Histone H3 Localizes to the Centromeric DNA in Budding Yeast
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{"title"=>"Histone H3 localizes to the centromeric DNA in budding yeast", "type"=>"journal", "authors"=>[{"first_name"=>"Berit", "last_name"=>"Lochmann", "scopus_author_id"=>"55309041700"}, {"first_name"=>"Dmitri", "last_name"=>"Ivanov", "scopus_author_id"=>"55426447800"}], "year"=>2012, "source"=>"PLoS Genetics", "identifiers"=>{"issn"=>"15537390", "scopus"=>"2-s2.0-84863638252", "sgr"=>"84863638252", "pui"=>"365220749", "isbn"=>"1553-7404 (Electronic)\\r1553-7390 (Linking)", "pmid"=>"22693454", "doi"=>"10.1371/journal.pgen.1002739"}, "id"=>"4e88fc3e-73f4-3d62-9fb0-6541896a8f47", "abstract"=>"During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.", "link"=>"http://www.mendeley.com/research/histone-h3-localizes-centromeric-dna-budding-yeast", "reader_count"=>46, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>5, "Researcher"=>16, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>9, "Student > Master"=>5, "Other"=>2, "Student > Bachelor"=>4, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>5, "Researcher"=>16, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>9, "Student > Master"=>5, "Other"=>2, "Student > Bachelor"=>4, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>8, "Agricultural and Biological Sciences"=>33, "Business, Management and Accounting"=>1, "Computer Science"=>3}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>33}, "Computer Science"=>{"Computer Science"=>3}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>8}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United States"=>1, "United Kingdom"=>1, "Germany"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/629503"], "description"=>"<p>A) The CEN-containing minichromosomes can be specifically co-immunoprecipitated with Cse4 and H3. Lysates from strains transformed with the minichromosomes 1021 (wt), 1498 (Cse4-HA6) and 1407 (H3-HA3) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed using a <sup>32</sup>P labeled <i>TRP1</i> probe. The map of the minichromosome is shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen.1002739.s001\" target=\"_blank\">Figure S1</a>. B) Experimental setup for the immunoprecipitation of minichromosomes digested with restriction enzyme. Chromatin is digested with BglII and incubated with anti-HA antibody recognizing tagged histones and protein A Dynabeads. Minichromosome digest with BglII produces three different fragments: a linearized full-length minichromosome (1), a CEN-less fragment (2) which can be detected with <i>TRP1</i> probe and a small CEN fragment (3) which can be detected with an LNA oligonucleotide. The red ellipse is depicting the centromeric nucleosome. C) Cse4 binding is restricted to minichromosomal CEN DNA. BglII-treated chromatin of strains carrying the minichromosome with BglII restriction sites 50 bp upstream and downstream of CEN boundaries was immunoprecipitated with anti-HA antibody. The strains were 1498 (Cse4-HA6), 1577 (H4-HA3), 1576 (H2A-HA3), 1587 (H2B-HA3), 1407 (H3-HA3), 1593 (Scm3-HA6), and 1021 (wt). DNA was analyzed as in (A) with <sup>32</sup>P labeled <i>TRP1</i> probe. D) H3 is associated with the CEN DNA. Top: Scheme of the excised CEN fragment. Double-DIG labeled LNA probe for CDEI/II is indicated. Bottom: Immunoprecipitated DNA from experiments shown in (C) was separated on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed using a double-DIG labeled LNA probe for CDEI/II. Western blots showing immunoprecipitation of the tagged proteins are shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen.1002739.s004\" target=\"_blank\">Figure S4A</a>.</p>", "links"=>[], "tags"=>["centromeric"], "article_id"=>300000, "categories"=>["Cell Biology"], "users"=>["Berit Lochmann", "Dmitri Ivanov"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002739.g001", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Composition_of_the_centromeric_nucleosome_/300000", "title"=>"Composition of the centromeric nucleosome.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-31 00:00:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/629839"], "description"=>"<p>A heterotetramer of H3, H2A, H2B and H4 is colored in green and a heterotetramer containing Cse4 instead of H3 is blue.1) A heterotypic octamer containing both Cse4 and H3. 2) A heterotypic octamer with additional Cse4 bound to it. 3) A Cse4 hemisome incorporated in the loop of a conventional nucleosome. A DNA fragment of 207 bp is sufficient to accommodate this arrangement (without spacer DNA). 