Identification of Dominant Transcripts in Oxidative Stress Response by a Full-Length Transcriptome Analysis

Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. ABSTRACT Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading concerning the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing.

[1]  K. Kinoshita,et al.  Landscape of electrophilic and inflammatory stress-mediated gene regulation in human lymphoblastoid cell lines. , 2020, Free radical biology & medicine.

[2]  H. Coller,et al.  Splicing Busts a Move: Isoform Switching Regulates Migration. , 2019, Trends in cell biology.

[3]  Masayuki Yamamoto,et al.  Cis-element architecture of Nrf2–sMaf heterodimer binding sites and its relation to diseases , 2019, Archives of Pharmacal Research.

[4]  K. Kinoshita,et al.  Construction and integration of three de novo Japanese human genome assemblies toward a population-specific reference , 2019, bioRxiv.

[5]  Masayuki Yamamoto,et al.  Direct and Specific Functional Evaluation of the Nrf2 and MafG Heterodimer by Introducing a Tethered Dimer into Small Maf-Deficient Cells , 2019, Molecular and Cellular Biology.

[6]  J. Hadfield,et al.  RNA sequencing: the teenage years , 2019, Nature Reviews Genetics.

[7]  Geo Pertea,et al.  Transcriptome assembly from long-read RNA-seq alignments with StringTie2 , 2019, Genome Biology.

[8]  Barbara J. Wold,et al.  A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification , 2019, bioRxiv.

[9]  R. Yamashita,et al.  Biobank Establishment and Sample Management in the Tohoku Medical Megabank Project. , 2019, The Tohoku journal of experimental medicine.

[10]  T. Derrien,et al.  Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules , 2019, Scientific Reports.

[11]  Angela N. Brooks,et al.  Nanopore native RNA sequencing of a human poly(A) transcriptome , 2018, bioRxiv.

[12]  Mark Gerstein,et al.  GENCODE reference annotation for the human and mouse genomes , 2018, Nucleic Acids Res..

[13]  Masayuki Yamamoto,et al.  The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis. , 2018, Physiological reviews.

[14]  Ali Mortazavi,et al.  TranscriptClean: variant-aware correction of indels, mismatches and splice junctions in long-read transcripts , 2018, Bioinform..

[15]  Junjun Li,et al.  Snail Driving Alternative Splicing of CD44 by ESRP1 Enhances Invasion and Migration in Epithelial Ovarian Cancer , 2017, Cellular Physiology and Biochemistry.

[16]  Heng Li,et al.  Minimap2: pairwise alignment for nucleotide sequences , 2017, Bioinform..

[17]  Albin Sandelin,et al.  The Landscape of Isoform Switches in Human Cancers , 2017, Molecular Cancer Research.

[18]  K. Zangger,et al.  Catalytic competence, structure and stability of the cancer-associated R139W variant of the human NAD(P)H: quinone oxidoreductase 1 (NQO1) , 2017, The FEBS journal.

[19]  Rob Patro,et al.  Salmon provides fast and bias-aware quantification of transcript expression , 2017, Nature Methods.

[20]  Leonard D. Goldstein,et al.  Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers. , 2016, Cell reports.

[21]  Tyson A. Clark,et al.  Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing , 2016, Nature Communications.

[22]  Michelle R. Campbell,et al.  A Polymorphic Antioxidant Response Element Links NRF2/sMAF Binding to Enhanced MAPT Expression and Reduced Risk of Parkinsonian Disorders. , 2016, Cell reports.

[23]  Lior Pachter,et al.  Near-optimal probabilistic RNA-seq quantification , 2016, Nature Biotechnology.

[24]  R. Nagatomi,et al.  Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism , 2016, Molecular and Cellular Biology.

[25]  Masayuki Yamamoto,et al.  Unique cistrome defined as CsMBE is strictly required for Nrf2-sMaf heterodimer function in cytoprotection. , 2016, Free radical biology & medicine.

[26]  Daniel J. Gaffney,et al.  A survey of best practices for RNA-seq data analysis , 2016, Genome Biology.

[27]  C. Mason,et al.  The impact of read length on quantification of differentially expressed genes and splice junction detection , 2015, Genome Biology.

