TdIF1 Recognizes a Specific DNA Sequence through Its Helix-Turn-Helix and AT-Hook Motifs to Regulate Gene Transcription
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{"title"=>"TdIF1 Recognizes a Specific DNA Sequence through Its Helix-Turn-Helix and AT-Hook Motifs to Regulate Gene Transcription", "type"=>"journal", "authors"=>[{"first_name"=>"Takashi", "last_name"=>"Kubota", "scopus_author_id"=>"56493886100"}, {"first_name"=>"Osamu", "last_name"=>"Koiwai", "scopus_author_id"=>"7004368429"}, {"first_name"=>"Katsutoshi", "last_name"=>"Hori", "scopus_author_id"=>"7403116279"}, {"first_name"=>"Nobuhisa", "last_name"=>"Watanabe", "scopus_author_id"=>"7403345664"}, {"first_name"=>"Kotaro", "last_name"=>"Koiwai", "scopus_author_id"=>"18634598900"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"23874396", "doi"=>"10.1371/journal.pone.0066710", "sgr"=>"84880026162", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "scopus"=>"2-s2.0-84880026162", "issn"=>"19326203", "pui"=>"369294494"}, "id"=>"c120b5de-2b24-362a-ab96-4f4d00a770bf", "abstract"=>"TdIF1 was originally identified as a protein that directly binds to DNA polymerase TdT. TdIF1 is also thought to function in transcription regulation, because it binds directly to the transcriptional factor TReP-132, and to histone deacetylases HDAC1 and HDAC2. Here we show that TdIF1 recognizes a specific DNA sequence and regulates gene transcription. By constructing TdIF1 mutants, we identify amino acid residues essential for its interaction with DNA. An in vitro DNA selection assay, SELEX, reveals that TdIF1 preferentially binds to the sequence 5'-GNTGCATG-3' following an AT-tract, through its Helix-Turn-Helix and AT-hook motifs. We show that four repeats of this recognition sequence allow TdIF1 to regulate gene transcription in a plasmid-based luciferase reporter assay. We demonstrate that TdIF1 associates with the RAB20 promoter, and RAB20 gene transcription is reduced in TdIF1-knocked-down cells, suggesting that TdIF1 stimulates RAB20 gene transcription.", "link"=>"http://www.mendeley.com/research/tdif1-recognizes-specific-dna-sequence-through-helixturnhelix-athook-motifs-regulate-gene-transcript", "reader_count"=>12, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>3, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Student > Bachelor"=>1, "Professor"=>1, "Student > Postgraduate"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>3, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Student > Bachelor"=>1, "Professor"=>1, "Student > Postgraduate"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>5, "Medicine and Dentistry"=>2, "Chemistry"=>1, "Decision Sciences"=>1, "Immunology and Microbiology"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>1}, "Decision Sciences"=>{"Decision Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"China"=>1, "Germany"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1116364"], "description"=>"<p>(A) TdIF1 promotes transcription. The pGL3-promoter-TdIF1-binding sequence and pRL-TK were co-transfected with pEGFP or pEGFP-TdIF1 into 293T cells, and the luciferase activity was assayed. The relative luciferase activity was normalized to the value obtained with the pGL3-promoter and pEGFP. Error bars represent S.E.M. Samples significantly different from the control are indicated by asterisks (p<0.01). (B) TdIF1 promotes transcription by directly binding to DNA. The pGL3-promoter-TdIF1-binding sequence and pRL-TK were co-transfected with pEGFP-TdIF1 truncated and point mutants, and the luciferase activity was assayed. Error bars represent S.E.M.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "DNA-binding proteins", "Computational biology", "Molecular genetics", "Gene regulation", "gene expression", "genetics", "DNA transcription", "Molecular cell biology", "transcription", "luciferase"], "article_id"=>743568, "categories"=>["Biological Sciences"], "users"=>["Takashi Kubota", "Osamu Koiwai", "Katsutoshi Hori", "Nobuhisa Watanabe", "Kotaro Koiwai"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066710.g004", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TdIF1_promotes_transcription_in_a_luciferase_reporter_assay_/743568", "title"=>"TdIF1 promotes transcription in a luciferase reporter assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-10 02:44:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1116366"], "description"=>"<p>(A) TdIF1 associates with the RAB20 promoter region. ChIP-qPCR was performed using 293T cells expressing Flag-TdIF1, an anti-Flag antibody, and the indicated