4) Two conventional nucleosomes flanking a Cse4 hemisome. The scissors indicate the BglII sites flanking the 214 bp fragment excised in our experiment. In case of model 4 this fragment would be tethered to non-centromeric DNA. The tethering was not observed in our experiments (<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen-1002739-g001\" target=\"_blank\">Figure 1C</a>). See text for discussion and additional details.</p>", "links"=>[], "tags"=>["h3", "cse4", "co-occupy", "centromeric"], "article_id"=>300336, "categories"=>["Cell Biology"], "users"=>["Berit Lochmann", "Dmitri Ivanov"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002739.g005", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Models_of_how_H3_and_Cse4_can_co_occupy_the_centromeric_DNA_/300336", "title"=>"Models of how H3 and Cse4 can co-occupy the centromeric DNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-31 00:05:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/629755"], "description"=>"<p>A) Cse4 nucleosome straddles the boundary between CDEII and CDEIII. Left: Map of the minichromosome utilized in the experiment. The construct contains 850 bp of pericentromeric sequence of chromosome IV, <i>TRP1</i> marker, <i>ARS1</i> and pUC19 sequence and has a size of 4.5 kb. There are two BglII sites: between CDEII and CDEIII in the CEN and in the <i>ARS1</i>. Right: BglII-treated chromatin of a strain 1498 (Cse4-HA6) carrying the minichromosome was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed with a <sup>32</sup>P labeled probe for the pericentric CEN4 sequence (to detect the CDEI/II containing fragment) and a <sup>32</sup>P labeled probe for the <i>TRP1</i> gene (to detect the CDEIII containing fragment). B) Both Cse4 and H3 are associated with the CDEI/II fragment. Left: Scheme of CDEI/II fragment excised from the minichromosome. Double-DIG labeled LNA probe for CDEI/II is indicated. Right: BglII-treated chromatin of strain 1498 (Cse4-HA6) and 1407 (H3-HA3) carrying the minichromosome with BglII sites between CDEII and CDEIII and 50 bp upstream of CDEI was cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Both the CDEI/II and the CDEIII fragments can be co-immunoprecipitated with Cse4 and H3. Strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carried the minichromosome where either the CDEI/II (left) or the CDEIII fragment (right) was flanked with BglII sites. BglII-treated chromatin was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated, and subjected to qPCR analysis. Bar graphs represent the average values from several independent experiments with SDs.</p>", "links"=>[], "tags"=>["cell biology"], "article_id"=>300261, "categories"=>["Cell Biology"], "users"=>["Berit Lochmann", "Dmitri Ivanov"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002739.g004", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cse4_association_with_CDEI_II_and_CDEIII_/300261", "title"=>"Cse4 association with CDEI/II and CDEIII.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-31 00:04:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/326276", "https://ndownloader.figshare.com/files/326344", "https://ndownloader.figshare.com/files/326404", "https://ndownloader.figshare.com/files/326454", "https://ndownloader.figshare.com/files/326509", "https://ndownloader.figshare.com/files/326572", "https://ndownloader.figshare.com/files/326644", "https://ndownloader.figshare.com/files/326689", "https://ndownloader.figshare.com/files/326754"], "description"=>"<div><p>During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.</p> </div>", "links"=>[], "tags"=>["histone", "h3", "localizes", "centromeric", "dna", "budding", "yeast"], "article_id"=>124405, "categories"=>["Cell Biology"], "users"=>["Berit Lochmann", "Dmitri Ivanov"], "doi"=>["https://dx.doi.org/10.1371/journal.pgen.1002739.s001", "https://dx.doi.org/10.1371/journal.pgen.1002739.s002", "https://dx.doi.org/10.1371/journal.pgen.1002739.s003", "https://dx.doi.org/10.1371/journal.pgen.1002739.s004", "https://dx.doi.org/10.1371/journal.pgen.1002739.s005", "https://dx.doi.org/10.1371/journal.pgen.1002739.s006", "https://dx.doi.org/10.1371/journal.pgen.1002739.s007", "https://dx.doi.org/10.1371/journal.pgen.1002739.s008", "https://dx.doi.org/10.1371/journal.pgen.1002739.s009"], "stats"=>{"downloads"=>7, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Histone_H3_Localizes_to_the_Centromeric_DNA_in_Budding_Yeast/124405", "title"=>"Histone