Mailand Group – University of Copenhagen

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CPR > Research > Protein Signaling > Mailand Group

Mailand group (Ubiquitin Signaling)

The Mailand group was established at CPR in 2009 and is headed by professor and group leader Niels Mailand.

First row from the left: Niels Mailand, Nikoline Borgermann, Katrine Lütken H Weischenfeldt, Jonas Damgaard Elsborg, Maxim Tollenaere, Divya Achuthankutty, Petra Schwertman, Saskia Hoffmann, Lisa Schubert, Robert Francis Shearer, Dimitris Typas, Teresa Ho, Claire Guerillon, Peter Haahr,

In the Mailand group at CPR we aim to address how regulatory signaling processes promote cellular responses to DNA damage and replication stress to protect chromosome stability in mammalian cells. While much is known about the core machineries underlying DNA repair pathways, the complex regulatory framework underpinning and coordinating these processes is less well understood. To remedy this knowledge gap, we are applying our expertise in functional characterization of cellular signaling processes in combination with innovative systems-wide proteomics- and functional genomics-based screening approaches, in order to identify and functionally characterize new factors and regulatory mechanisms that protect the integrity of the genome following genotoxic insults.

Research themes:

1. Systematic identification of new genome stability maintenance factors

To define the scope of genome stability maintenance responses, we are applying innovative proteomics-driven strategies to comprehensively identify the factors recruited to sites of DNA damage and replication stress in vertebrate cells. In collaboration with Prof. Matthias Mann (Max Planck Institute of Biochemistry, Munich, Germany) we recently developed and utilized a powerful new proteomics-based method termed CHROMASS, which allows systematic mapping of the dynamic protein landscape at damaged chromatin. Using this method, we comprehensively defined the inventory of cellular proteins that undergo enrichment at stalled replication forks, DNA interstrand crosslinks and DNA double-strand breaks (DSBs) (Raschle et al., Science 2015; Haahr et al., Nature Cell Biol 2016) (Fig. 1). We are thus able to obtain a detailed systems-level view of how cells respond to various types of DNA damage, thereby allowing unique new insights into the nature and dynamics of the factors acting directly in the context of genotoxic insults. These surveys are revealing a range of new factors with important functions in genome stability maintenance that we are now characterizing in detail. These include SLF1 and SLF2, which form a complex and recruits the SMC5/6 complex to genotoxic stress sites (Raschle et al., Science 2015); SCAI, a 53BP1-interacting protein that promotes 53BP1-dependent repair of heterochromatin-associated DSBs (Hansen et al., Nature Cell Biol 2016); and TRAIP and ETAA1, factors that promote ATR-dependent signaling after replication stress (Hoffmann et al., J Cell Biol 2016; Haahr et al., Nature Cell Biol 2016). We are currently studying additional new genome stability maintenance factors identified in CHROMASS experiments, and we are applying CHROMASS and related methods to survey chromatin composition after other types of DNA lesions. To complement these proteomics-driven screens, we are performing genome-wide CRISPR/Cas9 screens for synthetic viability or lethality relationships within genotoxic stress signaling pathways.

 

Fig. 1: CHROMASS enables comprehensive identification of the proteins undergoing enrichment at chromatin in response to DNA damage or replication stress. Such analyses are revealing new genome stability maintenance factors, such as SLF1 and SLF2, which physically link RAD18 and the SMC5/6 complex, thereby promoting SMC5/6 recruitment to damaged DNA (Raschle et al., Science 2015).

2. Mechanistic dissection of signaling responses to DNA damage and replication stress

Ubiquitin-dependent protein modification has been firmly established as a key regulatory mechanism in many critical DNA damage signaling and repair pathways. However, systematic exploration of the scope and extent of this involvement has been lacking. We are studying global protein ubiquitylation dynamics on a site- and linkage-specific level in response to different types of DNA damage (e.g. Povlsen et al., Nature Cell Biology 2012; Thorslund et al., Nature 2015). These studies, together with a range of related screens and work from many other labs, are illuminating the broad scope and sheer complexity of how dynamic genotoxic stress-regulated ubiquitylation processes orchestrate genome stability maintenance responses.

We have been particularly interested in ubiquitin-dependent responses to DNA double-strand breaks (DSBs), one of the most cytotoxic DNA lesions (Schwertman et al., Nature Rev Mol Cell Biol 2016). We pioneered the discovery and characterization of the RNF8 and RNF168 E3 ubiquitin ligases, whose sequential actions promote recruitment of many DNA repair factors to DSB-surrounding chromatin areas (Mailand et al., Cell 2007; Doil et al., Cell 2009). Subsequently, we have identified a number of factors whose accumulation at DSB sites and/or ubiquitylation status are regulated in an RNF8/RNF168-dependent manner (Bekker-Jensen et al., Nature Cell Biol 2010; Poulsen et al., J Cell Biol 2012; Raschle et al., Science 2015; Thorslund et al., Nature 2015). We are also exploring how protein SUMOylation regulates DNA damage signaling pathways, and we are currently interrogating the functional roles of a range of DNA damage- and replication stress-regulated ubiquitylation and SUMOylation processes that we identified in proteomic screens.