sets of primers (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066710#pone.0066710.s001\" target=\"_blank\">Table S1</a>). For negative controls, an anti-myc antibody (αNega) and 293T cells not expressing Flag-TdIF1 (noOE) were used. (B) Schematic structure of the RAB20 promoter region. The motif of 5′-GTTGCATG-3′ (underlined) is positioned at -1597 bp from ATG of RAB20 gene. An AT-tract (colored red) locates near the 5′-GTTGCATG-3′ motif. Primers used in ChIP-qPCR are shown below. HRE, hypoxia responsive element; TSS, transcriptional start site. (C) TdIF1 regulates RAB20 gene transcription. Total RNA was extracted from 293T cells transfected with siRNAs against TdIF1 (siTdIF1) or negative control siRNAs (siNega). The mRNA levels were calculated by the ΔΔCt method and normalized to each mRNA from siNega. <i>DNTTIP1</i> encodes TdIF1. Error bars represent S.E.M. Samples significantly different from the control sample are indicated by asterisks (p<0.01).</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "DNA-binding proteins", "Computational biology", "Molecular genetics", "Gene regulation", "gene expression", "genetics", "DNA transcription", "Molecular cell biology", "regulates", "rab20"], "article_id"=>743570, "categories"=>["Biological Sciences"], "users"=>["Takashi Kubota", "Osamu Koiwai", "Katsutoshi Hori", "Nobuhisa Watanabe", "Kotaro Koiwai"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066710.g005", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TdIF1_regulates_RAB20_gene_transcription_/743570", "title"=>"TdIF1 regulates RAB20 gene transcription.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-10 02:44:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1116367", "https://ndownloader.figshare.com/files/1116369", "https://ndownloader.figshare.com/files/1116370"], "description"=>"<div><p>TdIF1 was originally identified as a protein that directly binds to DNA polymerase TdT. TdIF1 is also thought to function in transcription regulation, because it binds directly to the transcriptional factor TReP-132, and to histone deacetylases HDAC1 and HDAC2. Here we show that TdIF1 recognizes a specific DNA sequence and regulates gene transcription. By constructing TdIF1 mutants, we identify amino acid residues essential for its interaction with DNA. An <i>in vitro</i> DNA selection assay, SELEX, reveals that TdIF1 preferentially binds to the sequence 5′-GNTGCATG-3′ following an AT-tract, through its Helix-Turn-Helix and AT-hook motifs. We show that four repeats of this recognition sequence allow TdIF1 to regulate gene transcription in a plasmid-based luciferase reporter assay. We demonstrate that TdIF1 associates with the RAB20 promoter, and RAB20 gene transcription is reduced in TdIF1-knocked-down cells, suggesting that TdIF1 stimulates RAB20 gene transcription.</p></div>", "links"=>[], "tags"=>["Biochemistry", "proteins", "DNA-binding proteins", "Computational biology", "Molecular genetics", "Gene regulation", "gene expression", "genetics", "DNA transcription", "Molecular cell biology", "recognizes", "dna", "helix-turn-helix", "at-hook", "motifs"], "article_id"=>743571, "categories"=>["Biological Sciences"], "users"=>["Takashi Kubota", "Osamu Koiwai", "Katsutoshi Hori", "Nobuhisa Watanabe", "Kotaro Koiwai"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0066710.s001", "https://dx.doi.org/10.1371/journal.pone.0066710.s002", "https://dx.doi.org/10.1371/journal.pone.0066710.s003"], "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TdIF1_Recognizes_a_Specific_DNA_Sequence_through_Its_Helix_Turn_Helix_and_AT_Hook_Motifs_to_Regulate_Gene_Transcription_/743571", "title"=>"TdIF1 Recognizes a Specific DNA Sequence through Its Helix-Turn-Helix and AT-Hook Motifs to Regulate Gene Transcription", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-07-10 02:44:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1116360"], "description"=>"<p>(A) Schematic representation of TdIF1 mutants and summary of their DNA binding activity. DNA binding regions which are previously determined <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066710#pone.0066710-Kubota1\" target=\"_blank\">[5]</a> are shown below the schematic representation of TdIF1. “DNA-binding” shows summary of GST pull-out assay shown in panels C and D: DNA fragments were held by TdIF1 or TdIF1 mutants until elution in buffer containing 300 mM NaCl (++++), 250 mM (+++), 200 mM (++) or 150 mM (+), or DNA fragments were not held (−). NLS, nuclear localization signal. (B) Schematic flowchart of GST pull-out