Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
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{"title"=>"Functional complementation of sir2Δ yeast mutation by the human orthologous gene SIRT1", "type"=>"journal", "authors"=>[{"first_name"=>"Davide", "last_name"=>"Gaglio", "scopus_author_id"=>"56006250800"}, {"first_name"=>"Anna", "last_name"=>"D'Alfonso", "scopus_author_id"=>"55314725500"}, {"first_name"=>"Giorgio", "last_name"=>"Camilloni", "scopus_author_id"=>"6604000307"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203", "doi"=>"10.1371/journal.pone.0083114", "pui"=>"372133163", "sgr"=>"84892562351", "scopus"=>"2-s2.0-84892562351", "pmid"=>"24349441", "issn"=>"19326203"}, "id"=>"5f5fbbfe-dfcf-3141-8139-65b87169cf5c", "abstract"=>"Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2Δ mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2Δ shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2Δ strains. By strong SIRT1 overexpression in sir2Δ cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2Δ mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.", "link"=>"http://www.mendeley.com/research/functional-complementation-sir2%CE%B4-yeast-mutation-human-orthologous-gene-sirt1", "reader_count"=>22, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>10, "Student > Master"=>1, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>10, "Student > Master"=>1, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>2, "Neuroscience"=>1, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}}, "reader_count_by_country"=>{"Canada"=>1, "United States"=>4}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1311376"], "description"=>"<div><p>ChIp analysis of Sirt1 enrichment at rDNA, HM loci, telomeric and sub telomeric regions in <i>sir2Δ</i>+ and <i>sir2Δ</i>- strains during <i>SIRT1</i> induction (galactose medium). Sirt1 enrichment for <i>sir2Δ</i>+ was referred to <i>sir2Δ</i>- levels =1. Histograms averages and Std. Dev. bars are representative of three technical replicates for at least three biological replicates performed. </p>\n\t\t\t\t\t\t<p>Statistical analysis as in Figure 3.</p></div>", "links"=>[], "tags"=>["enrichment"], "article_id"=>874869, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083114.g006", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sirt1_enrichment_in_specific_loci_/874869", "title"=>"Sirt1 enrichment in specific loci.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-11 03:33:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1311375"], "description"=>"<div><p>ChIp analysis of H4K16Ac (A) and H3K9Ac (B) at rDNA, HM loci, telomeric and sub telomeric regions in <i>sir2Δ</i>- and <i>sir2Δ</i>+ strains during <i>SIRT1</i> induction (galactose medium).</p>\n\t\t\t\t\t\t<p>(C) ChIp analysis of H4K16Ac and H3K9Ac at E-PRO in WT-, <i>sir2Δ</i>-, <i>sir2Δ</i>+ and <i>sir2Δ</i>+*.</p>\n\t\t\t\t\t\t<p>Acetylation enrichment for H4K16 and H3K9 were normalized to H4 C-terminal and H3 C-terminal, respectively, and referred to <i>sir2Δ</i>- levels =1. Histograms averages and Std. Dev. bars are representative of three technical replicates for at least three biological replicates performed. </p>\n\t\t\t\t\t\t<p>Statistical analysis as in Figure 3.</p></div>", "links"=>[], "tags"=>["deacetylates"], "article_id"=>874868, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083114.g005", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sirt1_deacetylates_specific_loci_/874868", "title"=>"Sirt1 deacetylates specific loci.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-11 03:33:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1311372"], "description"=>"<div><p>RT-PCR transcriptional analysis in WT and <i>sir2Δ</i> strains transformed with empty plasmid (-), <i>SIRT1</i> construct (+) or <i>SIRT1</i>-<i>H363Y</i> mutant construct (+*) both in repression (glucose) and induction (galactose) conditions. (A) rDNA locus: <i>NTS1r</i> and <i>NTS2</i>; (B) TEL VI locus: <i>YFR057W</i> and <i>IRC7</i>; (C) HM loci: <i>HMLalpha1</i>. Histograms indicate averages and Std. Dev. bars from at least three independent biological replicates. Two−tailed t−test was applied for statistical analysis. Asterisks indicate statistically significant differences between <i>sir2Δ</i>- and <i>sir2Δ</i>+ or between <i>sir2Δ</i>+* and <i>sir2Δ</i>+ in galactose medium; α = 0.05. (Percentages of p−value: *p< 5%, **p < 1%, ***p < 0.01%).</p>\n\t\t\t\t\t\t<p>(D) Alpha factor yeast spot test analysis to evaluate <i>HMLalpha1</i> silencing. Five-fold serial dilutions of mutant <i>sir2Δ</i> and WT cells transformed with <i>SIRT1</i>(+) or empty plasmid (−) were spotted onto minimal medium plates containing alpha-factor.</p></div>", "links"=>[], "tags"=>["overexpression", "restores", "transcriptional", "repression", "loci"], "article_id"=>874865, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083114.g003", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SIRT1_overexpression_partially_restores_the_transcriptional_repression_within_specific_loci_in_sir2_916_mutant_/874865", "title"=>"SIRT1 overexpression partially restores the transcriptional repression within specific loci in sir2Δ mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-11 03:33:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1311370"], "description"=>"<div><p>(A) Western blot analysis of H3K9, H4K16, H4K12 global acetylation after <i>SIRT1</i> overnight induction. <i>SIRT1</i>, H3Ct, H4Ct and α<i>TUB</i> hybridization were performed as induction or loading controls. (B) Western blot quantification of panel A. Histograms indicate averages and Std. Dev. bars from 4 biological replicates (H3K9Ac **p < 1%, H4K16Ac p = 10.3%, H4K12Ac p = 27.2%). (C) Western blot analysis of H3K9Ac on the same strains that in panels A and B but also with the strain <i>sir2Δ</i>+* containing the non catalytic mutant <i>SIRT1</i>-<i>H363Y</i> as control. (D) Western blot quantification of panel C. Histograms indicate averages and Std. Dev. bars from 4 biological replicates, (H3K9Ac, <i>sir2Δ</i>- <i>versus sir2Δ</i>+, ** p<1%; <i>sir2Δ</i>+* <i>versus sir2Δ</i>+, ** p<1%; Sirt1/Sirt1-H363Y, <i>sir2Δ</i>- <i>versus sir2Δ</i>+ or <i>sir2Δ</i>+ *, ** p<1%; Sirt1/Sirt1-H363Y, <i>sir2Δ</i>+* <i>versus sir2Δ</i>+, p>>5%).