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Transcription dynamics prevents RNA-mediated genomic instability through SRPK2-dependent DDX23 phosphorylation

ABSTRACT: Genomic instability is frequently caused by nucleic acids structures formed during transcription termed R-loops. Despite their harmful potential, mechanisms that sense, signal and suppress these structures remain elusive. Here we report that oscillations in transcription dynamics are a major sensor of R-loops. We show that pausing of RNA polymerase II (RNA Pol II) initiates a signaling cascade whereby the serine/arginine protein kinase 2 (SRPK2) phosphorylates the DDX23 helicase culminating in the suppression of R-loops. We show that in the absence of either SRPK2 or DDX23, accumulation of R-loops leads to massive genomic instability revealed by high levels of DNA double-strand breaks (DSBs). Importantly, we found DDX23 mutations in several cancers and detected homozygous deletions of the entire DDX23 locus in 10 (17%) adenoid cystic carcinoma samples. Our results unravel molecular details of a novel link between transcription dynamics and RNA-mediated genomic instability that may play important roles in cancer development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE76777 | GEO | 2016/12/22

SECONDARY ACCESSION(S): PRJNA308572

REPOSITORIES: GEO

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Homo sapiens

Project description:Transcription dynamics prevents RNA-mediated genomic instability through SRPK2-dependent DDX23 phosphorylation

| PRJNA308572 | ENA

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

ATAD5 regulates R-loop by maintaining RNA helicase level and unloading PCNA at the replication fork

Project description:R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflicts (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

2021-09-09 | PXD018207 | Pride

Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability [TT-seq]

Project description:Connections between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established however, the underlying mechanisms remain poorly understood. Here we used a mutant version of elongation factor TFIIS (TFIISmut) to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII. It affects mRNA splicing and termination as well. Remarkably, however, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed new light on the relationship between transcription stress and R-loops, and suggest that different classes of R-loops exist, potentially with distinct consequences for genome instability.

2019-06-13 | GSE132395 | GEO

Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability [ChIP-seq]

2019-06-13 | GSE132391 | GEO

Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability [ RNA-seq]

2019-06-13 | GSE132398 | GEO

Gene expression profile in mouse tumor RIF-1 cell line after knockdown of Bre1a (Rnf20) or Bre1b (Rnf40).

Project description:Mammalian Bre1 complexes (BRE1A/B (RNF20/40) in humans and Bre1a/b (Rnf20/40) in mice) function similarly to their yeast homolog Bre1 as ubiquitin ligases in monoubiquitination of histone H2B. This ubiquitination facilitates methylation of histone H3 at K4 and K79, and accounts for the roles of Bre1 and its homologs in transcriptional regulation. Recent studies by others suggested that Bre1 acts as a tumor suppressor, augmenting expression of select tumor suppressor genes and suppressing select oncogenes. In this study we present an additional mechanism of tumor suppression by Bre1 through maintenance of genomic stability. We track the evolution of genomic instability in Bre1-deficient cells from replication-associated double-strand breaks (DSBs) to specific genomic rearrangements that explain a rapid increase in DNA content and trigger breakage-fusion-bridge cycles. We show that aberrant RNA-DNA structures (R-loops) constitute a significant source of DSBs in Bre1-deficient cells. Combined with a previously reported defect in homologous recombination, generation of R-loops is a likely initiator of replication stress and genomic instability in Bre1-deficient cells. We propose that genomic instability triggered by Bre1 deficiency may be an important early step that precedes acquisition of an invasive phenotype, as we find decreased levels of BRE1A/B and dimethylated H3K79 in testicular seminoma and in the premalignant lesion in situ carcinoma. A cellular modification design type is where a modification of the transcriptome, proteome (not genome) is made, for example RNAi, antibody targeting. Knock down by RNA interference: gene knocked down in RIF-1 cell line cellular_modification_design

2013-01-09 | E-GEOD-34977 | biostudies-arrayexpress

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 that 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 at R-loops. One of the most consistently enriched proteins at R-loops was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP is necessary to suppress R-loops in vivo at its genomic targets and that it resolves R-loops in vitro. Furthermore, deletion of the 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 previously unappreciated role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

2021-06-03 | MSV000087568 | MassIVE

Mapping of TOP1cc and R-loops im MCF-7 after TOP1 KD and/or RNase H OE [CHiP_DRIP_Seq]

Project description:DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employed DRIP-seq and ChIP-seq to map the change in TOP1cc and R-loops after the depletion of TOP1 and R-loops, to comprehensively investigate the interplay between transcription, DSBs, Topoisomerase 1 (TOP1), and R-loops. Our findings revealed the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops, indicating their crucial involvement in transcription-associated genomic instability. Depletion of R-loops and TOP1 specifically reduced DSBs at highly expressed genes, uncovering their pivotal roles in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides novel insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development. Notably, our study highlights the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations, shedding light on the potential for targeted therapeutic strategies. Overall, these findings deepen our understanding of the regulatory mechanisms governing DSBs in hypertranscribed genes associated with carcinogenesis, opening avenues for future research and therapeutic interventions.

2024-05-22 | GSE241307 | GEO

Mapping of physiological DNA double stranded breaks in normal breast cells and breast cancer cells [BLISS]

Project description:DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employed the advanced BLISS techniques to map DSBs, with different genetic manipulations to comprehensively investigate the interplay between transcription, DSBs, Topoisomerase 1 (TOP1), and R-loops. Our findings revealed the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops, indicating their crucial involvement in transcription-associated genomic instability. Depletion of R-loops and TOP1 specifically reduced DSBs at highly expressed genes, uncovering their pivotal roles in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides novel insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development. Notably, our study highlights the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations, shedding light on the potential for targeted therapeutic strategies. Overall, these findings deepen our understanding of the regulatory mechanisms governing DSBs in hypertranscribed genes associated with carcinogenesis, opening avenues for future research and therapeutic interventions.

2024-05-22 | GSE241305 | GEO

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