Comparative genome- and proteome-wide screens yield large amounts of data. To efficiently present such datasets and to simplify the identification of hits, the results are often presented in a type ...of scatterplot known as a volcano plot, which shows a measure of effect size versus a measure of significance. The data points with the largest effect size and a statistical significance beyond a user-defined threshold are considered as hits. Such hits are usually annotated in the plot by a label with their name. Volcano plots can represent ten thousands of data points, of which typically only a handful is annotated. The information of data that is not annotated is hardly or not accessible. To simplify access to the data and enable its re-use, we have developed an open source and online web tool with R/Shiny. The web app is named VolcaNoseR and it can be used to create, explore, label and share volcano plots ( https://huygens.science.uva.nl/VolcaNoseR ). When the data is stored in an online data repository, the web app can retrieve that data together with user-defined settings to generate a customized, interactive volcano plot. Users can interact with the data, adjust the plot and share their modified plot together with the underlying data. Therefore, VolcaNoseR increases the transparency and re-use of large comparative genome- and proteome-wide datasets.
The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the ...dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.
Display omitted
•PARP1 recruits the chromatin remodeler CHD2 to DNA damage•CHD2 promotes chromatin expansion and H3.3 deposition at DNA breaks•CHD2 promotes the assembly of NHEJ repair complexes at DNA breaks•PARP1 drives CHD2- and H3.3-dependent DNA repair by NHEJ
Luijsterburg et al. define a PARP1-dependent mechanism that regulates NHEJ through localized chromatin expansion and deposition of the histone variant H3.3 by the nucleosome remodeler CHD2 at DNA breaks. Their data also show that these CHD2-mediated events promote DNA repair by facilitating the assembly of NHEJ complexes in chromatin.
A recent study by Fenstermaker et al. in Nature describes how transcriptionally active RNA polymerase II (Pol II) clings to the genomic tightrope during the passage of the replication fork and ...rapidly resumes transcription of immature RNA from both strands of nascent DNA, facilitated by protein-protein interactions between the replication and transcription machineries.
The accumulation of the human tumor suppressor 53BP1 at DNA damage sites requires the ubiquitin ligases RNF8 and RNF168. As 53BP1 recognizes dimethylated Lys20 in histone H4 (H4K20me2), the ...requirement for RNF8- and RNF168-mediated ubiquitylation has been unclear. Here we show that RNF8-mediated ubiquitylation facilitates the recruitment of the AAA-ATPase valosin-containing protein (VCP, also known as p97) and its cofactor NPL4 to sites of double-strand breaks. RIDDLE cells, which lack functional RNF168, also show impaired recruitment of VCP to DNA damage. The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. Consistent with this, nematodes lacking the VCP orthologs CDC-48.1 or CDC-48.2, or cofactors UFD-1 or NPL-4, are highly sensitive to ionizing radiation. Our data suggest that human RNF8 and RNF168 promote VCP-mediated displacement of L3MBTL1 to unmask 53BP1 chromatin binding sites.
DNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects in ...TCR cause severe clinical syndromes, including Cockayne syndrome (CS). The molecular mechanism of TCR and the molecular origin of CS have long remained enigmatic. Here we explore new advances in our understanding of how TCR complexes assemble through cooperative interactions between repair factors stimulated by RNAPII ubiquitylation. Mounting evidence suggests that RNAPII ubiquitylation activates TCR complex assembly during repair and, in parallel, promotes processing and degradation of RNAPII to prevent prolonged stalling. The fate of stalled RNAPII is therefore emerging as a crucial link between TCR and associated human diseases.
Recent advances in methods to isolate TCR complexes revealed a sequential and cooperative assembly of CSB, CSA, and UVSSA onto DNA damage-stalled RNAPII.The UV-induced ubiquitylation of a single lysine residue in the largest subunit of RNAPII (RPB1-K1268) by CRL4 E3 ligases, including CRL4CSA, is a key regulator of TCR and, together with the ubiquitylation of UVSSA (K414), promotes the transfer of TFIIH onto RNAPII to initiate DNA repair.The CS protein-dependent ubiquitylation of RPB1-K1268 acts as a molecular clock that promotes DNA repair at early time-points, but leads to the removal of DNA damage-stalled RNAPII when repair fails.Rather than a DNA repair disorder, emerging evidence suggests that Cockayne syndrome is caused by a deficiency in processing RNAPII, resulting in its prolonged arrest at DNA lesions, ultimately causing neurodegeneration.
DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in ...transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.
The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR ...proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.
The WD40-repeat protein DDB2 is essential for efficient recognition and subsequent removal of ultraviolet (UV)-induced DNA lesions by nucleotide excision repair (NER). However, how DDB2 promotes NER ...in chromatin is poorly understood. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a novel DDB2-associated factor. We demonstrate that DDB2 facilitated poly(ADP-ribosyl)ation of UV-damaged chromatin through the activity of PARP1, resulting in the recruitment of the chromatin-remodeling enzyme ALC1. Depletion of ALC1 rendered cells sensitive to UV and impaired repair of UV-induced DNA lesions. Additionally, DDB2 itself was targeted by poly(ADP-ribosyl)ation, resulting in increased protein stability and a prolonged chromatin retention time. Our in vitro and in vivo data support a model in which poly(ADP-ribosyl)ation of DDB2 suppresses DDB2 ubiquitylation and outline a molecular mechanism for PARP1-mediated regulation of NER through DDB2 stabilization and recruitment of the chromatin remodeler ALC1.
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA ...damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Display omitted
•RNAPII RPB1-K1268 ubiquitination is essential for transcription recovery and DNA repair•Sequential ubiquitination of RNAPII and UVSSA coordinates the recruitment of TFIIH•Strand-specific ChIPseq enables mapping of RNAPII and reveals genome-wide repair kinetics•RNAPII ubiquitination protects against neurodegeneration phenotype in Cockayne syndrome
Ubiquitination of RNA polymerase promotes recruitment of TFIIH and initiation of transcription-coupled repair, and dysregulation of this process in mice has neurological consequences akin to Cockayne syndrome.
PDF
