bims-ectoca Biomed News
on Epigenetic control of tolerance in cancer
Issue of 2022‒06‒26
23 papers selected by
Ankita Daiya
BITS Pilani

  1. Mol Cell. 2022 Jun 10. pii: S1097-2765(22)00490-7. [Epub ahead of print]
      Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.
    Keywords:  CyTOF; DIPG; H3-K27M; cancer heterogeneity; chromatin; epigenetic heterogeneity; glioma; histone modifications; oncohistone; single-cell
  2. Biochim Biophys Acta Gene Regul Mech. 2022 Jun 21. pii: S1874-9399(22)00054-2. [Epub ahead of print] 194839
      Enhancer is one kind of cis-elements regulating gene transcription, whose activity is tightly controlled by epigenetic enzymes and histone modifications. Active enhancers are classified into typical enhancers, super-enhancers and over-active enhancers, according to the enrichment and location of histone modifications. Epigenetic factors control the level of histone modifications on enhancers to determine their activity, such as histone methyltransferases and acetylases. Transcription factors, cofactors and mediators co-operate together and are required for enhancer functions. In turn, abnormalities in these trans-acting factors affect enhancer activity. Recent studies have revealed enhancer dysregulation as one of the important features for cancer. Variations in enhancer regions and mutations of enhancer regulatory genes are frequently observed in cancer cells, and altering the activity of onco-enhancers is able to repress oncogene expression, and suppress tumorigenesis and metastasis. Here we summarize the recent discoveries about enhancer regulation in cancer and discuss their potential application in diagnosis and treatment.
    Keywords:  Enhancer; Histone modification; Transcription factor; Transcription regulation; cancer
  3. Methods Mol Biol. 2022 ;2529 441-473
      The maintenance of transcriptional states regulated by histone modifications and controlled switching between these states are fundamental concepts in our understanding of nucleosome-mediated epigenetic memory. Any approach relying on genome-wide bioinformatic analyses alone offers limited scope for dissecting the molecular mechanisms involved in maintenance and switching. Mechanistic mathematical models-describing the dynamics of histone modifications at individual genomic loci-offer an alternative way to investigate these mechanisms. These models, in conjunction with quantitative experimental data-ChIP data, quantification of mRNA levels, and single-cell fluorescence tracking in clonal lineages-can generate predictions that drive more targeted experiments, allowing us to understand mechanisms that would be challenging to unravel by a purely experimental approach. In this chapter, we describe a generic stochastic modeling framework that can be used to capture histone modification dynamics and associated molecular processes-including transcription and read-write feedback by chromatin modifying complexes-at individual genomic loci. Using a specific example-transcriptional silencing by Polycomb-mediated H3K27 methylation-we demonstrate how to construct and simulate a stochastic histone modification model. We provide a step-by-step guide to programming simulations for such a model and discuss how to analyze the simulation output.
    Keywords:  Bistability; Gene expression states; Gillespie algorithm; Maintenance and switching; Mechanistic modeling; Nucleosome-mediated epigenetic memory; Polycomb; Stochastic histone modification model
  4. Cancers (Basel). 2022 Jun 09. pii: 2855. [Epub ahead of print]14(12):
      Euchromatin histone lysine methyltransferases (EHMTs) are epigenetic regulators responsible for silencing gene transcription by catalyzing H3K9 dimethylation. Dysregulation of EHMT1/2 has been reported in multiple cancers and is associated with poor clinical outcomes. Although substantial insights have been gleaned into the downstream targets and pathways regulated by EHMT1/2, few studies have uncovered mechanisms responsible for their dysregulated expression. Moreover, EHMT1/2 interacting partners, which can influence their function and, therefore, the expression of target genes, have not been extensively explored. As none of the currently available EHMT inhibitors have made it past clinical trials, understanding upstream regulators and EHMT protein complexes may provide unique insights into novel therapeutic avenues in EHMT-overexpressing cancers. Here, we review our current understanding of the regulators and interacting partners of EHMTs. We also discuss available therapeutic drugs that target the upstream regulators and binding partners of EHMTs and could potentially modulate EHMT function in cancer progression.
