Histone H3 Localizes to the Centromeric DNA in Budding Yeast

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

Histon H3 und seine zentromere Variante Cse4 co-okkupieren die zentromere DNS in Saccharomyces cerevisiae

For cell division, the replicated genetic information of a cell needs to be accurately segregated to the emerging daughter cells. This is achieved by packaging the DNA into sister chromatids, which are separated by the poleward pulling force of microtubules. Microtubules are attached to the centromeres of the sister chromatids by a multi-protein complex called the kinetochore. The assembly of the kinetochore at the centromere is directed by specialized centromeric chromatin. Conventional chromatin contains nucleosomes, which are comprised of two copies each of the histones H3, H4, H2A, and H2B. This protein octamer organizes 147 bp of DNA by wrapping it in 1.7 turns. In contrast to canonical nucleosomes it is believed that centromeric nucleosomes are devoid of histone H3 and contain in its place the variant CENP-A. CENP-A is an essential protein necessary for kinetochore formation and homologues have been identified in all eukaryotes studied so far. Whereas higher eukaryotes have long arrays (kilo- to megabases of DNA) of centromeric chromatin with CENP-A containing nucleosomes, the budding yeast centromeric DNA is approximately 125 bp with only a single Cse4 (budding yeast homologue of CENP-A) containing nucleosome that is sufficient to recruit and assemble the kinetochore. This so-called point centromere is thought to represent the smallest unit of larger centromeres. However, the exact composition of this special centromeric nucleosome is subject of intensive debate with contradicting models proposed. We investigated the exact composition of the centromeric nucleosome in budding yeast by developing a novel chromatin immunoprecipitation technique. This ChIP approach was based on the use of a centromeric DNA fragment that is too short to accommodate more than a single nucleosome. Not only did we observe the interaction of CENP-A (Cse4), histones H4, H2A, and H2B with the centromeric DNA, we also discovered a strong association of histone H3 with this fragment. By employing a sequential ChIP approach we could show that histone H3 and CENP-A (Cse4) are co-occupying the centromeric DNA. Our experimental evidence supports the view of a heterotypic H3/CENP-A (Cse4) nucleosome at the centromere and all future models need to account for the presence of histone H3 at the centromeric DNA.Für die Zellteilung muss die replizierte genetische Information einer Zelle korrekt auf die entstehenden Tochterzellen aufgeteilt werden. Die DNS wird dafür in Schwesterchromatiden verpackt, welche mit Hilfe von Mikrotubuli zu den entgegengesetzten Zellpolen gezogen werden. Die Mikrotubuli sind dafür mit den Zentromeren der Schwesterchromatiden über einen Multiproteinkomplex, dem Kinetochor, verbunden. Der Aufbau des Kinetochors findet gezielt an dem speziellen Chromatin des Zentromers statt. Kanonisches Chromatin besteht aus Nukleosomen, welche aus je zwei Molekülen der Histone H3, H4, H2A und H2B zusammengesetzt sind und diese Proteinoktamere sind von 147 bp DNS in 1.7 Windungen umwickelt. Im Vergleich dazu enthalten zentromere Nukleosomen die Histon H3 Variante CENP-A und es gibt die Auffassung, dass diese Nukleosomen kein Histon H3 beinhalten. CENP-A ist ein essentielles Protein, welches unerlässlich ist für den Aufbau des Kinetochors. Homologe Proteine von CENP-A wurden in allen untersuchten Eukaryoten entdeckt. Während höhere Eukaryoten lange Reihen (kbp bis Mbp DNS) von zentromeren Chromatin mit CENP-A Nukleosomen besitzen, hat die zentromere DNA der Sprosshefe nur eine länge von ungefähr 125 bp mit einem einzigen Cse4 (Sprosshefehomolog von CENP-A) beinhaltendem zentromeren Nukleosom. Dieses Nukleosom ist ausreichend um Kinetochorproteine zu rekrutieren und den Multiproteinkomplex aufzubauen. Bei diesem sogenannten Punktzentromer nimmt man an, dass es die kleinste Einheit von größeren Zentromeren darstellt. Allerdings wird der genaue Aufbau des speziellen zentromeren Nukleosoms mit verschiedenen, einander widersprechenden Modellen kontrovers diskutiert. Wir haben eine neuartige Chromatinimmunopräzipitationstechnik (ChIP) entwickelt um den exakten Aufbau des zentromeren Nukleosoms in Sprosshefe zu untersuchen. Dieser neue ChIP-Ansatz basiert auf der Anwendung eines zentromeren DNS-Fragments, das zu kurz ist um mehr als einem Nukleosom Platz zu bieten. Unsere Ergebnisse bestätigten die Assoziation von CENP-A (Cse4), Histon H4, H2A und H2B mit der zentromeren DNS. Jedoch entdeckten wir auch eine starke Interaktion von Histon H3 mit diesem DNS-Fragment. Mit der Durchführung eines sequenziellen ChIP-Ansatzes konnten wir zudem zeigen, dass Histon H3 und CENP-A (Cse4) gleichzeitig mit der zentromeren DNS assoziiert sind. Unsere Ergebnisse legen nahe, dass es sich bei dem zentromeren Nuklesom in Sprosshefe um ein heterotypisches Nukleosom, mit je einem Molekül Histon H3 und CENP-A (Cse4) handelt. Zukünftige Modelle müssen Histon H3 in den Aufbau des zentromeren Nukleosomes mit einbeziehen

Cse4 association with CDEI/II and CDEIII.

<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 ³²P labeled probe for the pericentric CEN4 sequence (to detect the CDEI/II containing fragment) and a ³²P 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

Co-occupancy of the centromeric DNA by histone H3 and Cse4.

<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

Models of how H3 and Cse4 can co-occupy the centromeric DNA.

<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

Composition of the centromeric nucleosome.

<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 ³²P 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 ³²P 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

Histone H3 localizes to the centromeric DNA.

<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 ³²P labeled <i>TRP1</i> probe.</p