ATM-Dependent Phosphorylation of Nemo SQ Motifs is Dispensable for Nemo-Mediated Gene Expression Changes in Response to DNA Double Strand Breaks
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ABSTRACT:
PROVIDER: PRJNA1101955 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28735482_1.fastq.gz | Fastqsanger.gz | |||
| SRR28735482_2.fastq.gz | Fastqsanger.gz | |||
| SRR28735483_1.fastq.gz | Fastqsanger.gz | |||
| SRR28735483_2.fastq.gz | Fastqsanger.gz | |||
| SRR28735484_1.fastq.gz | Fastqsanger.gz |
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Project description:In response to DNA double strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm where it promotes monoubiquitylation of Nemo and resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as lipopolysaccharides (LPS). To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy, fertile, and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.
2024-08-08 | GSE264315 | GEO
Project description:We previously demonstrated that IKKα binds to and phosphorylates ATM thus potentiating the non-homologous end joining DNA damage repair pathway in cancer cells. Hence, inhibiting IKKα enhances the efficacy of DNA damage-based anticancer therapy. Whether additional elements contribute to this resistance-related mechanism remains unknown. We here show that NEMO physically interacts with the ATM-IKKα complex before damage. Upon exposure to damaging agents, NEMO is dispensable for ATM activation, but it is required to drive active ATM and IKKα to the sites of damage thus enabling DNA damage resolution. Recognition of damaged DNA by this IKKα/NEMO/ATM complex is partially mediated by direct interaction of NEMO to histones but highly dependent on PARP1 activity. Finally, we detected increased ATR activity in NEMO-deficient cells, and that ATR inhibition potentiates the effect of chemotherapy upon NEMO or IKKα depletion. Bioinformatic analysis of public CRC datasets support the functional impact of the IKKα/NEMO/ATM pathway in patient prognosis, which could be therapeutically exploited.
2024-02-11 | PXD043552 | Pride
Project description:Lentiviral accessory genes enhance replication through diverse mechanisms. HIV-1 accessory protein Vpr modulates the host DNA damage response (DDR) at multiple steps through DNA damage, cell cycle arrest, the degradation of host proteins, and both the activation and repression of DDR signaling. Vpr also alters host and viral transcription; however, the connection between Vpr-mediated DDR modulation and transcriptional activation remains unclear. Here, we determined the cellular consequences of Vpr-induced DNA damage using Vpr mutants that allow us to separate the ability of Vpr to induce DNA damage from cell cycle arrest and other DDR phenotypes including host protein degradation and repression of DDR. RNA-sequencing of cells expressing Vpr or Vpr mutants identified that Vpr alters cellular transcription through mechanisms both dependent and independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), Vpr-induced DNA damage activates the ATM-NEMO pathway and alters cellular transcription via NF-κB/RelA signaling. HIV-1 infection of primary MDMs validated Vpr-dependent NF-κB transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional reprogramming can occur during early and late stages of viral replication. Together, our data identifies a mechanism by which Vpr activates NF-κB through DNA damage and the ATM-NEMO pathway, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral transcription and replication.
2024-08-23 | GSE253779 | GEO
Project description:Gene expression profiling reveals a potential role of SQ, Tri-SQ, and Tetra-SQ in stimulating hair growth in dermal papilla cells. Squalene (SQ) was found to exhibit a strong antioxidant and anti-inflammatory activity; however, its biological effects is underestimated because of its insolubility. To overcome this problem, new amphiphilic SQ derivatives were synthesized and were used in this study to check their potential effect in vitro and in vivo on inducing hair growth while exhibiting an anti-aging property.
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Project description:Expression data from SQ, TRI-SQ and Tetra-SQ-treated HFDPCs
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Project description:ADP-ribosylation is a dynamic post-translation modification that regulates the early phase of various DNA repair pathways by recruiting repair factors to chromatin. ADP-ribosylation levels are defined by the activities of transferases and hydrolases. Little is known about regulation of these enzymes except for that of the transferase ARTD1/PARP1. Among the hydrolases, MacroD2 contains a unique C-terminal region, which may regulate the enzymatic activity carried out by the N-terminal macrodomain. Our study shows that MacroD2 is exported from the nucleus upon DNA damage, and that this nuclear export is induced by ATM activity. In the MacroD2 C-terminal unstructured region we find two SQ/TQ motifs that are necessary for this regulation, arguing for a direct interaction with ATM. This study also shows that MacroD2 nuclear export restricts MacroD2 recruitment to the DNA damage site on the temporal scale, suggesting that the regulation of location determines a regulation of enzymatic activity.
2016-10-12 | PXD003712 | Pride
Project description:Gene expression profiling reveals functional difference between Sq and HH-Sq on differentiation, metabolism, and lipid droplot formation of dADSC Microarray gene expression profiling was conducted for technical replicates of adipocyte induced dADSC (IN), adipocyte induced and treated with Sq (Sq), adipocyte induced and treated with HH-Sq (HH-Sq) for 14 days (1 µM) to identify its effect in the regulation of adipocyte differentiation, metabolism, and lipid droplet accumulation. Technical replicates represent the same hybridization mixture applied to 3 independent arrays.
2020-06-27 | GSE153391 | GEO
Project description:Global gene expression profiling reveals a potential anti-melanoma effect of SQ-diEG in B16F10 lung colonization mice model. Squalene (SQ) was considered as a promising natural agent in anti-cancer treatment due to its strong antioxidant and anti-inflammatory activity; however, its pharmacological value has been largely underestimated because of its poor solubility and bioavailability. To adress this problem, a novel amphiphilic SQ derivative which bearing ethylene glycol oligomers was synthesized and was used as a permeation enhancer in this study to check its potential effect on anti-melanoma metastasis using B16F10 lung colonization mice model. Total 40 six years old male C57BL/6J mice were randomly divided into five groups (8 mice per group), including the control "(-)B16F10 injected and water treated"; the model "(+) B16F10 injected and water treated"; the positive control "(+) B16F10 injected and DTIC treated (Dacarbazine, 70 mg/kg/day)"; the SQ-diEG treatment "(+) B16F10 injected and SQ-diEG treated (5 mg/kg/day)"; the SQ-diEG treatment "(+) B16F10 injected and SQ-diEG treated (25 mg/kg/day)". After one week of acclimatization, B16F10 were murine melanoma cells line which intravenously injected (200000 tumor cells in 100 μl PBS, 0.2 ml/mouse) through the tail vein of mice. Mice was orally administrated with Dacarbazine (DTIC, positive control, 70mg/kg), and SQ-diEG (5mg/kg and 25mg/kg) for 20 days. On the last day of administration, mice was sacrificed and its lung was washed and collect for the further RNA extraction. Finally, the mice microarray gene expression profiling was conducted for two biological replicates.
2024-09-18 | GSE263339 | GEO
Project description:RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.
2022-04-14 | PXD031972 | Pride
Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).
2010-06-19 | E-GEOD-19841 | biostudies-arrayexpress