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1.
Oncogene ; 41(21): 2958-2972, 2022 05.
Article in English | MEDLINE | ID: mdl-35440714

ABSTRACT

The H3K4 demethylase KDM5B is overexpressed in multiple cancer types, and elevated expression levels of KDM5B is associated with decreased survival. However, the underlying mechanistic contribution of dysregulated expression of KDM5B and H3K4 demethylation in cancer is poorly understood. Our results show that loss of KDM5B in multiple types of cancer cells leads to increased proliferation and elevated expression of cancer stem cell markers. In addition, we observed enhanced tumor formation following KDM5B depletion in a subset of representative cancer cell lines. Our findings also support a role for KDM5B in regulating epigenetic plasticity, where loss of KDM5B in cancer cells with elevated KDM5B expression leads to alterations in activity of chromatin states, which facilitate activation or repression of alternative transcriptional programs. In addition, we define KDM5B-centric epigenetic and transcriptional patterns that support cancer cell plasticity, where KDM5B depleted cancer cells exhibit altered epigenetic and transcriptional profiles resembling a more primitive cellular state. This study also provides a resource for evaluating associations between alterations in epigenetic patterning upon depletion of KDM5B and gene expression in a diverse set of cancer cells.


Subject(s)
Jumonji Domain-Containing Histone Demethylases , Neoplasms , Cell Line, Tumor , Epigenesis, Genetic , Epigenomics , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Neoplasms/pathology , Nuclear Proteins/genetics , Repressor Proteins/genetics
2.
Cell ; 183(4): 1013-1023.e13, 2020 11 12.
Article in English | MEDLINE | ID: mdl-32970990

ABSTRACT

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , Antigen-Antibody Reactions , Binding Sites , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cricetinae , Cryoelectron Microscopy , Disease Models, Animal , Epitopes/chemistry , Epitopes/immunology , Female , Lung/pathology , Male , Molecular Dynamics Simulation , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protein Structure, Quaternary , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
3.
Cell ; 182(1): 73-84.e16, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32425270

ABSTRACT

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , B-Lymphocytes/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Single-Cell Analysis , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , COVID-19 , Convalescence , High-Throughput Nucleotide Sequencing , Humans , Mice , Pandemics , Sequence Analysis, RNA , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , VDJ Exons
4.
Hum Immunol ; 80(10): 848-854, 2019 Oct.
Article in English | MEDLINE | ID: mdl-30965079

ABSTRACT

Preeclampsia (PE) is one of the major causes of maternal and perinatal mortality worldwide. This study aimed to determine the immunological characteristics of PE patients and normal pregnancy at the T cell receptor beta-chain (TRB) level by using high-throughput sequencing. High-throughput sequencing was performed to analyze the expression of TRB-CDR3 in circulating T cells. T cells were isolated from 36 healthy pregnant women, 24 patients with severe PE, and 18 patients with moderate PE. Rearranged mRNA sequences were assigned to their germline V, D, and J counterparts, and translated into proper amino acids by the IMGT database. In general, PE samples had more TRB-CDR3 reads and types than those of normal pregnant woman in the circulation, but the mean number of TRB-CDR3 reads and unique TRB-CDR3 reads in severe group was lower than that in the moderate group. In PE patients, the V7_9 and V20_1 gene loci were more prevalent than in healthy pregnant women. In addition, 4 kinds of TRB-CDR3 peptides were found to be highly relevant to the pathogenesis of PE. Of them, peptides matched to herpes simplex virus antigen-specific T cells were much lower in PE samples.


