Publications by authors named "Kyungjae Myung"

97 Publications

SOCS3 is Related to Cell Proliferation in Neuronal Tissue: An Integrated Analysis of Bioinformatics and Experiments.

Front Genet 2021 27;12:743786. Epub 2021 Sep 27.

Department of Biomedical Informatics, School of Medicine, Pusan National University, Busan, South Korea.

Glioma is the most common primary malignant tumor that occurs in the central nervous system. Gliomas are subdivided according to a combination of microscopic morphological, molecular, and genetic factors. Glioblastoma (GBM) is the most aggressive malignant tumor; however, efficient therapies or specific target molecules for GBM have not been developed. We accessed RNA-seq and clinical data from The Cancer Genome Atlas, the Chinese Glioma Genome Atlas, and the GSE16011 dataset, and identified differentially expressed genes (DEGs) that were common to both GBM and lower-grade glioma (LGG) in three independent cohorts. The biological functions of common DEGs were examined using NetworkAnalyst. To evaluate the prognostic performance of common DEGs, we performed Kaplan-Meier and Cox regression analyses. We investigated the function of SOCS3 in the central nervous system using three GBM cell lines as well as zebrafish embryos. There were 168 upregulated genes and 50 downregulated genes that were commom to both GBM and LGG. Through survival analyses, we found that SOCS3 was the only prognostic gene in all cohorts. Inhibition of SOCS3 using siRNA decreased the proliferation of GBM cell lines. We also found that the zebrafish ortholog, socs3b, was associated with brain development through the regulation of cell proliferation in neuronal tissue. While additional mechanistic studies are necessary, our results suggest that SOCS3 is an important biomarker for glioma and that SOCS3 is related to the proliferation of neuronal tissue.
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http://dx.doi.org/10.3389/fgene.2021.743786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8502821PMC
September 2021

Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.

DNA Repair (Amst) 2021 Nov 8;107:103173. Epub 2021 Jul 8.

School of Life Sciences, Ulsan National Institute for Science and Technology (UNIST), Ulsan, 44919, Republic of Korea; Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea; Department of Biomedical Engineering, Ulsan National Institute for Science and Technology (UNIST), Ulsan, 44919, Republic of Korea. Electronic address:

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.
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http://dx.doi.org/10.1016/j.dnarep.2021.103173DOI Listing
November 2021

NSMF promotes the replication stress-induced DNA damage response for genome maintenance.

Nucleic Acids Res 2021 06;49(10):5605-5622

Department of Life Sciences, Ulsan National University of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.
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http://dx.doi.org/10.1093/nar/gkab311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191778PMC
June 2021

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

Nucleic Acids Res 2021 01;49(1):269-284

Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.
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http://dx.doi.org/10.1093/nar/gkaa1162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797050PMC
January 2021

O-GlcNAcylation regulates dopamine neuron function, survival and degeneration in Parkinson disease.

Brain 2020 12;143(12):3699-3716

School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

The dopamine system in the midbrain is essential for volitional movement, action selection, and reward-related learning. Despite its versatile roles, it contains only a small set of neurons in the brainstem. These dopamine neurons are especially susceptible to Parkinson's disease and prematurely degenerate in the course of disease progression, while the discovery of new therapeutic interventions has been disappointingly unsuccessful. Here, we show that O-GlcNAcylation, an essential post-translational modification in various types of cells, is critical for the physiological function and survival of dopamine neurons. Bidirectional modulation of O-GlcNAcylation importantly regulates dopamine neurons at the molecular, synaptic, cellular, and behavioural levels. Remarkably, genetic and pharmacological upregulation of O-GlcNAcylation mitigates neurodegeneration, synaptic impairments, and motor deficits in an animal model of Parkinson's disease. These findings provide insights into the functional importance of O-GlcNAcylation in the dopamine system, which may be utilized to protect dopamine neurons against Parkinson's disease pathology.
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http://dx.doi.org/10.1093/brain/awaa320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805798PMC
December 2020

The HLTF-PARP1 interaction in the progression and stability of damaged replication forks caused by methyl methanesulfonate.

Oncogenesis 2020 Dec 7;9(12):104. Epub 2020 Dec 7.

Department of Life Sciences, National Cheng Kung University, No.1 University Road, Tainan City, 701, Taiwan.

