TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution
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ABSTRACT:
PROVIDER: PRJNA741480 | ENA |
REPOSITORIES: ENA
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Project description:R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic “reader” Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.
2021-06-26 | GSE178905 | GEO
Project description:PARP inhibitor (PARPi)-resistant BRCA mutant (BRCAm) high-grade serous ovarian cancer (HGSOC) represents a new clinical challenge with unmet therapeutic needs. Quantitative high-throughput drug combination screen identifies that ATR inhibitor (ATRi) and AKT inhibitor (AKTi) combination is a rational treatment strategy for both PARPi-sensitive and PARPi-resistant BRCAm HGSOC by inducing DNA damage and R-loop-mediated replication stress (RS). Mechanistically, the kinase domain of AKT1 directly interacts with DHX9, thus facilitating recruitment of DHX9 to R-loops. AKTi increases ATRi-induced R-loop-mediated RS by mitigating recruitment of DHX9 to R-loops. Moreover, DHX9 is upregulated in tumors from PARPi-resistant BRCAm HGSOC patients and high co-expression of DHX9 and AKT1 correlates with worse survival. Our study reveals a previously unknown interaction between AKT1 and DHX9 in R-loop resolution and novel mechanisms of action of AKTi and ATRi combination. Our data also provide a rationale for the clinical development of ATRi and AKTi combination for BRCAm HGSOC irrespective of PARPi resistance status and a potential biomarker to predict the response of combination therapy.
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Project description:R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co- transcriptionally in the opposite orientation to replication fork progression. Recent studies have shown that replication forks stalled by co-transcription R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds RNA:DNA hybrids in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4 pathway for resolution of R-loop-mediated transcription- replication conflicts, which may be involved in R-loop unwinding.
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Project description:Successful R-loop homeostasis consists of its timely biogenesis and removal throughout the genome to regulate diverse cellular processes. Yet, they are considered hazardous source to our genome when their turnover goes awry. Here, we report HELZ, a novel player in R-loop homeostasis and DNA repair pathway. HELZ mediates resistance to several DNA damaging agents, and localizes to DSBs and its depletion increases R-loops fostering genomic instability. Loss of HELZ results in impaired homologous recombination (HR) due to R-loops accumulation, with an increase in classical-non-homologous end joining (c-NHEJ), suggesting a role for HELZ in regulating DSB repair pathway choice. Rad51 retention at DSBs is governed by HELZ mediated R-loop accumulation. Mechanistically, our data show that HELZ complexes with BRCA1 and facilitates its recruitment to DSBs in an R-loop dependent manner. Collectively, our data support a model in which HELZ recruits BRCA1 and facilitates R loop resolution at DSBs to promote HR and therefore maintains genome stability.
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Project description:The ATP-dependent DExH/D-box helicase DHX9 is a key participant in a number of gene regulatory steps, including transcriptional, translational, microRNA-mediated control, DNA replication, and maintenance of genomic stability. DHX9 has also been implicated in maintenance of the tumorigenic process and in drug response. Here, we report that inhibition of DHX9 expression is lethal to multiple human and mouse cancer cell lines. In contrast, using a novel conditional shDHX9 mouse model, we demonstrate that sustained and prolonged suppression of DHX9 is well tolerated at the organismal level. Our results demonstrate a robust tolerance for DHX9 knockdown in non-transformed cells and supports the targeting of DHX9 as an effective and specific chemotherapeutic approach. Comparison of gene expression in large intestine of mice with or without reduced expression of DHX9.
2016-05-19 | E-GEOD-76765 | biostudies-arrayexpress
Project description:In our study, we valided DHX9 and NPM1 interact with KIMAT1, whereas DHX9 also interacts with HIF1A-As2. DHX9 is a highly conserved DEAD-box protein expressed in the nucleus and the cytoplasm, involved in many processes including transcriptional activation, miRNA biogenesis and tumor cell maintenance. NPM1 is predominantly localized in the nucleoplasm, where it associates with active RNA polymerase II and transcriptionally activates genes involved in cancer. We silenced DHX9 and NPM1 and performed RNA-seq to examine the dysregulated genes by DHX9 and NPM1.
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Project description:Oesophageal cancer is a high malignant cancer and oesophageal squamous cell carcinoma (ESCC) is the most common subtype of oesophageal cancer. Here, we identified H/ACA box snoRNA42 (SNORA42) was upregulated in ESCC and can be applied as the diagnostic and prognostic marker. Specifically, overexpression of SNORA42 promoted ESCC proliferation, migration and invasion ability whereas knockdown of SNORA42 inhibited these phenotypes in vitro and in vivo. Using RNA pull-down assay combined with Mass Spectrometry technique, we identified protein DHX9 interacted with SNORA42. DHX9 protein expression was up-regulated in ESCC and had a positive correlation with the expression of SNORA42. Furthermore, the effects of SNORA42 overexpression on ESCC phenotypes can be reversed by knockdown of DHX9. Mechanically, SNORA42 promoted DHX9 stabilized by attenuating its ubiquitination and degradation. From KEGG analysis of next-generation sequencing, we identified the NF-kappa B pathway was one of the most regulated pathways by SNORA42. SNORA42 enhanced phosphorylation of p65 in the nucleus. Moreover, SNORA42 exerted its function through promoting DHX9 interacted with p-65, inducing transcription of NF-kappa B target genes. These findings suggest that SNORA42 promotes ESCC tumorgenesis via SNORA42/DHX9/p65 axis
2023-02-28 | GSE146195 | GEO
Project description:The ATP-dependent DExH/D-box helicase DHX9 is a key participant in a number of gene regulatory steps, including transcriptional, translational, microRNA-mediated control, DNA replication, and maintenance of genomic stability. DHX9 has also been implicated in maintenance of the tumorigenic process and in drug response. Here, we report that inhibition of DHX9 expression is lethal to multiple human and mouse cancer cell lines. In contrast, using a novel conditional shDHX9 mouse model, we demonstrate that sustained and prolonged suppression of DHX9 is well tolerated at the organismal level. Our results demonstrate a robust tolerance for DHX9 knockdown in non-transformed cells and supports the targeting of DHX9 as an effective and specific chemotherapeutic approach.
2016-05-19 | GSE76765 | GEO
Project description:Cell division ensures that both genetic information and non-genetic contents are inherited by daughter cells. Whereas considerable detail has been learned about the processing of intact or damaged DNA during the cell cycle (Branzei & Foiani, 2008; Klaasen et al., 2022),(Bakhoum & Cantley, 2018),(Hustedt & Durocher, 2016), how daughter cells deal with other forms of inherited damage is unknown. Here we identified a special kind of cytoplasmic granules responsible for the compartmentalisation of parental RNA damage. We found that ultraviolet (UV)-induced RNA, but not DNA, damage triggered assembly of this unique type of granules characterized by the presence of RNA helicase DHX9. By developing a novel methodology, FANCI, we discovered that DHX9 granules are enriched in damaged intron RNA and pre-mRNA-binding proteins, which is in contrast to other classical stress granules (SGs) that are composed of mature mRNA. Intron damage impeded proper splicing and intron decay, and induced generation of circRNA and dsRNA in the granules. Moreover, we showed that intron damage induced DHX9 granules assembled specifically in postmitotic daughter cells and triggered a cellular dsRNA immune response. Condensation with dsRNA is crucial for DHX9 localization to the granules and the modulation of dsRNA in these granules by DHX9 was crucial for daughter cell survival. Our observations revealed that DHX9 granules constitute a dedicated non-membrane-bound cytoplasmic compartment that protects daughter cells from parental damaged RNA.
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