FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function

Tang, Mengfan; Feng, Xu; Pei, Guangsheng; Srivastava, Mrinal; Wang, Chao; Chen, Zhen; Li, Siting; Zhang, Huimin; Zhao, Zhongming; Xu, Li; Chen, Junjie

doi:10.1016/j.celrep.2020.108018

Cited by 16 publications

(21 citation statements)

References 54 publications

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“…FOXK1, a member of the Forkhead Box K transcription factor family, is implicated in a variety of biologic processes relevant to UF pathogenesis. For example, FOXK1 promotes muscle progenitor cell proliferation and represses myogenic differentiation (31,32), activates the Wnt/b-catenin pathway to promote cell growth (62), regulates metabolism to favor aerobic glycolysis (61), and interacts with TP53BP1 (identified in this study as a Mediator kinase substrate) to dictate the choice of DNA repair pathway following DNA double-strand breaks (70). The role of FOXK1 in the regulation of both Wnt/b-catenin signaling and myogenic cell fate is particularly notable.…”

Section: Discussionmentioning

confidence: 93%

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

et al. 2021

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Objective: To identify, in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediatorassociated cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12). Design: Experimental study. Setting: Academic research laboratory. Patient(s): Women undergoing hysterectomy for uterine fibroids. Intervention(s): Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells. Main Outcome Measure(s): High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an empirical Bayes analysis and validated orthogonally by in vitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase-regulated transcripts identified by RNA sequencing were linked to Mediator kinase substrates by computational analyses. Result(s): A total of 296 unique phosphosites in 166 proteins were significantly decreased (R twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells. Conclusion(s): These studies identify a new catalog of pathologically and biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12 mutation-positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase

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Section: Discussionmentioning

confidence: 93%

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

et al. 2021

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“…CRISPR–Cas9 technology was used to knock in SFB tag to the N terminus of endogenous RIF1 as previously reported ( 72 ). 293T cells were cotransfected with RIF1 knock-in sgRNA and a donor vector containing puromycin resistance selection gene, P2A self-cleavage site, and SFB sequence, flanked by approximately 1 kb of homology arms in PUC19 backbone.…”

Section: Methodsmentioning

confidence: 99%

“…293T RIF1 SFB knock-in cells and 293T cells with overexpression of N-terminal SFB-tagged ASF1A or ASF1B were collected for TAP. For the analysis of endogenous RIF1-associated proteins, cells were first lysed in NETN (250 mM NaCl, 1 mM EDTA, 20 mM Tris–HCl [pH 8.0], 0.5% NP-40) buffer and separated into soluble and chromatin fraction as previously described ( 72 ). For the analysis of ASF1A- and ASF1B-associated proteins, cells were lysed in NETN buffer with turbonuclease for 1 h and centrifuged at 13,000 rpm for 15 min at 4 °C to get the whole cell lysates.…”

Section: Methodsmentioning

confidence: 99%

Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells

Tang¹,

Chen²,

Wang

et al. 2022

Journal of Biological Chemistry

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“…DSB repair efficiency by HR was measured in U2OS cells stably expressing HR reporter DR‐GFP reporter as previous described ( 34 ). Briefly, CCNC knocking down U2OS DR-GFP cells were generated by infection with CCNC g1 Lenti-virus.…”

Section: Methodsmentioning

confidence: 99%

Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

Tang

Pei

et al. 2021

Nucleic Acids Research

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Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.

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FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function

Cited by 16 publications

References 54 publications

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells

Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

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