Human HLTF participates in the lesion-bypass mechanism through the fork reversal structure, known as template switching of post-replication repair. However, the mechanism by which HLTF promotes the replication progression and fork stability of damaged forks remains unclear. Here, we identify a novel protein-protein interaction between HLTF and PARP1. The depletion of HLTF and PARP1 increases chromosome breaks, further reduces the length of replication tracks, and concomitantly increases the number of stalled forks after methyl methanesulfonate treatment according to a DNA fiber analysis. The progression of replication also depends on BARD1 in the presence of MMS treatment. By combining 5-ethynyl-2'-deoxyuridine with a proximity ligation assay, we revealed that the HLTF, PARP1, and BRCA1/BARD1/RAD51 proteins were initially recruited to damaged forks. However, prolonged stalling of damaged forks results in fork collapse. HLTF and PCNA dissociate from the collapsed forks, with increased accumulation of PARP1 and BRCA1/BARD1/RAD51 at the collapsed forks. Our results reveal that HLTF together with PARP1 and BARD1 participates in the stabilization of damaged forks, and the PARP1-BARD1 interaction is further involved in the repair of collapse forks.
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http://dx.doi.org/10.1038/s41389-020-00289-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719709PMC
December 2020

FRZB as a key molecule in abdominal aortic aneurysm progression affecting vascular integrity.

Biosci Rep 2021 01;41(1)

Department of Anatomy, School of Medicine, Pusan National University, Yangsan, Republic of Korea.

Abdominal aortic aneurysm (AAA), when ruptured, results in high mortality. The identification of molecular pathways involved in AAA progression is required to improve AAA prognosis. The aim of the present study was to assess the key genes for the progression of AAA and their functional role. Genomic and clinical data of three independent cohorts were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (GSE57691, GSE7084, and GSE98278). To develop AAA diagnosis and progression-related differentially expressed genes (DEGs), we used a significance analysis of microarray (SAM). Spearman correlation test and gene set analysis were performed to identify potential enriched pathways for DEGs. Only the Frizzled-related protein (FRZB) gene and chromosome 1 open reading frame 24 (C1orf24) exhibited significant down-regulation in all analyses. With FRZB, the pathways were associated with RHO GTPase and elastin fiber formation. With C1orf24, the pathways were elastic fiber formation, extracellular matrix organization, and cell-cell communication. Since only FRZB was evolutionally conserved in the vertebrates, function of FRZB was validated using zebrafish embryos. Knockdown of frzb remarkably reduced vascular integrity in zebrafish embryos. We believe that FRZB is a key gene involved in AAA initiation and progression affecting vascular integrity.
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http://dx.doi.org/10.1042/BSR20203204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789806PMC
January 2021

Haematopoietic stem cell-dependent Notch transcription is mediated by p53 through the Histone chaperone Supt16h.

Nat Cell Biol 2020 12 23;22(12):1411-1422. Epub 2020 Nov 23.

Department of Cellular and Molecular Medicine and Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.
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http://dx.doi.org/10.1038/s41556-020-00604-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092813PMC
December 2020

Exploring the Role of a Novel Peptide from Larvae in Ameliorating Lipid Metabolism in Obesity.

Int J Mol Sci 2020 Nov 12;21(22). Epub 2020 Nov 12.

Department of Food Science and Nutrition, Dong-A University, Busan 49315, Korea.

The aim of this study was to identify an anti-obesity peptide from and investigate the lipid metabolic mechanism. Enzymatically hydrolyzed larvae were further separated using tangential flow filtration and consecutive chromatographic processes. Finally, an anti-obesity peptide that showed the highest inhibitory effect on lipid accumulation was obtained, and the sequence was Glu-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu (EIA10). EIA10 decreased lipid aggregation in vitro and significantly reduced the accumulation of body weight gain, liver weight, and adipose tissue weight in high-fat-fed mice. Compared with the control group, the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL), insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) in the high-fat diet (HFD) group increased significantly, and the content of high-density lipoprotein cholesterol (HDL) in the serum decreased significantly. On the contrary, the levels of TC, TG, and insulin in the EIA10 group decreased significantly, and the HDL content increased significantly compared with the HFD group. Additionally, EIA10 dramatically decreased mRNA and protein levels of transcription factors involved in lipid adipogenesis. Taken together, our results suggest that EIA10 could be a promising agent for the treatment and prevention of obesity.
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http://dx.doi.org/10.3390/ijms21228537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698306PMC
November 2020

AML poor prognosis factor, TPD52, is associated with the maintenance of haematopoietic stem cells through regulation of cell proliferation.