Subject(s)
Complementarity Determining Regions/genetics , Genes, T-Cell Receptor beta/genetics , Genetic Variation/genetics , Pre-Eclampsia/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Adult , Amino Acid Sequence/genetics , Base Sequence/genetics , Female , Genetic Loci/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Multiplex Polymerase Chain Reaction/methods , Pre-Eclampsia/pathology , Pregnancy , T-Lymphocytes/immunology , Young Adult
5.
Sci Rep ; 8(1): 10775, 2018 Jul 17.
Article in English | MEDLINE | ID: mdl-30018329

ABSTRACT

Pluripotent stem cells within the inner cell mass and epiblast of mammalian embryos have the capacity to form all lineages in the adult organism, while multipotent trophoblast stem (TS) cells derived from the trophectoderm are capable of differentiating into fetal lineages of the placenta. While mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) exhibit distinct expression patterns and utilize distinct external signaling pathways for self-renewal, because mouse EpiSCs resemble human ES cells they are a useful model to investigate mechanisms of human ES cell self-renewal and differentiation. Recent studies have shown that haploid embryos and ES cells can be generated from chemically-activated unfertilized mouse oocytes. However, it is unclear whether EpiSCs or TS cells can be derived from haploid embryos. Here, we describe the derivation of EpiSCs from haploid blastocyst-stage embryos using culture conditions that promote TS cell self-renewal. Maternal (parthenogenetic/gynogenetic) EpiSCs (maEpiSCs) functionally and morphologically resemble conventional EpiSCs. Established maEpiSCs and conventional EpiSCs are diploid and exhibit a normal number of chromosomes. Moreover, global expression analyses and epigenomic profiling revealed that maEpiSCs and conventional EpiSCs exhibit similarly primed transcriptional programs and epigenetic profiles, respectively. Altogether, our results describe a useful experimental model to generate EpiSCs from haploid embryos, provide insight into self-renewal mechanisms of EpiSCs, and suggest that FGF4 is not sufficient to derive TS cells from haploid blastocyst-stage embryos.


Subject(s)
Blastocyst/cytology , Germ Layers/cytology , Stem Cells/cytology , Animals , Embryo Culture Techniques , Epigenomics , Gene Expression Profiling , Haploidy , Mice , Multipotent Stem Cells , Pluripotent Stem Cells , Signal Transduction
6.
Sci Rep ; 7(1): 16360, 2017 11 27.
Article in English | MEDLINE | ID: mdl-29180818

ABSTRACT

Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.


Subject(s)
Cell Differentiation , Cell Self Renewal , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Leukemia Inhibitory Factor/genetics , Octamer Transcription Factor-3/metabolism , Animals , Biomarkers , Cell Differentiation/genetics , Cell Line , Cell Self Renewal/genetics , Cells, Cultured , Epigenesis, Genetic , Gene Expression Profiling , Gene Regulatory Networks , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Histones/metabolism , Humans , Immunohistochemistry , Leukemia Inhibitory Factor/metabolism , Mice , Octamer Transcription Factor-3/genetics , Transcription, Genetic , Transcriptome
7.
Cancer Res ; 77(23): 6729-6745, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28951459

ABSTRACT

Epigenetic regulation of chromatin states is thought to control gene expression programs during lineage specification. However, the roles of repressive histone modifications, such as trimethylated histone lysine 20 (H4K20me3), in development and genome stability are largely unknown. Here, we show that depletion of SET and MYND domain-containing protein 5 (SMYD5), which mediates H4K20me3, leads to genome-wide decreases in H4K20me3 and H3K9me3 levels and derepression of endogenous LTR- and LINE-repetitive DNA elements during differentiation of mouse embryonic stem cells. SMYD5 depletion resulted in chromosomal aberrations and the formation of transformed cells that exhibited decreased H4K20me3 and H3K9me3 levels and an expression signature consistent with multiple human cancers. Moreover, dysregulated gene expression in SMYD5 cancer cells was associated with LTR and endogenous retrovirus elements and decreased H4K20me3. In addition, depletion of SMYD5 in human colon and lung cancer cells results in increased tumor growth and upregulation of genes overexpressed in colon and lung cancers, respectively. These findings implicate an important role for SMYD5 in maintaining chromosome integrity by regulating heterochromatin and repressing endogenous repetitive DNA elements during differentiation. Cancer Res; 77(23); 6729-45. ©2017 AACR.