    Keywords:  interactome; lysine methyltransferases; post-translational modifications; therapeutics; upstream regulators
  5. Methods Mol Biol. 2022 ;2529 63-88
      Dynamic histone methylation regulates gene activation and repression. It is involved in proliferation, differentiation, lineage specification, and development. Histone demethylase assays are invaluable in studying histone demethylation substrate recognition, kinetics, regulation, and inhibition by small molecules, many of which are potential therapeutics. Here we describe general procedures to purify recombinant enzymes from different expression hosts, and to prepare a broad range of substrates, as well as to set up a variety of in vitro histone demethylase assays. These assays provide useful tools for discoveries from enzymes to drugs.
    Keywords:  Chromatin; Demethylase assays; Gene transcription; Histone demethylases; Histone methylation; Histone modifications; Histones; Nucleosome
  6. Cell Mol Life Sci. 2022 Jun 25. 79(7): 382
      BACKGROUND: Osteosarcoma is one of the five leading causes of cancer death among all pediatric malignancies. Recent advances in non-coding RNAs suggested that many long noncoding RNAs (lncRNAs) are dysregulated in cancer tissues and play important roles in carcinogenesis. We aimed to further explore the mechanisms of Long Intergenic Non-Protein Coding RNA 313 (LINC00313)-promoted malignant phenotypes of osteosarcoma.METHODS: The mRNA expressions were determined by quantitative real-time PCR. Protein levels were detected using Western blotting or immunohistochemistry staining. Protein binding to genomic DNA and RNA were measured using chromatin and RNA immunoprecipitation assay, respectively. CCK-8 and EdU incorporation assay were adopted to detect cell proliferation. Transwell assay was employed to assess the capacity of cell migration and invasion. The roles of LINC00313 and its target genes in tumorigenesis and metastasis of osteosarcoma were evaluated using subcutaneous xenograft models and tail vein inoculation models.
    RESULTS: LINC00313 was elevated in osteosarcoma tissues compared with adjacent normal tissues. Higher LINC00313 was associated with advanced grades of osteosarcoma. LINC00313 promoted cell proliferation, migration, invasion in vitro and tumor growth as well as metastasis in vivo through inhibiting PTEN expression to promote AKT phosphorylation. Mechanistically, LINC00313 favored the interaction between FUS and EZH2, leading to the prolonged half-life of EZH2 mRNA, thereby in turn up-regulating EZH2 proteins and increasing EZH2-mediated epigenetic silence of PTEN.
    CONCLUSION: LINC00313 exerted oncogene-like actions through increasing EZH2 mRNA stability, leading to PTEN deficiency in osteosarcoma.
    Keywords:  EZH2; LINC00313; Metastasis; Osteosarcoma; PTEN/AKT signaling
  7. Front Cell Dev Biol. 2022 ;10 917662
      Cells are exposed and respond to various mechanical forces and physical cues stemming from their environment. This interaction has been seen to differentially regulate various cellular processes for maintenance of homeostasis, of which autophagy represents one of the major players. In addition, autophagy has been suggested to regulate mechanical functions of the cells including their interaction with the environment. In this minireview, we summarize the state of the art of the fascinating interplay between autophagy and the mechanotransduction machinery associated with cell adhesions, that we name ¨Mechanoautophagy¨.
    Keywords:  Autophagy; Extracellular Matrix; cadherin mediated adhesion; focal adhesion; mechanoautophagy
  8. Genes (Basel). 2022 Jun 02. pii: 1002. [Epub ahead of print]13(6):
      Chromatin organization is essential to maintain a correct regulation of gene expression and establish cell identity. However, during cell division, the replication of the genetic material produces a global disorganization of chromatin structure. In this paper, we describe the new scientific breakthroughs that have revealed the nature of the post-replicative chromatin and the mechanisms that facilitate its restoration. Moreover, we highlight the implications of these chromatin alterations in gene expression control and their impact on key biological processes, such as cell differentiation, cell reprogramming or human diseases linked to cell proliferation, such as cancer.