J Cell Biochem 2021 04 9;122(3-4):403-412. Epub 2020 Nov 9.

Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, Republic of Korea.

Acute myeloid leukaemia (AML) is a blood cancer where undifferentiated myeloid cells are increased in the bone marrow and peripheral blood. As AML is dangerous and shows poor prognosis, many researchers categorised the relevant cytogenetic factors according to risk and prognosis. However, the specific reasons for poor cytogenetic factors remain unknown. We analysed a large data set from AML patients and found that TPD52 expression is elevated in patient groups with poor cytogenetic factors. As the amino acid sequence of TPD52 is evolutionally conserved in vertebrates, zebrafish embryos were used to investigate the function of TPD52. Since myeloid-biased haematopoietic stem cells (HSCs) are relevant to AML, the function of TPD52 in the development of HSCs was investigated. We determined that the zebrafish paralog, tpd52, is important for the maintenance of HSCs through regulation of cell proliferation. As tpd52 is linked to cell proliferation in zebrafish embryos, the proliferation-related gene, CD59, was correlated to TPD52 in every AML cohort with a high correlation coefficient. We suggest that TPD52 can be a novel therapeutic target for AML patients with poor cytogenetic factors. Additionally, more studies between TPD52 and CD59 will further increase the value of TPD52 as a novel target.
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http://dx.doi.org/10.1002/jcb.29869DOI Listing
April 2021

Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin.

EMBO Rep 2020 11 1;21(11):e48676. Epub 2020 Oct 1.

Center for Genomic Integrity, Institute for Basic Science, Ulsan, Korea.

Poly(ADP-ribose) polymerase 1 (PARP1) facilitates DNA damage response (DDR). While the Ewing's sarcoma breakpoint region 1 (EWS) protein fused to FLI1 triggers sarcoma formation, the physiological function of EWS is largely unknown. Here, we investigate the physiological role of EWS in regulating PARP1. We show that EWS is required for PARP1 dissociation from damaged DNA. Abnormal PARP1 accumulation caused by EWS inactivation leads to excessive Poly(ADP-Ribosy)lation (PARylation) and triggers cell death in both in vitro and in vivo models. Consistent with previous work, the arginine-glycine-glycine (RGG) domain of EWS is essential for PAR chain interaction and PARP1 dissociation from damaged DNA. Ews and Parp1 double mutant mice do not show improved survival, but supplementation with nicotinamide mononucleotides extends Ews-mutant pups' survival, which might be due to compensatory activation of other PARP proteins. Consistently, PARP1 accumulates on chromatin in Ewing's sarcoma cells expressing an EWS fusion protein that cannot interact with PARP1, and tissues derived from Ewing's sarcoma patients show increased PARylation. Taken together, our data reveal that EWS is important for removing PARP1 from damaged chromatin.
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http://dx.doi.org/10.15252/embr.201948676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645264PMC
November 2020

Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore.

Genome Res 2020 09 4;30(9):1306-1316. Epub 2020 Sep 4.

School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

The higher-order structural organization and dynamics of the chromosomes play a central role in gene regulation. To explore this structure-function relationship, it is necessary to directly visualize genomic elements in living cells. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. This feature enables the application of CRISPR-based imaging to loci throughout the genome and opens up new possibilities for the study of nuclear processes in living cells.
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http://dx.doi.org/10.1101/gr.260018.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545142PMC
September 2020

ATAD5 suppresses centrosome over-duplication by regulating UAF1 and ID1.

Cell Cycle 2020 08 28;19(15):1952-1968. Epub 2020 Jun 28.

Center for Genomic Integrity, Institute for Basic Science , Ulsan, Korea.

Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.
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http://dx.doi.org/10.1080/15384101.2020.1785724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469630PMC
August 2020

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork.

Nucleic Acids Res 2020 07;48(13):7218-7238

Center for Genomic Integrity, Institute for Basic Science, Ulsan, Korea.