Subject(s)
Cell Differentiation/genetics , Chromosomes/physiology , Colonic Neoplasms/genetics , Embryonic Stem Cells/cytology , Heterochromatin/physiology , Lung Neoplasms/genetics , Methyltransferases/genetics , A549 Cells , Animals , Base Sequence , Cell Line, Tumor , Colonic Neoplasms/pathology , DNA Copy Number Variations/genetics , Epigenesis, Genetic , HCT116 Cells , Histone Code/genetics , Histones/metabolism , Humans , Lung Neoplasms/pathology , MCF-7 Cells , Methylation , Mice , RNA Interference , RNA, Small Interfering/genetics , Sequence Analysis, DNA , Xenograft Model Antitumor Assays
8.
Nucleic Acids Res ; 45(11): 6427-6441, 2017 Jun 20.
Article in English | MEDLINE | ID: mdl-28402433

ABSTRACT

Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell self-renewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these alternatively splice exons. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells.


Subject(s)
Alternative Splicing , DNA-Binding Proteins/physiology , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/physiology , Mouse Embryonic Stem Cells/metabolism , Transcription Elongation, Genetic , Animals , Cell Line , Epigenesis, Genetic , Methylation , Mice , Promoter Regions, Genetic , Protein Binding , Protein Processing, Post-Translational , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
9.
Nat Commun ; 8: 15057, 2017 04 19.
Article in English | MEDLINE | ID: mdl-28422092

ABSTRACT

In animal cells, the centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored. The centriole subdistal appendages (SDAs) are the key structures that anchor microtubules in interphase cells, but the composition and assembly mechanisms of SDAs are not well understood. Here, we reveal that centrosome-binding proteins, coiled-coil domain containing (CCDC) 120 and CCDC68 are two novel SDA components required for hierarchical SDA assembly in human cells. CCDC120 is anchored to SDAs by ODF2 and recruits CEP170 and Ninein to the centrosome through different coiled-coil domains at its N terminus. CCDC68 is a CEP170-interacting protein that competes with CCDC120 in recruiting CEP170 to SDAs. Furthermore, CCDC120 and CCDC68 are required for centrosome microtubule anchoring. Our findings elucidate the molecular basis for centriole SDA hierarchical assembly and microtubule anchoring in human interphase cells.


Subject(s)
Centrioles/metabolism , Centrosome/metabolism , Nerve Tissue Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Binding, Competitive , Cytoskeletal Proteins/metabolism , Heat-Shock Proteins/metabolism , Humans , Interphase , Mice , Microtubule-Associated Proteins , Microtubules/metabolism , Models, Biological , Nerve Tissue Proteins/chemistry , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , Protein Domains , Protein Transport
10.
Water Sci Technol ; 72(8): 1411-20, 2015.
Article in English | MEDLINE | ID: mdl-26465313

ABSTRACT

This paper taking a full-scale coking wastewater (CWW) treatment plant as a case study aimed to characterize removal behaviors of dissolved organic matter (DOM) by UV spectra and fluorescence excitation-emission matrix-parallel factor analysis (PARAFAC), and investigate the correlations between spectroscopic indices and water quality parameters. Efficient removal rates of chemical oxygen demand (COD), dissolved organic carbon (DOC) and total nitrogen (TN) after the bio-treatment were 91.3%, 87.3% and 69.1%, respectively. UV270 was proven to be a stable UV absorption peak of CWW that could reflect the mixture of phenols, heterocyclics, polynuclear aromatic hydrocarbons and their derivatives. Molecular weight and aromaticity were increased, and also the content of polar functional groups was greatly reduced after bio-treatment. Three fluorescent components were identified by PARAFAC: C1 (tyrosine-like), C2 (tryptophan-like) and C3 (humic-like). The removal rate of protein-like was higher than that of humic-like and C1 was identified as biodegradable substance. Correlation analysis showed UV270 had an excellent correlation with COD (r=0.921, n=60, P<0.01) and DOC (r=0.959, n=60, P<0.01) and significant correlation (r=0.875, n=60, P<0.01) was also found between C2 and TN. Therefore, spectroscopic characterization could provide novel insights into removal behaviors of DOM and potential to monitor water quality real-time during CWW bio-treatment.


