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EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association

ABSTRACT: The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by posttranslational modifications, we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress, EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE115643 | GEO | 2018/06/12

REPOSITORIES: GEO

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EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association

Project description:EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association

| PRJNA475639 | ENA

EVI1-phosphorylation at S436 regulates interactions with CtBP1 and DNMT3A and promotes self-renewal

Project description:The transcriptional regulator EVI1 has an essential role in early development and haematopoiesis. However, acute myeloid leukaemia (AML) driven by aberrantly high EVI1 expression has very poor prognosis. To investigate effects of posttranslational modifications on EVI1 function, we carried out a mass spectrometry (MS) analysis of EVI1 in AML and detected dynamic phosphorylation at serine 436 (S436). EVI1-WT (wild type), with S436 available for phosphorylation, but not EVI1-S436A, conferred haematopoietic progenitor cell self-renewal associated with significantly higher organised transcriptional patterns. In silico modelling of EVI1-S436 phosphorylation showed affinity reduction to CtBP1, and EVI1-WT showed reduced interaction with CtBP1 compared with EVI1-S463A. The target the motif harbouring S436 is targeted by CDK2 and CDK3 kinases, which both interacted with EVI1-WT. The methyltransferase DNMT3A bound preferentially to EVI1-WT compared to EVI1-S436A, and a hypo-methylated cell population associated with EVI1-WT expression is not maintained with EVI1-S436A. These data point to a role of the EVI1-S436 phosphorylation for directing functional protein interactions for haematopoietic self-renewal. Targeting EVI1-S436 phosphorylation may benefit therapy for EVI1-driven leukaemia.

2020-11-01 | GSE157315 | GEO

Deeply conserved gene-regulatory functions of carboxy-terminal domains in dictyostelid C-module-binding factors

Project description:We describe the gene expression pattern of a Dictyostelium discoideum mutant depleted in the C-module-binding factor (CbfA) and autonomous gene-regulatory activities of CbfA carboxy-terminal domains (CbfA-CTDs)

2012-06-03 | GSE38418 | GEO

Deeply conserved gene-regulatory functions of carboxy-terminal domains in dictyostelid C-module-binding factors

2012-06-03 | E-GEOD-38418 | biostudies-arrayexpress

Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress

Project description:The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. Our results highlight the intricate relationships between these PIKKs in regulating the DDR.

2020-11-25 | PXD020884 | Pride

Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress

Project description:The DNA damage response (DDR) is a complex signaling network that is robustly mobilized by double-strand breaks (DSBs) in the DNA. This network relies on extensive cascades of protein phosphorylation and dephosphorylation, which are initiated by three apical protein kinases that belong to the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR and DNA-PK. Loss of ATM leads to a prototypic genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the DSB-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique selected reaction monitoring (SRM). We uncovered redundant as well as non-redundant roles of the PIKKs following genotoxic stress and obtained evidence of a DNA-PK-dependent attenuation of the ATM response. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics to each of them. Our results shed light on the intricate relationships between the three PIKKs in regulating the DDR.

2020-11-25 | PXD015455 | Pride

Concerted roles of PTEN and ATM in controlling hematopoietic stem cell fitness and dormancy

Project description:In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal and differentiation. Ablation of the kinase ATM, a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor PTEN, which also regulates HSC function. We generated and analyzed a knock-in mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/-, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSC.

2021-01-11 | GSE164388 | GEO

Expression profiling of rpb1-12XWTCTD and rpb1-12XS2ACTD fission yeast strains.

Project description:In fission yeast, the nuclear-localized Lsk1p-Lsc1p-Lsg1p cyclin dependent kinase complex is required for the reliable execution of cytokinesis and is also required for Ser-2 phosphorylation RNA pol II carboxy terminal domain. To address whether alterations in CTD phosphorylation might selectively alter expression of cytokinesis genes, expression profiling of site-directed CTD mutants was performed.

2011-09-01 | GSE31369 | GEO

Tyrosine kinase c-Abl couples RNA polymerase II transcription to an RNA-dependent DNA damage response

Project description:Various modes of DNA repair counteract genotoxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilises damage-induced small RNA for efficient repair of DSBs. However, production and processing of RNA is poorly understood. Here we show that localised induction of DSBs triggers phosphorylation of RNA polymerase II (RNAPII) on carboxy-terminal domain (CTD) residue tyrosine-1 in an Mre11-Rad50-Nbs1 (MRN) complex-dependent manner. CTD Tyr1-phosphorylated RNAPII synthetises, strand-specific, damage-responsive transcripts (DARTs). DART synthesis occurs via formation of transient RNA-DNA hybrid (R-loop) intermediates. Impaired R-loop formation attenuates DART synthesis, impairs recruitment of repair factors and delays the DDR. Collectively, we provide mechanistic insight in RNA-dependent DSB repair.

2019-02-04 | GSE96825 | GEO

Transcriptome of MEFs with BMP2 and DNA damage reagents

Project description:The tightly controlled BMP-Smad1 pathway is essential for embryonic development and postnatal tissue homeostasis. Dysfunction of BMP-Smad1 signaling also leads to tumor development such as juvenile polyposis and Cowden syndromes and various tumors in mouse models, with unknown pathological mechanisms. Here we establish a link between the BMP-Smad pathway and the prominent tumor suppressor Atm-p53 pathway. We identify activated nuclear Smad1 as an Atm substrate under genotoxic stress. Atm-mediated Smad1 S239 phosphorylation disrupts Smad1 interaction with protein phosphatase PPM1A and enhances Smad1 activation and up-regulation, which not only turns on target genes including Cdk1nc but also interacts with p53 and inhibits Mdm2-mediated p53 ubiquitination, leading to p53 stabilization. Functionally, Smad1 acts like a tumor suppressor in DNA damage response, cell transformation and tumorigenesis in a p53-dependent manner. Sequencing of the gastric cancer samples revealed that Smad1 is frequently mutated, with S239 as mutational hotspot. This study thus establishes the BMP-Smad1 pathway as an integral part of DNA damage response, which can suppresses tumorigenesis via p53. Transformed MEFs (Smad1f/f, Smad1f/f Cre) treated with 50ng/ml BMP2, 1 ug/ml doxorubincin, or both for different periods of time. The mRNA levels of all genes were compared using microarray analysis.

2012-12-31 | E-GEOD-36843 | biostudies-arrayexpress

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