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.
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http://dx.doi.org/10.1093/nar/gkaa501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367208PMC
July 2020

Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca homeostasis.

Exp Mol Med 2020 06 5;52(6):940-950. Epub 2020 Jun 5.

Department of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 689-798, Korea.

The endoplasmic reticulum (ER) stress response is an adaptive mechanism that is activated upon disruption of ER homeostasis and protects the cells against certain harmful environmental stimuli. However, critical and prolonged cell stress triggers cell death. In this study, we demonstrate that Flightless-1 (FliI) regulates ER stress-induced apoptosis in colon cancer cells by modulating Ca homeostasis. FliI was highly expressed in both colon cell lines and colorectal cancer mouse models. In a mouse xenograft model using CT26 mouse colorectal cancer cells, tumor formation was slowed due to elevated levels of apoptosis in FliI-knockdown (FliI-KD) cells. FliI-KD cells treated with ER stress inducers, thapsigargin (TG), and tunicamycin exhibited activation of the unfolded protein response (UPR) and induction of UPR-related gene expression, which eventually triggered apoptosis. FliI-KD increased the intracellular Ca concentration, and this upregulation was caused by accelerated ER-to-cytosolic efflux of Ca. The increase in intracellular Ca concentration was significantly blocked by dantrolene and tetracaine, inhibitors of ryanodine receptors (RyRs). Dantrolene inhibited TG-induced ER stress and decreased the rate of apoptosis in FliI-KD CT26 cells. Finally, we found that knockdown of FliI decreased the levels of sorcin and ER Ca and that TG-induced ER stress was recovered by overexpression of sorcin in FliI-KD cells. Taken together, these results suggest that FliI regulates sorcin expression, which modulates Ca homeostasis in the ER through RyRs. Our findings reveal a novel mechanism by which FliI influences Ca homeostasis and cell survival during ER stress.
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http://dx.doi.org/10.1038/s12276-020-0448-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338537PMC
June 2020

Role of , A Gene Related to ALL Relapse, in Embryonic Hematopoiesis in Zebrafish.

Int J Mol Sci 2020 Apr 28;21(9). Epub 2020 Apr 28.

Department of Anatomy, School of Medicine, Pusan National University, Yangsan 50612, Korea.

Relapse of acute lymphoblastic leukemia (ALL) is dangerous and it worsens the prognosis of patients; however, prognostic markers or therapeutic targets for ALL remain unknown. In the present study, using databases such as TARGET, GSE60926 and GSE28460, we determined that and its binding partner, are overexpressed in patients with relapsed ALL compared to that in patients diagnosed with ALL for the first time. As 50% of the residues are exactly the same and the signature domain of is highly conserved between human and zebrafish, we used zebrafish embryos as a model to investigate the function of in vivo. We determined that is necessary for lymphopoiesis in zebrafish embryos. Additionally, we observed that is not related to differentiation of HSCs; however, it is important for the maintenance of HSCs as it provides survival signals to HSCs. These results imply that the ALL relapse-related gene is linked to the survival of HSCs. In conclusion, we suggest that can serve as a novel therapeutic target for relapsed ALL.
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http://dx.doi.org/10.3390/ijms21093127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246619PMC
April 2020

FAM213A is linked to prognostic significance in acute myeloid leukemia through regulation of oxidative stress and myelopoiesis.

Hematol Oncol 2020 Aug 18;38(3):381-389. Epub 2020 Mar 18.

Department of Anatomy, School of Medicine, Pusan National University, Yangsan, Republic of Korea.

Accurate prediction of malignancies is important in choosing therapeutic strategies. Although there are many genetic and cytogenetic prognostic factors for acute myeloid leukemia (AML), prognosis is difficult to predict because of the heterogeneity of AML. Prognostic factors, including messenger RNA (mRNA) expression, have been determined for other malignancies, but not for AML. A total of 402 patients from The Cancer Genome Atlas, GSE12417 (GPL96, 97), and GSE12417 (GPL570) were included in this study. In Kaplan-Meier curve analyses, high expression of family with sequence similarity 213 member A (FAM213A), which activates antioxidant proteins, was associated with worse prognosis of AML. Similar to the results of the survival curve, C-indices and area under the curve values were high. Current prognostic factors of AML, unlike those of other cancers, do not take mRNA expression into consideration. Thus, the development of mRNA-based prognostic factors would be beneficial for accurate prediction of the survival of AML patients. Additionally, in vivo validation using zebrafish revealed that fam213a is important for myelopoiesis at the developmental stage and is a negative regulator of the p53 tumor suppressor gene. The findings implicate fam213a as a novel prognostic factor for AML patients.
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http://dx.doi.org/10.1002/hon.2728DOI Listing
August 2020

PCNA Unloading Is Negatively Regulated by BET Proteins.

