weakly suppressed by TBP mutants in which K239 was changed to hydrophobic residues. However, this mutant TFIIB was strongly suppressed by conservative substitutions in the DNA binding surface of TBP. Biochemical characterization showed that these TBP mutants had increased affinity for a TATA element. The TBPs with increased affinity could not suppress TFIIB(G204D), leading us to propose a two-step model for the interaction between TFIIB and the TBP-DNA complex..
Downs, J.A.
Lowndes, N.F.
Jackson, S.P.
(2000). A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature,
Vol.408
(6815),
pp. 1001-1004.
Downs, J.A.
Jackson, S.P.
(1999). Involvement of DNA End-Binding Protein Ku in Ty Element Retrotransposition. Molecular and cellular biology,
Vol.19
(9),
pp. 6260-6268.
show abstract
ABSTRACT
Saccharomyces cerevisiae
Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses. They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome. Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process. Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition. Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku. In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair. We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo. These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems.
.
Foster, E.R.
Downs, J.A.
Histone H2A phosphorylation in DNA double-strand break repair. Febs journal,
Vol.272
(13),
pp. 3231-3240.
Harvey, A.C.
Downs, J.A.
What functions do linker histones provide?. Molecular microbiology,
Vol.53
(3),
pp. 771-775.
Chambers, A.L.
Brownlee, P.M.
Durley, S.C.
Beacham, T.
Kent, N.A.
Downs, J.A.
The Two Different Isoforms of the RSC Chromatin Remodeling Complex Play Distinct Roles in DNA Damage Responses. Plos one,
Vol.7
(2),
pp. e32016-e32016.
Somaiah, N.
Yarnold, J.
Anbalagan, S.
Harrington, K.
Downs, J.
Wilkins, A.
McBay, D.
TP53 MODULATES RADIOTHERAPY FRACTION SIZE SENSITIVITY IN NORMAL AND MALIGNANT CELLS. Scientific reports,
.
Wilkins, A.
Fontana, E.
Nyamundanda, G.
Ragulan, C.
Patil, Y.
Mansfield, D.
Kingston, J.
Errington-Mais, F.
Bottomley, D.
von Loga, K.
Bye, H.
Carter, P.
Tinkler-Hundal, E.
Noshirwani, A.
Downs, J.
Dillon, M.
Demaria, S.
Sebag-Montefiore, D.
Harrington, K.
West, N.
Melcher, A.
Sadanandam, A.
Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer. J immunother cancer,
Vol.9
(3).
show abstract
BACKGROUND: Rectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer. METHODS: We generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses. RESULTS: ΔTCD scores ranged from 12.4% to -47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders. CONCLUSION: This study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune "cold" towards an immunologically "hot" phenotype on treatment with radiotherapy..