Subject(s)
Organic Chemicals/analysis , Waste Management , Wastewater/analysis , Water Pollutants, Chemical/analysis , Biological Oxygen Demand Analysis , Coke , Factor Analysis, Statistical , Industrial Waste , Nitrogen/analysis , Phenols/analysis , Spectrometry, Fluorescence , Spectrum Analysis , Water Quality
11.
Cell Rep ; 4(6): 1100-7, 2013 Sep 26.
Article in English | MEDLINE | ID: mdl-24035387

ABSTRACT

During interphase, centrosomes are connected by a proteinaceous linker between the proximal ends of the centrioles, which is important for the centrosomes to function as a single microtubule-organizing center. However, the composition and regulation of centrosomal linker remain largely unknown. Here, we show that LRRC45 is a centrosome linker that localizes at the proximal ends of the centrioles and forms fiber-like structures between them. Depletion of LRRC45 results in centrosome splitting during interphase. Moreover, LRRC45 interacts with both C-Nap1 and rootletin and is phosphorylated by Nek2A at S661 during mitosis. After phosphorylation, both LRRC45 centrosomal localization and fiber-like structures are significantly reduced, which subsequently leads to centrosome separation. Thus, LRRC45 is a critical component of the proteinaceous linker between two centrioles and is required for centrosome cohesion.


Subject(s)
Centrosome/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Centrosome/physiology , Cytoskeletal Proteins/metabolism , HeLa Cells , Humans , Interphase/physiology , NIMA-Related Kinases , Nerve Tissue Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Receptors, Cell Surface/genetics , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
12.
J Biol Chem ; 288(20): 14384-14390, 2013 May 17.
Article in English | MEDLINE | ID: mdl-23569207

ABSTRACT

Cytokinesis is the final stage of cell division in which the cytoplasm of a cell is divided into two daughter cells after the segregation of genetic material, and the central spindle and midbody are considered to be the essential structures required for the initiation and completion of cytokinesis. Here, we determined that the centrosome protein Cep57, which is localized to the central spindle and midbody, acts as a spindle organizer and is required for cytokinesis. Depletion of Cep57 disrupted microtubule assembly of the central spindle and further led to abnormal midbody localization of MKLP1, Plk1, and Aurora B, which resulted in cytokinesis failure and the formation of binuclear cells. Furthermore, we found that Cep57 directly recruited Tektin 1 to the midbody matrix to regulate microtubule organization. Thus, our data reveal that Cep57 is essential for cytokinesis via regulation of central spindle assembly and formation of the midbody.


Subject(s)
Carrier Proteins/physiology , Cytokinesis/physiology , Gene Expression Regulation , Microtubule-Associated Proteins/physiology , Microtubules/metabolism , Nuclear Proteins/physiology , Spindle Apparatus/metabolism , Animals , Cell Cycle Proteins , Centrosome/metabolism , HEK293 Cells , HeLa Cells , Humans , Mice , Microtubule Proteins/metabolism , Mitosis , Plasmids/metabolism , Protein Binding
13.
Cell Res ; 22(9): 1390-401, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22508265

ABSTRACT

Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle integrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, centrosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle microtubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1-binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture.


Subject(s)
Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Spindle Apparatus/metabolism , Animals , CHO Cells , Centrosome/metabolism , Cricetinae , Cricetulus , HeLa Cells , Humans , Mice , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Xenopus/metabolism
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