Cell Rep 2019 12;29(13):4632-4645.e5

Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea. Electronic address:

Proliferating cell nuclear antigen (PCNA) is a DNA clamp essential for DNA replication. During DNA synthesis, PCNA is continuously loaded onto and unloaded from DNA. PCNA recruits various proteins to nascent DNA to facilitate chromosome duplication. Therefore, timely PCNA unloading is crucial for high-fidelity DNA replication. The ATAD5-RFC-like complex (ATAD5-RLC) unloads PCNA from replicated DNA. It is unclear how ATAD5-RLC activity is regulated to prevent premature PCNA unloading. Here, we find that BRD4, an acetyl-histone-binding chromatin reader, inhibits the PCNA-unloading activity of ATAD5-RLC. The BRD4 ET domain interacts with a region upstream of the ATAD5 PCNA-unloading domain. BRD4-ATAD5 binds to acetyl-histones in nascent chromatin. BRD4 release from chromatin correlates with PCNA unloading. Disruption of the interaction between BRD4 and acetyl-histones or between BRD4 and ATAD5 reduces the PCNA amount on chromatin. In contrast, the overexpression of BRD4 increases the amount of chromatin-bound PCNA. Thus, acetyl-histone-bound BRD4 fine-tunes PCNA unloading from nascent DNA.
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http://dx.doi.org/10.1016/j.celrep.2019.11.114DOI Listing
December 2019

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Nat Commun 2019 12 16;10(1):5718. Epub 2019 Dec 16.

Center for Genomic Integrity, Institute for Basic Science, Ulsan, Korea.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.
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http://dx.doi.org/10.1038/s41467-019-13667-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914801PMC
December 2019

Simple Method To Characterize the Ciliary Proteome of Multiciliated Cells.

J Proteome Res 2020 01 18;19(1):391-400. Epub 2019 Nov 18.

Center for Genomic Integrity , Institute for Basic Science , Ulsan 44919 , Republic of Korea.

Motile cilia of multiciliated epithelial cells have important roles in animal development and cell homeostasis. Although several studies have identified and reported proteins localized in this complex organelle and the related immotile primary cilia from various cell types, it is still challenging to isolate high quantities of ciliary proteins for proteomic analysis. In this study, African clawed frog () embryos, which have many multiciliated cells in the epidermis, were treated with a simple ionic buffer to identify 1009 proteins conserved across vertebrates; these proteins were putatively localized in motile cilia. Using two ciliary proteome databases, we confirmed that previously validated cilia-associated proteins are highly enriched in our ciliary proteome. Proteins localized at the transition zone and Ellis-van Creveld zone, which are distinct regions at the base of cilia, near the junction with the apical cell surface, were isolated using our method. Among the newly identified ciliary proteins, we report that KRT17 may have an unrecognized function in motile cilia. Hence, the method developed in this study would be useful for understanding the ciliary proteome.
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http://dx.doi.org/10.1021/acs.jproteome.9b00589DOI Listing
January 2020

TonEBP Regulates PCNA Polyubiquitination in Response to DNA Damage through Interaction with SHPRH and USP1.

iScience 2019 Sep 19;19:177-190. Epub 2019 Jul 19.