    Keywords:  cell identity; chromatin organization; chromatin replication; epigenetic maintenance; parental histone recycling; transcription regulation
  9. BMC Genom Data. 2022 Jun 21. 23(1): 47
      OBJECTIVES: Functional genetic variation plays an important role in predicting patients' response to chemotherapeutic agents. A growing catalogue of mitochondrial DNA (mtDNA) alterations in various cancers point to their important roles in altering the drug responsiveness and survival of cancer cells. In this work, we report the mtDNA sequences, obtained using a nanopore sequencer that can directly sequence unamplified DNA, and the transcriptomes of oral squamous cell carcinoma (OSCC) cell lines with differing responses to cisplatin, to explore the interplay between mtDNA alterations, epigenetic regulation of gene expression, and cisplatin response in OSCC.DATA DESCRIPTION: Two human OSCC cell lines, namely H103 and SAS, and drug-resistant stem-like cells derived from SAS were used in this work. To validate our hypothesis that cisplatin sensitivity is linked to mtDNA changes, we sequenced their mtDNA using a nanopore sequencer, MinION. We also obtained the whole transcriptomic profiles of the cells from a microarray analysis. The mtDNA mutational and whole transcriptomic profiles that we provide can be used alongside other similar datasets to facilitate the identification of new markers of cisplatin sensitivity, and therefore the development of effective therapies for OSCC.
    Keywords:  Cisplatin response; Gene expression; Human Clariom S array; Mitochondrial DNA; Oral squamous cell carcinoma; Oxford Nanopore Technologies
  10. Methods Mol Biol. 2022 ;2529 91-107
      In eukaryotic cells, chromatin plays an important role in gene regulation by controlling the access of the transcription machinery to DNA. In this chapter, we will describe methods for generating different chromatin templates to investigate the impact of histone variants and chromatin structure on histone methyltransferase activities. For this purpose, we take Polycomb Repressive Complex 2 (PRC2) as an example and investigate how its activity on H3K27me3 is regulated by the histone variants H3.3 and H2A.Z and higher-order chromatin structure.
    Keywords:  Chromatin structure; H2A.Z; H3.3; Histone variant; PRC2
  11. Front Cell Dev Biol. 2022 ;10 882698
      Different cancer types not only have common characteristics but also have their own characteristics respectively. The mechanism of these specific and common characteristics is still unclear. Pan-cancer analysis can help understand the similarities and differences among cancer types by systematically describing different patterns in cancers and identifying cancer-specific and cancer-common molecular biomarkers. While long non-coding RNAs (lncRNAs) are key cancer modulators, there is still a lack of pan-cancer analysis for lncRNA methylation dysregulation. In this study, we integrated lncRNA methylation, lncRNA expression and mRNA expression data to illuminate specific and common lncRNA methylation patterns in 23 cancer types. Then, we screened aberrantly methylated lncRNAs that negatively regulated lncRNA expression and mapped them to the ceRNA relationship for further validation. 29 lncRNAs were identified as diagnostic biomarkers for their corresponding cancer types, with lncRNA AC027601 was identified as a new KIRC-associated biomarker, and lncRNA ACTA2-AS1 was regarded as a carcinogenic factor of KIRP. Two lncRNAs HOXA-AS2 and AC007228 were identified as pan-cancer biomarkers. In general, the cancer-specific and cancer-common lncRNA biomarkers identified in this study may aid in cancer diagnosis and treatment.