[28]  Alfonso Valencia,et al.  Most highly expressed protein-coding genes have a single dominant isoform. , 2015, Journal of proteome research.

[29]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[30]  Leena Salmela,et al.  LoRDEC: accurate and efficient long read error correction , 2014, Bioinform..

[31]  H. Aburatani,et al.  Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. , 2012, Cancer cell.

[32]  ENCODEConsortium,et al.  An Integrated Encyclopedia of DNA Elements in the Human Genome , 2012, Nature.

[33]  Michelle R. Campbell,et al.  Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha , 2012, Nucleic acids research.

[34]  Auinash Kalsotra,et al.  Functional consequences of developmentally regulated alternative splicing , 2011, Nature Reviews Genetics.

[35]  Colin N. Dewey,et al.  RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome , 2011, BMC Bioinformatics.

[36]  M. McMahon,et al.  Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway. , 2010, Antioxidants & redox signaling.

[37]  M. Takagi,et al.  Irradiation-induced p53 expression is attenuated in cells with NQO1 C465T polymorphism. , 2010, Journal of medical and dental sciences.

[38]  M. McMahon,et al.  p62/SQSTM1 Is a Target Gene for Transcription Factor NRF2 and Creates a Positive Feedback Loop by Inducing Antioxidant Response Element-driven Gene Transcription* , 2010, The Journal of Biological Chemistry.

[39]  Mihee M. Kim,et al.  The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1 , 2010, Nature Cell Biology.

[40]  M. McMahon,et al.  NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. , 2009, Trends in biochemical sciences.

[41]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[42]  K. Itoh,et al.  Nrf2 regulates the alternative first exons of CD36 in macrophages through specific antioxidant response elements. , 2008, Archives of biochemistry and biophysics.

[43]  Ok-Hee Lee,et al.  An Auto-regulatory Loop between Stress Sensors INrf2 and Nrf2 Controls Their Cellular Abundance* , 2007, Journal of Biological Chemistry.

[44]  Martin S. Taylor,et al.  Genome-wide analysis of mammalian promoter architecture and evolution , 2006, Nature Genetics.

[45]  J. Conboy,et al.  From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 28, 2013. For personal use only. , 2004 .

[46]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Tsujimoto,et al.  Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: Role of NF‐E2‐related factor‐2 , 2005, Journal of cellular physiology.

[48]  James Douglas Engel,et al.  Nrf2 Transcriptionally Activates the mafG Gene through an Antioxidant Response Element* , 2005, Journal of Biological Chemistry.

[49]  Kengo Kinoshita,et al.  eF-site and PDBjViewer: database and viewer for protein functional sites , 2004, Bioinform..

[50]  J. D. Engel,et al.  Small Maf proteins serve as transcriptional cofactors for keratinocyte differentiation in the Keap1-Nrf2 regulatory pathway. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Paul Talalay,et al.  Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Hofker.  Faculty Opinions recommendation of PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. , 2003 .

[53]  Ken Itoh,et al.  Transcription Factor Nrf2 Coordinately Regulates a Group of Oxidative Stress-inducible Genes in Macrophages* , 2000, The Journal of Biological Chemistry.

[54]  H. Ishihara,et al.  Alternative Promoters Regulate Transcription of the Mouse GATA-2 Gene* , 1998, The Journal of Biological Chemistry.

[55]  K. Itoh,et al.  An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. , 1997, Biochemical and biophysical research communications.

[56]  G. Powis,et al.  An alternatively spliced form of NQO1 (DT-diaphorase) messenger RNA lacking the putative quinone substrate binding site is present in human normal and tumor tissues. , 1995, Cancer research.

[57]  C. B. Pickett,et al.  Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-acetate. , 1993, The Journal of biological chemistry.

[58]  J. D. Engel,et al.  Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis , 1993, Nature.

[59]  Heidemarie Neitzel,et al.  A routine method for the establishment of permanent growing lymphoblastoid cell lines , 1986, Human Genetics.

[60]  W. Gilbert.  Why genes in pieces? , 1978, Nature.

[61]  Haruki Nakamura,et al.  Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive. , 2017, Methods in molecular biology.

[62]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[63]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[64]  J. D. Engel,et al.  Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. , 1999, Genes & development.