School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea. Electronic address:

Polyubiquitination of proliferating cell nuclear antigen (PCNA) regulates the error-free template-switching mechanism for the bypass of DNA lesions during DNA replication. PCNA polyubiquitination is critical for the maintenance of genomic integrity; however, the underlying mechanism is poorly understood. Here, we demonstrate that tonicity-responsive enhancer-binding protein (TonEBP) regulates PCNA polyubiquitination in response to DNA damage. TonEBP was recruited to DNA damage sites with bulky adducts and sequentially recruited E3 ubiquitin ligase SHPRH, followed by deubiquitinase USP1, to DNA damage sites, in correlation with the dynamics of PCNA polyubiquitination. Similarly, TonEBP was found to be required for replication fork protection in response to DNA damage. The Rel-homology domain of TonEBP, which encircles DNA, was essential for the interaction with SHPRH and USP1, PCNA polyubiquitination, and cell survival after DNA damage. The present findings suggest that TonEBP is an upstream regulator of PCNA polyubiquitination and of the DNA damage bypass pathway.
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http://dx.doi.org/10.1016/j.isci.2019.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677787PMC
September 2019

CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks.

Nucleic Acids Res 2019 09;47(17):9160-9179

Department of Life Sciences and Cellulomics Institute Ajou University, Suwon 16499, Korea.

The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11-CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.
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http://dx.doi.org/10.1093/nar/gkz639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753481PMC
September 2019

Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes.

Nat Commun 2019 06 3;10(1):2420. Epub 2019 Jun 3.

Center for Genomic Integrity, Institute for Basic Science, Ulsan, 44919, Republic of Korea.

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.
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http://dx.doi.org/10.1038/s41467-019-10376-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546911PMC
June 2019

The structure of human EXD2 reveals a chimeric 3' to 5' exonuclease domain that discriminates substrates via metal coordination.

Nucleic Acids Res 2019 07;47(13):7078-7093

Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, 50 UNIST-gil, Ulsan 44919, Republic of Korea.

EXD2 (3'-5' exonuclease domain-containing protein 2) is an essential protein with a conserved DEDDy superfamily 3'-5' exonuclease domain. Recent research suggests that EXD2 has two potential functions: as a component of the DNA double-strand break repair machinery and as a ribonuclease for the regulation of mitochondrial translation. Herein, electron microscope imaging analysis and proximity labeling revealed that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. Crystal structures of the exonuclease domain in complex with Mn2+/Mg2+ revealed a domain-swapped dimer in which the central α5-α7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Combined structural and biochemical analyses demonstrated that the unusual dimeric organization stabilizes the active site, facilitates discrimination between DNA and RNA substrates based on divalent cation coordination and generates a positively charged groove that binds substrates.
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http://dx.doi.org/10.1093/nar/gkz454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648332PMC
July 2019

Chemoselective Trifluoroethylation Reactions of Quinazolinones and Identification of Photostability.

J Org Chem 2019 Jun 13;84(11):6737-6751. Epub 2019 May 13.

School of Energy and Chemical Engineering , Ulsan National Institute of Science and Technology (UNIST) , 50 UNIST-gil , Ulsan 44919 , Republic of Korea.

Herein, we report chemoselective trifluoroethylation routes of unmasked 2-arylquinazolin-4(3 H)-ones using mesityl(2,2,2-trifluoroethyl)iodonium triflate at room temperature. Homologous C-, O-, and N-functionalized subclasses are accessed in a straightforward manner with a wide substrate scope. These chemoselective branching events are driven by Pd-catalyzed ortho-selective C-H activation at the pendant aryl ring and base-promoted reactivity modulation of the amide group, leveraging the intrinsic directing capability and competing pronucleophilicity of the quinazolin-4(3 H)-one framework. Furthermore, outstanding photostability of the quinazolin-4(3 H)-one family associated with nonradiative decay is presented.
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http://dx.doi.org/10.1021/acs.joc.9b00470DOI Listing
June 2019

GCA links TRAF6-ULK1-dependent autophagy activation in resistant chronic myeloid leukemia.

Autophagy 2019 12 30;15(12):2076-2090. Epub 2019 Mar 30.

Leukemia Research Institute, The Catholic University of Korea, Seoul, Republic of Korea.

