Regulatory and atomic context of histone H3 serine 57.

<p>A. Theoretical scheme imbricating the acetylation cycle of H3-K56 with the putative phosphorylation cycle of H3-S57. Double point mutations introduced to constitutively mimic the 4 possible modification states are indicated in red. The observed fitness upon induction of S-phase double strand breaks by MMS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone-0010851-g003" target="_blank">Figure 3A</a>) is shown as smileys next to the mutations. B. Ribbon view of one nucleosomal molecule of histone H3 (blue) and of histone H4 (green) as well as the first 14 base pairs of nucleosomal DNA, displaying the H3-K56, H3-S57 and H4-R40 residue atoms as ball and stick. H3-K56 makes a water mediated contact with bp 9 of the DNA and H3-S57 is linked by a hydrogen bridge to H4-R40 on histone H4 helix 1. This figure was built using Yasara (<a href="http://www.yasara.org" target="_blank">http://www.yasara.org</a>) and PDB file 1ID3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone.0010851-White1" target="_blank">[2]</a>. The same hydrogen bridges are visible in all the nucleosome crystal structures we examined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone.0010851-Luger1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone.0010851-White1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone.0010851-Davey1" target="_blank">[54]</a>.</p

Interplay between H3-K56 and H3-S57 mutations.

<p>A. Five-fold serial dilutions of the indicated mutant strains were analyzed on the indicated YEPD plates. One representative experiment out of at least three is shown. Note the formamide sensitivity of both single H3-S57 mutants and also the inverse MMS, HU and 6-AU hypersensitivity relations between the H3-S57A and H3-S57E mutations in the contexts of the H3-K56R and H3-K56Q point mutations. B. Wild type H3, H3-S57A and H3-57E were tested as in (A) but using higher concentrations of MMS and of HU.</p

Prolonged G2/M delay upon brief exposure of H3-K56R/A/Q-S57E mutants to MMS in G1.

<p>The indicated yeast strains were arrested in G1 with mating pheromone and divided into two samples that were exposed (bottom) or not (top) to 0.1% MMS for 20 minutes prior to release from a G1 arrest into complete growth medium. Cell cycle progression was measured as a function of cellular DNA content in samples collected at the indicated time.</p

Lack of dominant negative effects of the H3-K56/H3S57 mutant histone genes.

<p>The plasmid-borne H3-K56 (Q, R) and -S57 (A, E) <i>hht2</i> point mutations were analyzed for dominant negative effects in YN1038, a strain harboring wild type chromosomal copies of the <i>HHT2</i> and <i>HHT1</i> yeast histone H3 genes. Neither growth rates measured at 30°C (A) and nor clone sizes determined on plates containing methyl methanesulfonate, hydroxyurea or formamide (B) revealed any dominant effects of the mutant histone H3 genes when wild type yeast histone H3 was present.</p

Recovery rates of yeast clones harboring H3-K56/S57 point mutations.

<p>YN1375-derived yeast strains harboring the wild type allele of the yeast histone H3 gene <i>HHT2</i> on a <i>URA3</i> gene-bearing plasmid as well as the indicated <i>hht2</i> allele on a <i>HIS3</i>-gene bearing plasmid were streaked-out on 5-FOA plates to select for cells that had lost the <i>URA3</i>-bearing plasmid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone.0010851-Ozdemir1" target="_blank">[3]</a>. Note that 5-FOA resistant clones emerged at similar frequencies for all the strains except for the positive control strain (<i>WT</i>, <i>ura3</i>) that lacked a <i>URA3</i> gene to begin with and the negative control strain that lacked a histone H3 gene borne on a <i>HIS3</i> plasmid (<i>hht1</i>Δ, <i>hht2</i>Δ, <i>pURA-HHT2</i>). The <i>hht2-K56R</i> allele is not shown here but an identical experiment was described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010851#pone.0010851-Ozdemir1" target="_blank">[3]</a>.</p

<i>Saccharomyces cerevisiae</i> strains used in this study.

<p><i>Saccharomyces cerevisiae</i> strains used in this study.</p

Orthologs of known SWI/SNF complex subunits in human, fly and yeast.

<p>Orthologs of known SWI/SNF complex subunits in human, fly and yeast.</p

Abundance of purified proteins^‡ in each TAP-tag preparation.

‡<p>Protein abundance is represented by the exponentially modified Protein Abundance Index <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834-Ishihama1" target="_blank">[96]</a>.</p>*<p>mRNA abundance was estimated from probe set fluorescence signal intensities, as recommended by Affymetrix (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834.s002" target="_blank">Data S2</a>).</p

Complex walking.

<p>(A) Silver-stained gels of the actual purified protein preparations that were analyzed by mass spectrometry. The respective TAP-tag fusion proteins are designated by black triangles. Size markers (Da) are indicated for every gel. Banding patterns differ because the gels were not all run under the same conditions. (B) Osprey interaction network <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834-Breitkreutz1" target="_blank">[131]</a> based on the mass spectrometry results obtained with the material shown in panel A. Blue name labels indicate the TAP-tag fusion employed here. The thickness of the lines reflect purification yield and are proportional to the emPAI values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834-Ishihama1" target="_blank">[96]</a> shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone-0033834-t001" target="_blank">Table 1</a>. The presence of orthologs in yeast and <i>Drosophila</i> genomes of the human factors that are displayed is indicated on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone-0033834-t002" target="_blank">Table 2</a>.</p

SS18 and the animal-specific SWI/SNF subunits.

<p>(A) Domain organization of the human proteins DPF1,-2,-3; PHF10; SS18 and its paralog SS18l1/crest; and BCL7A,-B,-C. We note that while CG2682, the <i>Drosophila melanogaster</i> ortholog of DPF2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone-0033834-t002" target="_blank">Table 2</a>), lacks the C2H2 domain, this domain is present in the <i>Tribolium</i> ortholog D6WFQ9_TRICA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834-Richards1" target="_blank">[125]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834-Punta1" target="_blank">[132]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033834#pone.0033834-Hunter1" target="_blank">[133]</a>, suggesting conservation of this domain in protostome and in deuterostome animals (B) Co-immunoprecipitation of SS18-SSX1^MYC by antibodies directed against human BRM. (C) Co-immunoprecipitation of SS18^MYC by antibodies directed against DPF2 and BRD9.</p