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R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability.

ABSTRACT: Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

SUBMITTER: Mosler T

PROVIDER: S-EPMC8677849 | biostudies-literature |

REPOSITORIES: biostudies-literature

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R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instabilityR-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

Project description:Transcription can pose a threat to genomic stability through the formation of R-loops that obstruct the progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RDProx to identify proteins that regulate R-loops in human cells. RDProx relies on the expression of the hybrid-binding domain (HBD) of Ribonuclease H1 (RNaseH1) fused to the ascorbate peroxidase (APEX2), which permits mapping of the R-loop proximal proteome using quantitative mass spectrometry. We associated R-loop regulation with different cellular proteins and identified a role of the tumor suppressor DEAD box protein 41 (DDX41) in opposing R-loop-dependent genomic instability. Depletion of DDX41 resulted in replication stress, double strand breaks and increased inflammatory signaling. Furthermore, DDX41 opposes accumulation of R-loops at gene promoters and its loss leads to upregulated expression of TGFβ and NOTCH signaling genes. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia (AML) in adulthood. We propose that accumulation of co-transcriptional R-loops, associated gene expression changes and inflammatory response contribute to the development of familial AML with mutated DDX41.

2021-11-25 | PXD024517 | Pride

ATM pathway activation limits R-loop-associated genomic instability in Werner syndrome cells.

Project description:Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.

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Project description:Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12') in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.

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Checkpoint Defects Elicit a WRNIP1-Mediated Response to Counteract R-Loop-Associated Genomic Instability.

Project description:Conflicts between replication and transcription are a common source of genomic instability, a characteristic of almost all human cancers. Aberrant R-loops can cause a block to replication fork progression. A growing number of factors are involved in the resolution of these harmful structures and many perhaps are still unknown. Here, we reveal that the Werner interacting protein 1 (WRNIP1)-mediated response is implicated in counteracting aberrant R-loop accumulation. Using human cellular models with compromised Ataxia-Telangiectasia and Rad3-Related (ATR)-dependent checkpoint activation, we show that WRNIP1 is stabilized in chromatin and is needed for maintaining genome integrity by mediating the Ataxia Telangiectasia Mutated (ATM)-dependent phosphorylation of Checkpoint kinase 1 (CHK1). Furthermore, we demonstrated that loss of Werner Syndrome protein (WRN) or ATR signaling leads to formation of R-loop-dependent parental ssDNA upon mild replication stress, which is covered by Radiorestistance protein 51 (RAD51). We prove that Werner helicase-interacting protein 1 (WRNIP1) chromatin retention is also required to stabilize the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall, our findings suggest a novel function for WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication-transcription conflicts are handled.

| S-EPMC7072626 | biostudies-literature

Proximity labeling identifies a repertoire of site-specific R-loop modulators.

Project description:R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

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Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability.

Project description:Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.

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Metabolic regulation by p53 prevents R-loop-associated genomic instability [RNA-Seq]

Project description:To investigate the role of p53 in maintaining genome integrity we deleted p53 via doxycycline-inducible short hairpin (sh)RNA targeting p53 (KPshp53 cells), and we treat the cells with the DNA demethylating molecule 5-Aza-2′-deoxycytidine (AZA or dC)

2022-08-24 | GSE207879 | GEO

Metabolic regulation by p53 prevents R-loop-associated genomic instability [ATAC-Seq]

2022-08-24 | GSE207878 | GEO

Genetic instability associated with loop or stem-loop structures within transcription units can be independent of nucleotide excision repair.

Project description:Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem-loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them.

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BRCA1 prevents R-loop-associated centromeric instability.

Project description:Centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive α-satellite sequences, which are actively transcribed throughout the cell cycle. Centromeres play an essential role in chromosome inheritance and genome stability through coordinating kinetochores assembly during mitosis. Structural and functional alterations of the centromeres cause aneuploidy and chromosome aberrations which can induce cell death. In human cells, the tumor suppressor BRCA1 associates with centromeric chromatin in the absence of exogenous damage. While we previously reported that BRCA1 contributes to proper centromere homeostasis, the mechanism underlying its centromeric function and recruitment was not fully understood. Here, we show that BRCA1 association with centromeric chromatin depends on the presence of R-loops, which are non-canonical three-stranded structures harboring a DNA:RNA hybrid and are frequently formed during transcription. Subsequently, BRCA1 counteracts the accumulation of R-loops at centromeric α-satellite repeats. Strikingly, BRCA1-deficient cells show impaired localization of CENP-A, higher transcription of centromeric RNA, increased breakage at centromeres and formation of acentric micronuclei, all these features being R-loop-dependent. Finally, BRCA1 depletion reveals a Rad52-dependent hyper-recombination process between centromeric satellite repeats, associated with centromere instability and missegregation. Altogether, our findings provide molecular insights into the key function of BRCA1 in maintaining centromere stability and identity.

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