H3 Localizes to the Centromeric DNA in Budding Yeast", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-05-31 01:13:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/629594"], "description"=>"<p>A) H3 is associated with CEN DNA throughout the cell cycle. Strains carrying the minichromosomes with BglII restriction sites 50 bp upstream and downstream of CEN boundaries, 1498 (Cse4-HA6), 1407 (H3-HA3), and 1587 (H2B-HA3) were arrested in G1 with alpha factor and in G2 with nocodazole/benomyl. Chromatin was treated with BglII and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. The FACS profiles are shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen.1002739.s004\" target=\"_blank\">Figure S4B</a>. B) H3 is associated with the CEN DNA on a native chromosome IV. BglII-treated chromatin of strains with BglII sites 50 bp upstream and downstream of CEN boundaries on chromosome IV 2059 (wt), 2043 (Cse4-HA3), and 2042 (H3-HA3) was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads, separated on a 6% denaturing TBE polyacrylamide gel and analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Minichromosome-bound histone H3 does not turn over during the immunoprecipitation procedure. Lysates of strains 1021 (wt, carrying the minichromosome), 1407 (H3-HA, carrying the minichromosome), 1407 (H3-HA3, without the minichromosome), and mixed lysate of 1021 (wt with minichromosome) and 1407 (H3-HA3, without the minichromosome) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads, separated on a 1% agarose gel and analyzed using a <sup>32</sup>P labeled <i>TRP1</i> probe.</p>", "links"=>[], "tags"=>["h3", "localizes", "centromeric"], "article_id"=>300092, "categories"=>["Cell Biology"], "users"=>["Berit Lochmann", "Dmitri Ivanov"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002739.g002", "stats"=>{"downloads"=>4, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Histone_H3_localizes_to_the_centromeric_DNA_/300092", "title"=>"Histone H3 localizes to the centromeric DNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-31 00:01:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/629684"], "description"=>"<p>A) Only the 214 bp BglII CEN4 fragment and no full-length minichromosome is detected in the ChIP/qPCR assay. DNA isolated from untreated and BglII-treated lysates was size-fractionated on 2% agarose gel and analyzed by qPCR. A PCR product after 30 cycles of amplification in a conventional PCR reaction with the same primers that were used for qPCR is shown below. B) Minichromosomal CEN DNA can be co-immunoprecipitated with H3 and Cse4. BglII-treated chromatin of the strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carrying the minichromosome was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified and size fractionated and subjected to qPCR analysis. C) CEN DNA of the native chromosome IV can be co-immunoprecipitatd with H3 and Cse4. BglII-treated chromatin of the strains 2059 (wt), 2042 (H3-HA3), and 2043 (Cse4-HA6) with CEN DNA of the native chromosome IV flanked with BglII was either not cross-linked or cross-linked with formaldehyde followed by immunoprecipitation as in (B). D) Flowchart of the sequential Cse4-H3 ChIP. E) Sequential ChIP of minichromosomal CEN DNA. BglII-treated chromatin of the strains 1923 (Cse4-Myc6) and 2300 (H3-HA3, Cse4-Myc6) carrying the minichromosome was cross-linked with formaldehyde and immunoprecipitated with anti-Myc or anti-HA antibody as indicated in the figure, the DNA was eluted off the beads and re-immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated on a 2% agarose gel and subjected to qPCR analysis. F) The same as in (E) but performed with the native CEN DNA. The strains, 2562 (Cse4-Myc6), and 2561 (H3-HA3, Cse4-Myc6) had CEN DNA of the native chromosome IV flanked with BglII. The bar graphs represent the average values from several independent experiments with SDs.</p>", "links"=>[], "tags"=>["centromeric", "dna", "histone", "h3"], "article_id"=>300181, "categories"=>["Cell Biology"], "users"=>["Berit Lochmann", "Dmitri Ivanov"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002739.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Co_occupancy_of_the_centromeric_DNA_by_histone_H3_and_Cse4_/300181", "title"=>"Co-occupancy of the centromeric DNA by histone H3 and Cse4.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-31 00:03:01"}

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Relative Metric

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