Jannie Rendtlew Danielsen

A Role for Small RNAs in DNA Double-Strand Break Repair

Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.

HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes

Regulatory ubiquitylation is emerging as an important mechanism to protect genome integrity in cells exposed to DNA damage. However, the spectrum of known ubiquitin regulators of the DNA damage response (DDR) is limited and their functional interplay is poorly understood. Here, we identify HERC2 as a factor that regulates ubiquitin-dependent retention of repair proteins on damaged chromosomes. In response to ionising radiation (IR), HERC2 forms a complex with RNF8, a ubiquitin ligase involved in the DDR. The HERC2–RNF8 interaction requires IR-inducible phosphorylation of HERC2 at Thr 4827, which in turn binds to the forkhead-associated (FHA) domain of RNF8. Mechanistically, we provide evidence that HERC2 facilitates assembly of the ubiquitin-conjugating enzyme Ubc13 with RNF8, thereby promoting DNA damage-induced formation of Lys 63-linked ubiquitin chains. We also show that HERC2 interacts with, and maintains the levels of, RNF168, another ubiquitin ligase operating downstream of RNF8 (Refs 7, 8). Consequently, knockdown of HERC2 abrogates ubiquitin-dependent retention of repair factors such as 53BP1, RAP80 and BRCA1. Together with the increased radiosensitivity of HERC2-depleted cells, these results uncover a regulatory layer in the orchestration of protein interactions on damaged chromosomes and they underscore the role of ubiquitin-mediated signalling in genome maintenance.

FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis

The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

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).

Human Xip1 (C2orf13) Is a Novel Regulator of Cellular Responses to DNA Strand Breaks

DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we identify the previously uncharacterized human protein Xip1 (C2orf13) as a novel component of the checkpoint response to DNA strand breaks. Green fluorescent protein-tagged Xip1 was rapidly recruited to sites of DNA breaks, and this accumulation was dependent on a novel type of zinc finger motif located in the C terminus of Xip1. The initial recruitment kinetics of Xip1 closely paralleled that of XRCC1, a central organizer of single strand break (SSB) repair, and its accumulation was both delayed and sustained when the detection of SSBs was abrogated by inhibition of PARP-1. Xip1 and XRCC1 stably interacted through recognition of CK2 phosphorylation sites in XRCC1 by the Forkhead-associated (FHA) domain of Xip1, and XRCC1 was required to maintain steady-state levels of Xip1. Moreover, Xip1 was phosphorylated on Ser-116 by ataxia telangiectasia-mutated in response to ionizing radiation, further underscoring the potential importance of Xip1 in the DNA damage response. Finally, depletion of Xip1 significantly decreased the clonogenic survival of cells exposed to DNA SSB- or double strand break-inducing agents. Collectively, these findings implicate Xip1 as a new regulator of genome maintenance pathways, which may function to organize DNA strand break repair complexes at sites of DNA damage.

DNA damage–inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

SUMOylation of the ubiquitin ligase HERC2 promotes efficient chromatin licensing in the vicinity of DNA double-strand breaks.

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.

Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities.

A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

Centriolar satellites are small, granular structures that cluster around centrosomes, but whose biological function and regulation are poorly understood. We show that centriolar satellites undergo striking reorganization in response to cellular stresses such as UV radiation, heat shock, and transcription blocks, invoking acute and selective displacement of the factors AZI1/CEP131, PCM1, and CEP290 from this compartment triggered by activation of the stress‐responsive kinase p38/MAPK14. We demonstrate that the E3 ubiquitin ligase MIB1 is a new component of centriolar satellites, which interacts with and ubiquitylates AZI1 and PCM1 and suppresses primary cilium formation. In response to cell stress, MIB1 is abruptly inactivated in a p38‐independent manner, leading to loss of AZI1, PCM1, and CEP290 ubiquitylation and concomitant stimulation of ciliogenesis, even in proliferating cells. Collectively, our findings uncover a new two‐pronged signalling response, which by coupling p38‐dependent phosphorylation with MIB1‐catalysed ubiquitylation of ciliogenesis‐promoting factors plays an important role in controlling centriolar satellite status and key centrosomal functions in a cell stress‐regulated manner.

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.