</p>\n\t\t\t\t\t\t<p>Statistical analysis as in Figure 3.</p></div>", "links"=>[], "tags"=>["histone", "acetylation", "influenced"], "article_id"=>874863, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083114.g002", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Global_histone_acetylation_is_influenced_by_Sirt1_/874863", "title"=>"Global histone acetylation is influenced by Sirt1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-11 03:33:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1311369"], "description"=>"<p>(A) Construct for yeast expression with <i>SIRT1</i> under the inducible promoter GAL1 in pYES2 background. (+: SIRT1 construct; −: empty plasmid). (B) Yeast spot test analysis of growth phenotypes during plasmid repression and induction conditions (glucose and galactose, respectively). For each strain five-fold serial dilutions were made and 5 μl were spotted onto minimal medium plates.</p>", "links"=>[], "tags"=>["toxicity", "yeast"], "article_id"=>874862, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083114.g001", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SIRT1_construct_and_its_toxicity_in_yeast_cells_/874862", "title"=>"SIRT1 construct and its toxicity in yeast cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-11 03:33:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1311379", "https://ndownloader.figshare.com/files/1311380"], "description"=>"<div><p>Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in <i>S.cerevisiae sir2Δ</i> mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an <i>in vivo</i> substrate. Mutant <i>sir2Δ</i> shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of <i>sir2Δ</i> strains. By strong <i>SIRT1</i> overexpression in <i>sir2Δ</i> cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) <i>SIRT1</i> gene is able to complement different molecular phenotypes of the <i>sir2Δ</i> mutant at rDNA ii) the <i>in vivo</i> screening of Sirt1 activity is possible in yeast.</p> </div>", "links"=>[], "tags"=>["complementation", "yeast", "mutation", "orthologous"], "article_id"=>874872, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083114.s001", "https://dx.doi.org/10.1371/journal.pone.0083114.s002"], "stats"=>{"downloads"=>18, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Functional_Complementation_of_sir2_916_Yeast_Mutation_by_the_Human_Orthologous_Gene_SIRT1_/874872", "title"=>"Functional Complementation of <i>sir2Δ</i> Yeast Mutation by the Human Orthologous Gene <i>SIRT1</i>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-12-11 03:33:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1311373"], "description"=>"<div><p>A) Southern blot analysis of ERCs species in WT and <i>sir2Δ</i> with <i>SIRT1</i> construct (+), <i>SIRT1</i>-<i>H363Y</i> mutant construct (+*), empty plasmid (−) or without plasmid (/) as control. Strains were grown both in <i>SIRT1</i> repression and induction conditions (glucose or galactose, respectively). DNA was isolated from the specified yeast strains and probed with a radiolabeled rDNA sequence shown in panel (A). ERCs are indicated by an arrow. B) Quantification of ERCs amount in galactose condition: band intensities corresponding to ERCs were normalized to the hybridized bulk rDNA and referred to WT- levels. Histograms indicate averages and Std. Dev. bars from at least 4 biological replicates .</p>\n\t\t\t\t\t\t<p>Statistical analysis as in Figure 3.</p></div>", "links"=>[], "tags"=>["decreases", "complemented"], "article_id"=>874866, "categories"=>["Biological Sciences"], "users"=>["Davide Gaglio", "Anna D’Alfonso", "Giorgio Camilloni"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083114.g004", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ERCs_level_decreases_in_complemented_strain_/874866", "title"=>"ERCs level decreases in complemented strain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-11 03:33:56"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"8", "full-text"=>"6", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"11", "full-text"=>"12", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}
  • {"unique-ip"=>"14", "full-text"=>"13", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}
  • {"unique-ip"=>"9", "full-text"=>"8", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2019", "month"=>"12"}
  • {"unique-ip"=>"6", "full-text"=>"3", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2020", "month"=>"2"}
  • {"unique-ip"=>"9", "full-text"=>"5", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2020", "month"=>"3"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}

Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[269, 466, 588, 697, 800, 896, 988, 1076, 1165, 1254, 1340, 1417]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Biology and life sciences/Genetics", "average_usage"=>[284, 491, 620, 738, 843, 945, 1043, 1137, 1225, 1315, 1400, 1479, 1555]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[281, 484, 611, 728, 835, 934, 1030, 1123, 1214, 1299, 1383, 1464]}, {"subject_area"=>"/Physical sciences/Chemistry", "average_usage"=>[247, 429, 544, 647, 747, 842, 929, 1012, 1099, 1179, 1263, 1339, 1409]}]}
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