Dorthe Helena Larsen

RNF168 Binds and Amplifies Ubiquitin Conjugates on Damaged Chromosomes to Allow Accumulation of Repair Proteins

DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

Site-specific Phosphorylation Dynamics of the Nuclear Proteome during the DNA Damage Response

To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

The CHD4 helicase is identified as a new component of the genome surveillance machinery in a proteomic screen for factors enriched on chromatin after ionizing radiation (see also related paper by Smeenk et al. in this issue).

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8‐mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double‐strand breaks. Interestingly, RNF8‐mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin‐ligase activity of RNF8, but involved a non‐canonical interaction with the forkhead‐associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling‐assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.

The NBS1–Treacle complex controls ribosomal RNA transcription in response to DNA damage

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.

HCLK2 is required for activity of the DNA damage response kinase ATR.

ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G2 checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.

Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response.

Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell’s exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

Nucleolar responses to DNA double-strand breaks

Maintenance of cellular homeostasis is key to prevent transformation and disease. The cellular response to DNA double-strand breaks, primarily orchestrated by the ATM/ATR kinases is one of many mechanisms that serve to uphold genome stability and homeostasis. Upon detection of double-strand breaks (DSBs), several signaling cascades are activated to halt cell cycle progression and initiate repair. Furthermore, the DNA damage response (DDR) controls cellular processes such as transcription, splicing and metabolism. Recent studies have uncovered aspects of how the DDR operates within nucleoli. It appears that the DDR controls transcription in the nucleoli, not only when DNA breaks occur in the rDNA repeats, but also when a nuclear DDR is activated. In addition, we have gained first insights into how repair of DSBs is organized in the nucleolus. Collectively, these recent studies provide a more comprehensive picture of how the DDR regulates basic cellular functions to maintain cellular homeostasis. In this review we will summarize recent findings and discuss their implications for our understanding of how the DDR regulates transcription and repair in the nucleolus.

DNA damage-induced dynamic changes in abundance and cytosol-nuclear translocation of proteins involved in translational processes, metabolism, and autophagy

ABSTRACT Ionizing radiation (IR) causes DNA double-strand breaks (DSBs) and activates a versatile cellular response regulating DNA repair, cell-cycle progression, transcription, DNA replication and other processes. In recent years proteomics has emerged as a powerful tool deepening our understanding of this multifaceted response. In this study we use SILAC-based proteomics to specifically investigate dynamic changes in cytoplasmic protein abundance after ionizing radiation; we present in-depth bioinformatics analysis and show that levels of proteins involved in autophagy (cathepsins and other lysosomal proteins), proteasomal degradation (Ubiquitin-related proteins), energy metabolism (mitochondrial proteins) and particularly translation (ribosomal proteins and translation factors) are regulated after cellular exposure to ionizing radiation. Downregulation of no less than 68 ribosomal proteins shows rapid changes in the translation pattern after IR. Additionally, we provide evidence of compartmental cytosol-nuclear translocation of numerous DNA damage related proteins using protein correlation profiling. In conclusion, these results highlight unexpected cytoplasmic processes actively orchestrated after genotoxic insults and protein translocation from the cytoplasm to the nucleus as a fundamental regulatory mechanism employed to aid cell survival and preservation of genome integrity.