We are currently devoting a strong focus to understanding the complex signaling responses that protect genome stability following replication stress, a major driver of genome instability that may lead to cancer and other severe pathologies. We recently discovered that an uncharacterized human protein, ETAA1, acts as a direct activator of the ATR kinase, the master organizer of cellular responses to replication stress (Haahr et al., Nature Cell Biol 2016) (Fig. 2). In addition, we identified a new PCNA-associated E3 ubiquitin ligase, TRAIP, whose activity is critical for promoting ATR-dependent signaling after replication stress (Hoffmann et al., J Cell Biol 2016). Through our affiliation with the Center for Chromosome Stability at University of Copenhagen (http://ccs.ku.dk/), headed by Prof. Ian Hickson, we are collaborating with a number of experts to explore in detail how these and other new factors protect chromosome stability on a mechanistic and physiological level following perturbations to the DNA replication process.

 

Fig 2.: ETAA1 activates ATR kinase signaling in response to replication stress. ETAA1 is recruited to RPA-coated ssDNA regions by means of dual RPA1- and RPA2-binding motifs and directly stimulates ATR kinase activity via an ATR-activating domain (AAD), in parallel with but independently of the canonical ATR activation pathway mediated by TopBP1 and the 9-1-1 complex (Haahr et al., Nature Cell Biol 2016).

 

3. Roles of linker histones in genome stability maintenance

We seek to understand how H1-type linker histones and their post-translational modifications (PTMs) function in the ‘histone code’ governing DNA repair and other chromosomal transactions. The histone code is the process by which specific PTMs on core histones are laid down and subsequently recognized by specific effector proteins to specify a multitude of distinct functional outputs of the genome. We recently discovered that linker histone PTMs can also contribute to the histone code for DNA repair, by showing that DNA damage-associated ubiquitylation of histone H1 generates a binding platform for RNF168, a central E3 ubiquitin ligase in the signaling response to DNA double-strand breaks (DSBs) (Thorslund et al., Nature 2015) (Fig. 3). Given the myriad PTMs that have been mapped on H1 subtypes, it is conceivable that the role of H1 in the histone code is not restricted to DSB repair but extends to many other aspects of genome stability maintenance. By combining systems-wide and focused approaches, we are currently investigating how H1-type histones and their PTMs facilitate critical chromatin-associated responses to DNA damage and replication stress.

 

Fig. 3: RNF8 promotes UBC13-dependent K63-linked polyubiquitylation of H1-type linker histones after DNA double-strand breaks (DSBs), generating a recruitment platform for RNF168, which subsequently amplifies DSB-induced chromatin ubiquitylation leading to recruitment of downstream repair factors (Thorslund et al., Nature 2015).

Funding

Work in the Mailand group is generously supported by The Novo Nordisk Foundation, European Research Council (ERC), EMBO, The Danish Council for Independent Research, The Danish Cancer Society, and The Lundbeck Foundation.

For more information and inquiries about available positions, please contact Prof. Niels Mailand (email: niels.mailand@cpr.ku.dk).

Selected recent publications:

Haahr P, Hoffmann S, Tollenaere M, Ho T, Toledo L, Mann M, Bekker-Jensen S, Raschle M, Mailand N. (2016). Activation of the ATR kinase by the RPA-binding protein ETAA1. Nature Cell Biol 18, 1196-1207.

Hansen RK, Mund A, Poulsen SL, Sandoval M, Tsouroula K, Klement K, Tollenaere MAX, Räschle M, Soria R, Offermanns S, Worzfeld T, Grosse R, Brandt DT, Rozell B, Mann M, Cole F, Soutoglou E, Goodarzi AA, Daniel J, Mailand N, Bekker-Jensen S. (2016). SCAI promotes DNA double-strand break repair in distinct chromosomal contexts. Nature Cell Biol 18, 1357-1366.

Saredi G, Huang H, Hammond CM, Alabert C, Bekker-Jensen S, Forne I, Reverón-Gómez N, Foster BM, Mlejnkova L, Bartke T, Mailand N, Imhof A, Patel D, Groth A. (2016). H4K20me0 marks post-replicative chromatin and recruits the TONSL-MMS22 DNA repair complex. Nature 534, 714-718.