    Keywords:  DNA methylation; biomarker; ceRNA; lncRNA; pan-cancer
  12. Front Oncol. 2022 ;12 913669
      Background: Despite the effectiveness of chemotherapy and targeted therapy for colorectal cancer, drug resistance drives therapy failure and tumor relapse. Increasing evidence has suggested that cancer cells can enter a reversible drug-tolerant persister state to survive chemotherapy or targeted agents. However, the traits and treatable vulnerabilities of anti-colorectal cancer drug-tolerant persister cells is not yet known.Methods: In this study, we established 5-fluorouracil and AZ628-tolerant persister cell models in two colorectal cancer cell lines, namely HCT116 and SW620, and revealed the characteristics of colorectal cancer persister cells by cell viability assay and flow cytometry. We investigated the efficacy and mechanism of ferroptosis inducers RSL3 and FIN56 on persister cells, which are glutathione peroxidase 4 inhibitors. In the xenograft mouse model, we further evaluated the inhibitory effect of RSL3 on tumor regrowth.
    Results: Colorectal cancer persister cells, which were enriched in the residual cancer cell population, exhibited reduced drug sensitivity, were largely quiescent and expressed high levels of stem cell-related genes and mesenchymal markers but not epithelial markers. The persister cells were more sensitive and underwent ferroptosis induced by glutathione peroxidase 4 inhibitors. Mechanistically, glutathione peroxidase 4 and ferrous iron, which are pivotal ferroptosis regulators, were upregulated in residual cells or tumors, and were hence potential therapeutic targets of persister cells. In the xenograft model, we confirmed that inhibition of glutathione peroxidase 4 restrained tumor regrowth after discontinuation of anti-cancer drug treatment. Moreover, biopsies obtained from patients with colorectal cancer undergoing neoadjuvant chemoradiotherapy revealed upregulated glutathione peroxidase 4 and ferritin heavy chain 1. High glutathione peroxidase 4 expression correlates with a worse prognosis in colorectal cancer patients.
    Conclusions: Our work reveals that the upregulated glutathione peroxidase 4 and ferrous iron in anti-colorectal cancer drug-tolerant persister cells were potential therapeutic targets. Glutathione peroxidase 4 inhibition combined with chemotherapy or targeted therapy may be a promising therapy for colorectal cancer.
    Keywords:  colorectal cancer; drug tolerance; ferroptosis; ferrous iron; glutathione peroxidase 4 (GPX4)
  13. Life (Basel). 2022 May 26. pii: 792. [Epub ahead of print]12(6):
      The Hippo-YAP pathway serves as a central signalling hub in epithelial tissue generation and homeostasis. Yes-associated protein (YAP) is an essential downstream transcription cofactor of this pathway, with its activity being negatively regulated by Hippo kinase-mediated phosphorylation, leading to its cytoplasmic translocation or degradation. Our recent study showed phospho-YAP complexes with Desmoglein-3 (Dsg3), the desmosomal cadherin known to be required for junction assembly and cell-cell adhesion. In this study, we show that YAP inhibition by Verteporfin (VP) caused a significant downregulation of desmosomal genes and a remarkable reduction in desmosomal proteins, including the Dsg3/phospho-YAP complex, resulting in attenuation of cell cohesion. We also found the desmosomal genes, along with E-cadherin, were the YAP-TEAD transcriptional targets and Dsg3 regulated key Hippo components, including WWTR1/TAZ, LATS2 and the key desmosomal molecules. Furthermore, Dsg3 and phospho-YAP exhibited coordinated regulation in response to varied cell densities and culture durations. Overexpression of Dsg3 could compensate for VP mediated loss of adhesion components and proper architecture of cell junctions. Thus, our findings suggest that Dsg3 plays a crucial role in the Hippo network and regulates junction configuration via complexing with phospho-YAP.