assay. (C) DNA-binding activities of TdIF1 mutants. <i>Hae</i> III-digested pcDNA3.1 plasmid was incubated with TdIF1 mutants, and then DNA fragments bound to TdIF1 mutants were sequentially eluted with buffer A containing 150, 200, 250, or 300 mM NaCl, separated by PAGE, and detected by silver staining. Lanes 1 and 19 contained a 200-bp DNA ladder marker. Lanes 2 and 20 contained 1/5 of the amount of pcDNA3.1/<i>Hae</i> III used in the reaction. Asterisk indicates higher molecular weight bands, which are probably a complex of DNA and protein eluted. (D) DNA-binding activity of full-length TdIF1 containing point mutations. DNA fragments bound to TdIF1 mutants were sequentially eluted with buffer containing 200, 250, 300, or 350 mM NaCl, and analysed as in (C).</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "DNA-binding proteins", "Computational biology", "Molecular genetics", "Gene regulation", "gene expression", "genetics", "DNA transcription", "Molecular cell biology", "amino", "acids", "residues", "hth", "tdif1", "dna-binding"], "article_id"=>743564, "categories"=>["Biological Sciences"], "users"=>["Takashi Kubota", "Osamu Koiwai", "Katsutoshi Hori", "Nobuhisa Watanabe", "Kotaro Koiwai"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066710.g001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Basic_amino_acids_in_residues_1_8211_75_an_AT_hook_and_an_HTH_of_TdIF1_are_required_for_its_DNA_binding_activity_/743564", "title"=>"Basic amino acids in residues 1–75, an AT-hook, and an HTH of TdIF1 are required for its DNA-binding activity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-10 02:44:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1116361"], "description"=>"<p>(A) Flow chart of the SELEX experiment. (B) EMSA results in the 7th cycle. DNA was detected by EtBr staining. The arrow indicates the TdIF1/oligoDNA complex. (C) Oligonucleotides selected by SELEX aligned around the identified consensus 5′-GNTGCATG-3′, underlined in each case. Sequences of six or more continuous As or Ts are in red. Flanking linker sequence shown in italics. (D) Conserved nucleotides drawn as a sequence logo <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066710#pone.0066710-Crooks1\" target=\"_blank\">[20]</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "DNA-binding proteins", "Computational biology", "Molecular genetics", "Gene regulation", "gene expression", "genetics", "DNA transcription", "Molecular cell biology", "dna", "sequences", "recognized", "tdif1"], "article_id"=>743565, "categories"=>["Biological Sciences"], "users"=>["Takashi Kubota", "Osamu Koiwai", "Katsutoshi Hori", "Nobuhisa Watanabe", "Kotaro Koiwai"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066710.g002", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_the_DNA_sequences_recognized_by_TdIF1_by_SELEX_/743565", "title"=>"Identification of the DNA sequences recognized by TdIF1 by SELEX.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-10 02:44:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1116363"], "description"=>"<p>(A) Sequences of the biotin-labelled AT-rich probe and the competitors used. Asterisks indicate replaced residues in the AT-tract or 5′-GNTGCATG-3′ motifs. (B) Competitive EMSA. All reaction mixtures contained 5 pmol of biotin-labelled AT-rich probe. Purified His-TdIF1 (100 ng) was incubated in the reaction mixture alone or with competitors (5, 15, or 45 pmol) as indicated (lanes 2–14). (C) Competitive EMSA using TdIF1mtHTH2, which has mutations in the HTH. TdIF1mtHTH2 (200 ng) or wild-type TdIF1 (100 ng) was incubated with biotin-labelled AT-rich probe alone or with competitors (15 or 45 pmol), as indicated.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "DNA-binding proteins", "Computational biology", "Molecular genetics", "Gene regulation", "gene expression", "genetics", "DNA transcription", "Molecular cell biology", "preferentially", "recognizes"], "article_id"=>743567, "categories"=>["Biological Sciences"], "users"=>["Takashi Kubota", "Osamu Koiwai", "Katsutoshi Hori", "Nobuhisa Watanabe", "Kotaro Koiwai"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0066710.g003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TdIF1_preferentially_recognizes_5_8242_GNTGCATG_3_8242_following_an_AT_tract_/743567", "title"=>"TdIF1 preferentially recognizes 5′-GNTGCATG-3′ following an AT-tract.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-07-10 02:44:29"}

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Relative Metric

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