Imatinib is the first molecularly targeted compound for chronic myeloid leukemia (CML) capable to inhibit BCR-ABL kinase activity. However, recent clinical evidence indicates that a substantial proportion of CML patients exhibit BCR-ABL-dependent or independent resistance to imatinib. Despite the importance of imatinib resistance in CML, the underlying molecular mechanisms of this resistance are largely unknown. Here, we identified GCA (grancalcin) as a critical regulator of imatinib resistance in chronic phase CML via activation of autophagy. Mechanistically, we demonstrated that GCA activates TRAF6 ubiquitin ligase activity to induce Lys63 ubiquitination of ULK1, a crucial regulator of autophagy, resulting in its stabilization and activation. We also highlighted the role of GCA-TRAF6-ULK1 autophagy regulatory axis in imatinib resistance. Our findings represent the basis for novel therapeutic strategies against CML. ACTB/β-actin: actin beta; ADM: adrenomedullin; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ANXA5: annexin A5; CP: cytogenetic response; CML: chronic myeloid leukemia; CUL3: cullin 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCA: grancalcin; Dx: at diagnosis; E-64-d: (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; IMres: Imatinib resistance; KLHL20: Kelch-like protein 20; LRMP: lymphoid-restricted membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMR: major molecular response; NHCl: ammonium chloride; PBMCs: peripheral blood mononuclear cells; PTPRC: protein tyrosine phosphatase, receptor type, C; SQSTM1/p62: sequestosome 1; SYK: spleen associated tyrosine kinase; TAP1: transporter 1, ATP binding cassette subfamily B member; TKIs: ABL-specific tyrosine kinase inhibitors; TLR9: toll- like receptor 9; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1.
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http://dx.doi.org/10.1080/15548627.2019.1596492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844495PMC
December 2019

Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia.

Am J Hum Genet 2019 03 14;104(3):439-453. Epub 2019 Feb 14.

Department of Bio and Brain Engineering, Korean Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.
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http://dx.doi.org/10.1016/j.ajhg.2019.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407524PMC
March 2019

TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC.

Nat Commun 2018 10 19;9(1):4354. Epub 2018 Oct 19.

Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, 92093, USA.

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit.
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http://dx.doi.org/10.1038/s41467-018-06774-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195577PMC
October 2018

The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPARγ.

Exp Mol Med 2018 10 15;50(10):1-11. Epub 2018 Oct 15.

Department of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 689-798, Korea.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor that regulates adipocyte differentiation and glucose homeostasis. The transcriptional activity of PPARγ is regulated not only by ligands but also by post-translational modifications (PTMs). In this study, we demonstrate that a novel E3 ligase of PPARγ, tripartite motif-containing 25 (TRIM25), directly induced the ubiquitination of PPARγ, leading to its proteasome-dependent degradation. During adipocyte differentiation, both TRIM25 mRNA and protein expression significantly decreased and negatively correlated with the expression of PPARγ. The stable expression of TRIM25 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 cells. In contrast, the specific knockdown of TRIM25 increased PPARγ protein levels and stimulated adipocyte differentiation. Furthermore, TRIM25-knockout mouse embryonic fibroblasts (MEFs) exhibited an increased adipocyte differentiation capability compared with wild-type MEFs. Taken together, these data indicate that TRIM25 is a novel E3 ubiquitin ligase of PPARγ and that TRIM25 is a novel target for PPARγ-associated metabolic diseases.
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http://dx.doi.org/10.1038/s12276-018-0162-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189217PMC
October 2018

ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis.

Sci Transl Med 2018 10;10(462)

School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea.

Developing and mature chondrocytes constantly interact with and remodel the surrounding extracellular matrix (ECM). Recent research indicates that integrin-ECM interaction is differentially regulated during cartilage formation (chondrogenesis). Integrin signaling is also a key source of the catabolic reactions responsible for joint destruction in both rheumatoid arthritis and osteoarthritis. However, we do not understand how chondrocytes dynamically regulate integrin signaling in such an ECM-rich environment. Here, we found that developing chondrocytes express integrin-β-like 1 () at specific stages, inhibiting integrin signaling and promoting chondrogenesis. Unlike cytosolic integrin inhibitors, ITGBL1 is secreted and physically interacts with integrins to down-regulate activity. We observed that expression was strongly reduced in the damaged articular cartilage of patients with osteoarthritis (OA). Ectopic expression of protected joint cartilage against OA development in the destabilization of the medial meniscus-induced OA mouse model. Our results reveal ITGBL1 signaling as an underlying mechanism of protection against destructive cartilage disorders and suggest the potential therapeutic utility of targeting ITGBL1 to modulate integrin signaling in human disease.
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http://dx.doi.org/10.1126/scitranslmed.aam7486DOI Listing
October 2018
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