    Keywords:  YAP; desmoglein 3; desmosomes; junction formation; keratinocyte; verteporfin
  14. Curr Pharm Biotechnol. 2022 Jun 17.
      BACKGROUND: Dysregulated Yes-associated protein 1 (YAP1) is closely associated with cancer progression and chemo-resistance. We aim to explore the role of YAP1/Hippo pathway in regulating doxorubicin (ADM)-resistance in acute myeloid leukemia (AML).METHODS: In this study, we established two ADM-resistant cell lines (THP-1/ ADM and K562/ ADM). Then cell viability and apoptosis were detected by MTT assay and FCM assay, respectively. Real time PCR were performed to examine the expression of genes in the AML/ADM cells and the clinic BM samples. The levels of all related proteins were examined by Western blot.
    RESULTS: We found that the YAP1 and its downstream target genes, including EGFR, SOX2, and OCT4, were associated with ADM-resistance, evidenced by the increased expression in ADM-resistant AML/ADM cells and clinical BM specimens. Additionally, YAP1 ablation enhanced the promoting effects of ADM treatment on cell death in AML/ADM cells. Conversely, YAP1 increased ADM-a resistance in the original ADM-sensitive AML cells. These results may provide important new insights into understanding this role of YAP1 regulates AML resistance by affecting CSCs characteristics.
    CONCLUSION: In summary, we evidenced that the dysregulated YAP1/Hippo pathway influenced ADM-resistance in AML. YAP1 might be novel biomarkers for treatment of drug-resistance in AML.
    Keywords:  AML; Hippo signal pathway; Yes-associated protein 1; acute myeloid leukemia; cancer stem cells; drug resistance
  15. Cancer Lett. 2022 Jun 21. pii: S0304-3835(22)00288-9. [Epub ahead of print] 215804
      Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells.
    Keywords:  Chemoresistance; E2F; Intra S-Phase checkpoint; NRF2
  16. Methods Mol Biol. 2022 ;2529 229-251
      Chromatin immunoprecipitation (ChIP) enables the study of DNA-protein interactions. When coupled with high-throughput sequencing (ChIP-seq), this method allows the generation of genome-wide profiles of the distribution of specific proteins in a given cellular context. Typical ChIP-seq experiments require millions of cells as input material and thus are not ideal to study many in vivo cell populations. Here, we describe an ultra-low-input native ChIP-seq method, ULI-NChIP-seq, to profile histone modification patterns in as low as 150 cells.
    Keywords:  Chromatin; Chromatin immunoprecipitation; Embryo; Epigenetics; Histone modifications; Low-input; Methylation; Oocyte
  17. Methods Mol Biol. 2022 ;2529 267-293
      The field of genomics and genome-wide analysis has exploded since around 2008 with the development of high-throughput omics approaches, largely driven by the emergence of the next-generation sequencing technologies. Among the different biological applications supported by recent sequencing technologies, ChIP-seq (Chromatin ImmunoPrecipitation followed by Sequencing) is one of the most powerful techniques which has dramatically changed our view of the epigenetics landscape of cells.In this chapter, I will present and discuss the main steps of bioinformatic and biostatistical analysis of ChIP-seq data (Fig. 1). While this technique has been widely used to study transcription factor binding sites, I will focus here on the analysis of histone modifications.
    Keywords:  Bioinformatics; ChIP-seq; Differential binding; Peak calling; Quality controls
  18. Methods Mol Biol. 2022 ;2529 43-61
      Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).
    Keywords:  Chromatin; Histone methylation; Histone posttranslational modification; Nucleosomes; S-adenosylhomocysteine; S-adenosylmethionine; Transcription
  19. Cell Chem Biol. 2022 Jun 18. pii: S2451-9456(22)00198-2. [Epub ahead of print]
      Histone lysine methylation is an epigenetic mark that can control gene expression. In particular, H3K9me3 contributes to transcriptional repression by regulating chromatin structure. Successful mitotic progression requires correct timing of chromatin structure changes, including epigenetic marks. However, spatiotemporal information on histone modifications in living cells remains limited. In this study, we created an FRET-based probe for live-cell imaging based on the HP1α chromodomain (HP1αCD), which binds to H3K9me3. The probe was incorporated into chromatin and the emission ratio decreased after treatment with histone methyltransferase inhibitors, indicating that it successfully traced dynamic changes in H3K9me3. Upon entry into mitosis, the probe's emission ratio transiently increased with a concomitant increase in H3K9me3, then exhibited a stepwise decrease, probably due to loss of HP1αCD binding caused by phosphorylation of H3S10 and demethylation of H3K9me3. This probe will be a useful tool for detecting dynamic changes in chromatin structure associated with HP1α.
