Gene expression profile of human ARPE-19 cells subjected to mitochondrial DNA double stranded breaks through expression of mitochondrial TALENs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE164979 | GEO | 2021/02/24
REPOSITORIES: GEO
Similar Datasets
Project description:Gene expression profile of human ARPE-19 cells subjected to mitochondrial DNA double stranded breaks through expression of mitochondrial TALENs
| PRJNA692724 | ENA
Project description:Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.
2023-05-05 | GSE222763 | GEO
Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.
2022-03-03 | E-MTAB-11497 | biostudies-arrayexpress
Project description:We set out to develop an unbiased, genome-wide method to screen for proteins that locate to the OCT4 proximal enhancer (PE) in hESCs; for this purpose, we developed an optimized locus-specific proteomics approach in hESCs. First, we designed TALEN (Transcription Activator-Like Effector Nuclease) constructs to target the sequences that are near the OCT4 PE, located in a region of DNaseI hypersensitivity. TALEN constructs with the highest cutting efficiency were chosen for locus-specific proteomics. We then made modifications to the original TALEN protein to transform it into a catalytically-dead TALE (dTALE) protein that is optimized for locus-specific proteomics in hESCs via three steps: 1) The nuclease-domain FokI at the C-terminus was replaced by a GFP (green fluorescence protein); 2) a 3X FLAG tag at the N-terminus was included for following pull-down analysis; 3) the existing CMV promoter was replaced with an EF1alpha promoter that has robust expression in hESCs. This dTALE protein could then be chemically crosslinked to the OCT4 locus together with all the other proteins that bind to the locus. We verified that dTALE protein binds to the targeted locus by chromatin immunoprecipitation (ChIP)-qPCR. Following crosslinking, chromatin was sheared, and all the associated proteins were immunoprecipitated using an anti-FLAG antibody. Immunoprecipitation pulled down the dTALE protein as well as other proteins and complexes that are also attached to that region. Crosslinking was then reversed, and the samples were subjected to mass spectrometry to enable generation of a list of proteins that potentially bind to OCT4 PE locus.
2019-01-07 | PXD010963 | Pride
Project description:Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts capable of forming long double-stranded RNA structures. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here, we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single cell level and identify keyroles for the degradosome components, mitochondrial dsRNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms evolved to protect vertebrates against microbial and viral attack.
2018-05-03 | GSE94957 | GEO
Project description:Mitochondrial DNA (mtDNA) breaks are deleterious lesions that lead to degradation of mitochondrial genomes and subsequent reduction in mtDNA copy number. The signaling pathways activated in response to mtDNA damage remain ill-defined. Using mitochondrial targeted restriction enzymes, we show that cells with mtDNA breaks exhibit reduced respiratory complexes, loss of membrane potential, and mitochondrial protein import defect. Furthermore, mtDNA damage activates the integrated stress response (ISR) through phosphorylation of eIF2α by the OMA1-DELE1-HRI pathway. Electron microscopy reveals concomitant defects in mitochondrial membranes and cristae ultrastructure. Notably, inhibition of the ISR exacerbates mitochondrial defects and delays the recovery of mtDNA copy number, thereby implicating this stress response in mitigating mitochondrial dysfunction following mtDNA damage. Last, we provide evidence suggesting that ATAD3A, a membrane-anchored protein that interacts with nucleoids, relays the signal from mtDNA breaks to the inner mitochondrial membrane. Altogether, our study fully delineates the sequence of events linking damaged mitochondrial genomes with the cytoplasm and uncovers an unanticipated role for the ISR in response to mitochondrial genome instability.
2022-10-04 | GSE214512 | GEO
Project description:Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Second, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.
2019-08-09 | GSE135548 | GEO
Project description:Single Centre, open label assignment phase II clinical study.
To evaluate the effect of oral 200mg Methylene Blue tablets (administered 8x25mg) prior to endoscopy on double stranded DNA breaks in colonic biopsy samples assessed by histone gamma H2AX analysis, compared to control biopsies.
| 2178654 | ecrin-mdr-crc
Project description:In this study on human Tousled-Like-Kinase 2 (TLK2) mass spectrometry-based analysis was applied as part of a molecular characterization of TLK2 in the aim to increase the understanding of the mode of activation of this protein. Specifically, different TLK2 constructs were analyzed to generate a “phosphorylation map” of the TLK2 protein. To analyse the phosphorylation state of TLK2 we used recombinant protein purified from Escherichia coli. Samples of different TLK2 constructs were used for in-gel digestion and analysed by mass spectrometry. All samples were prepared and run in triplicates for label-free quantification. Explanation for MS raw files with TLK2 construct included: Ac: TLK2 construct consisting of amino acid residues 191-772 (Ac indicates active form). KD: TLK2 construct consisting of amino acid residues 191-772 with mutation D613A (KD indicates kinase-dead form). FusionKD: Kinase-dead part of a heterodimeric TLK2 construct (amino acid residues 191-772) with mutation D613A (KD indicates kinase-dead form). FusionAC: Active part of a heterodimeric TLK2 construct (amino acid residues 191-772) (Ac indicates kinase-dead form). KDlong: Kinase domain of TLK2 with C-tail (amino acid residues 450-772). KDshort: Kinase domain of TLK2 without C-tail (amino acid residues 450-753).
2018-10-23 | PXD007675 | Pride
Project description:DallePazze2014 - Cellular senescene-induced
mitochondrial dysfunction
This model is described in the article:
Dynamic modelling of
pathways to cellular senescence reveals strategies for targeted
interventions.
Dalle Pezze P, Nelson G, Otten EG,
Korolchuk VI, Kirkwood TB, von Zglinicki T, Shanley DP.
PLoS Comput. Biol. 2014 Aug; 10(8):
e1003728
Abstract:
Cellular senescence, a state of irreversible cell cycle
arrest, is thought to help protect an organism from cancer, yet
also contributes to ageing. The changes which occur in
senescence are controlled by networks of multiple signalling
and feedback pathways at the cellular level, and the interplay
between these is difficult to predict and understand. To
unravel the intrinsic challenges of understanding such a highly
networked system, we have taken a systems biology approach to
cellular senescence. We report a detailed analysis of
senescence signalling via DNA damage, insulin-TOR, FoxO3a
transcription factors, oxidative stress response, mitochondrial
regulation and mitophagy. We show in silico and in vitro that
inhibition of reactive oxygen species can prevent loss of
mitochondrial membrane potential, whilst inhibition of mTOR
shows a partial rescue of mitochondrial mass changes during
establishment of senescence. Dual inhibition of ROS and mTOR in
vitro confirmed computational model predictions that it was
possible to further reduce senescence-induced mitochondrial
dysfunction and DNA double-strand breaks. However, these
interventions were unable to abrogate the senescence-induced
mitochondrial dysfunction completely, and we identified
decreased mitochondrial fission as the potential driving force
for increased mitochondrial mass via prevention of mitophagy.
Dynamic sensitivity analysis of the model showed the network
stabilised at a new late state of cellular senescence. This was
characterised by poor network sensitivity, high signalling
noise, low cellular energy, high inflammation and permanent
cell cycle arrest suggesting an unsatisfactory outcome for
treatments aiming to delay or reverse cellular senescence at
late time points. Combinatorial targeted interventions are
therefore possible for intervening in the cellular pathway to
senescence, but in the cases identified here, are only capable
of delaying senescence onset.
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2024-09-02 | BIOMD0000000582 | BioModels