Schwertman P, Bekker-Jensen S, Mailand N. (2016). Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers. Nature Reviews Mol Cell Biol 17, 379-394.

Hoffmann S, Smedegaard S, Nakamura K, Mortuza GB, Raschle M, Ibanez de Opakua A, Oka Y, Feng Y, Blanco FJ, Mann M, Montoya G, Groth A, Bekker-Jensen S, Mailand N. (2016). TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress. J Cell Biol 212, 63-75.

Bekker-Jensen S, Mailand N. (2015). RNF138 joins the HR team. Nature Cell Biol 17, 1375-1377.

Thorslund T, Ripplinger A, Hoffmann S, Wild T, Uckelmann M, Villumsen B, Narita T, Sixma TK, Choudhary C, Bekker-Jensen S, Mailand N. (2015). Histone H1 couples initiation and amplification of ubiquitin signaling after DNA damage. Nature 527, 389-393.

Tollenaere M, Villumsen B, Blasius M, Wagner SA, Borisova M, Bartek J, Choudhary C, Beli P, Mailand N, Bekker-Jensen S. (2015). p38-, MK2- and 14-3-3-dependent sequestration of CEP131/AZI1 promotes stress-induced remodeling of centriolar satellites. Nature Commun, 6, 10075.

Raschle M, Smeenk G, Hansen RK, Temu T, Oka Y, Hein MY, Nagaraj N, Long DT, Walter JC, Hofmann K, Storchova Z, Bekker-Jensen S, Mailand N, Mann M. (2015). Proteomics reveals dynamic assembly of repair complexes during bypass of DNA crosslinks. Science 384, 1253671.

Oka Y, Bekker-Jensen S, Mailand N. (2015). Ubiquitin-like protein UBL5 is required for the functional integrity of the Fanconi anemia DNA repair pathway. EMBO J 34, 1385-1398.

Gibbs-Seymour I, Mailand N. (2015). SLX4: Not SIMply a nuclease scaffold? Mol Cell 57, 3-5.

Gibbs-Seymour I, Oka Y, Rajendra E, Weinert BT, Passmore LA, Patel KJ, Olsen JV, Choudhary C, Bekker-Jensen S, Mailand N. (2014). Ubiquitin-SUMO circuitry controls activated Fanconi Anemia ID complex dosage in response to DNA damage. Mol Cell 57, 150-164.

Oka Y, Varmark H, Vitting-Seerup K, Beli P, Waage J, Hakobyan A, Mistrik M, Choudhary C, Rohde M, Bekker-Jensen S, Mailand N. (2014). UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells. EMBO Rep 15, 956-964.

Villumsen BH, Povlsen LK, Danielsen JR, Sylvestersen KB, Merdes A, Beli P, Yang Y, Choudhary C, Nielsen ML, Mailand N, Bekker-Jensen S. (2013). A new cell stress response that triggers centriolar satellite reorganization and ciliogenesis. EMBO J 32, 3029-3040.

Toledo L, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK, Bekker-Jensen S, Mailand N, , Bartek J, Lukas J. (2013). ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088-1103.

Poulsen SL, Hansen RK, Wagner SA, Choudhary C, Bekker-Jensen S, Mailand N. (2013). RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response. J Cell Biol 201, 797-807.

Mailand N, Gibbs-Seymour I, Bekker-Jensen S. (2013). Regulation of PCNA-protein interactions during replication of damaged DNA. Nature Rev Mol Cell Biol 14, 269-282.

Mosbech A, Gibbs-Seymour I, Kagias K, Thorslund T, Beli P, Povlsen LK, Nielsen SV, Smedegaard S, Sedgwick G, Lukas C, Petersen RH, Lukas J, Choudhary C, Pocock R, Bekker-Jensen S, Mailand N. (2012). DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks. Nature Struct Mol Biol 19, 1084-1092.

Povlsen, LK, Beli P, Wagner SA, Poulsen SL, Sylvestersen KB, Poulsen JW, Nielsen ML, Bekker-Jensen S, Mailand N, Choudhary C. (2012). Systems-wide analysis of ubiquitylation dynamics reveals a key role of PAF15 ubiquitylation in DNA damage bypass. Nature Cell Biol 14, 1089-1098.

Poulsen M, Lukas C, Lukas J, Bekker-Jensen S, Mailand N. (2012). Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks. J Cell Biol 197, 189-199.

Danielsen JR, Povlsen LK, Villumsen BH, Streicher W, Nilsson J, Wikström M, Bekker-Jensen S, Mailand N. (2012). DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger. J Cell Biol 197, 179-187.