    Keywords:  FRET; H3K9me3; H3S10p; HP1α chromodomain; chromatin; live-cell imaging; mitosis
  20. Biochim Biophys Acta Rev Cancer. 2022 Jun 15. pii: S0304-419X(22)00074-9. [Epub ahead of print]1877(4): 188749
      Dormancy occurs when cells preserve viability but stop proliferating, which is considered an important cause of tumor relapse, which may occur many years after clinical remission. Since the life cycle of dormant cancer cells is affected by both intracellular and extracellular factors, gene mutation or epigenetic regulation of tumor cells may not fully explain the mechanisms involved. Recent studies have indicated that redox signaling regulates the formation, maintenance, and reactivation of dormant cancer cells by modulating intracellular signaling pathways and the extracellular environment, which provides a molecular explanation for the life cycle of dormant tumor cells. Indeed, redox signaling regulates the onset of dormancy by balancing the intrinsic pathways, the extrinsic environment, and the response to therapy. In addition, redox signaling sustains dormancy by managing stress homeostasis, maintaining stemness and immunogenic equilibrium. However, studies on dormancy reactivation are still limited, partly explained by redox-mediated activation of lipid metabolism and the transition from the tumor microenvironment to inflammation. Encouragingly, several drug combination strategies based on redox biology are currently under clinical evaluation. Continuing to gain an in-depth understanding of redox regulation and develop specific methods targeting redox modification holds the promise to accelerate the development of strategies to treat dormant tumors and benefit cancer patients.
    Keywords:  Cancer therapy; Dormancy; Drug resistance; ROS; Redox signaling
  21. APL Bioeng. 2022 Jun;6(2): 021504
      Two meters of DNA in each of our cells must be protected against many types of damage. Mechanoprotection is increasingly understood to be conferred by the nuclear lamina of intermediate filament proteins, but very different patterns of expression and regulation between different cells and tissues remain a challenge to comprehend and translate into applications. We begin with a tutorial style presentation of "tissue blueprints" of lamin expression including single-cell RNA sequencing in major public datasets. Lamin-A, C profiles appear strikingly similar to those for the mechanosensitive factors Vinculin, Yap1, and Piezo1, whereas datasets for lamin-B1 align with and predict regulation by the cell cycle transcription factor, FOXM1, and further predict poor survival across multiple cancers. Various experiments support the distinction between the lamin types and add mechanistic insight into the mechano-regulation of lamin-A, C by both matrix elasticity and externally imposed tissue strain. Both A- and B-type lamins, nonetheless, protect the nucleus from rupture and damage. Ultimately, for mechanically active tissue constructs and organoids as well as cell therapies, lamin levels require particular attention as they help minimize nuclear damage and defects in a cell cycle.
  22. Immunology. 2022 Jun 24.
      Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1-/- deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1-/- deletions eliminated G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a-GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1-/- deletions. G9a-GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a-GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a-GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents. This article is protected by copyright. All rights reserved.
    Keywords:  Sendai virus induced gene transcription; lysine methyltransferase recruitment; moderation of transcription; sentinel cell gene regulation; stability of chromatin binding; virus induced chromatin modification
  23. Neuro Oncol. 2022 Jun 20. pii: noac156. [Epub ahead of print]
      BACKGROUND: We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the non-receptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities.METHODS: Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms.
    RESULTS: The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK.
    CONCLUSIONS: We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.
    Keywords:  FRK; GBM; Siah1; YAP; ubiquitination