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Results: 7

1.
Figure 1

Figure 1. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Three models of the budding yeast centromeric nucleosome. The cartoon schematic represents each of the proposed centromeric nucleosomes wrapped by DNA. The DNA is represented by the blue lines. Actual nucleosome/DNA contact points are unknown for all three models. A. The octamer model proposes that Cse4 is found in a canonical-like octameric configuration. B. The hemisome model predicts that the centromeric nucleosomes consist of a monomer of each Cse4, H2A, H2B, and H4. The hemisome is predicted to wrap less DNA than an octameric or hexameric nucleosome (~120bp). C. The hexamer model proposes that the centromeric nucleosome is comprised of a tetramer of Cse4-H4 and two molecules of Scm3.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.
2.
Figure 2

Figure 2. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Genome-wide localization of Cse4. Cultures expressing Cse4-12Myc (SBY617) or pGAL1-10-Cse4-12Myc (SBY1071) were grown to mid-log phase and arrested in metaphase with nocodazole. XChIP-chip and qPCR was performed using two independent biological samples and the results were averaged. A. PeakFinder display (Glynn et al., 2004) of the pattern of Cse4-12Myc and pGAL1-10-Cse4-12Myc on Chromosome 1. The centromere is indicated by the circle. B) Box plot representing Cse4-12Myc and pGAL1-10-Cse4-12Myc localization at all centromeres (CEN), centromeres along with 3 array spots on both sides (CEN_7pt), the sequence at the rDNA locus, Ty elements, the telomeres, 3 array spots proximal to all telomeres (Tel_3Pt) and all other array spots (Background). The span of each box represents a range where 95% of the array spots fall. The line in the box represents the median of each sample. The whiskers represent the 1.5 interquartile range. C. XChIP/qPCR was carried out for several non-centromeric regions. Cse4-12Myc ChIP/total chromatin signal was divided by the no antibody/total chromatin control for each region to obtain the fold-enrichment. Error bars represent +/− the average deviation of biological replicates.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.
3.
Figure 5

Figure 5. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Suppression of the scm3Δ strain. The SCM3/scm3Δ (RC204) or CSE4/cse4Δ (RC203) heterozygous diploid knockout “Magic Marker” strain was transformed with plasmids from the HIP overexpression library. A. Sporulated cultures were pinned in quadruplicate onto medium which allowed the recovery of haploid yeast containing either scm3Δ or cse4Δ and the plasmid indicated. B. Dilution assay comparing the growth of scm3Δ covered by either pHIP-Scm3 or pHIP-Cse4 on Gal-Ura medium. C. FACscan analysis and DAPI staining of the scm3Δ strain covered by pHIP-Scm3 and scm3Δ covered by pHIP-Cse4. DAPI stained DNA is shown in white. Arrows indicate cells with abnormal morphology (elongated buds). D. qPCR analysis of pHIP-Myc-Cse4 localization was performed in both a wild type (WT) Scm3 strain containing the pHIP-Myc-Cse4 plasmid (RC205) and a scm3Δ strain containing the pHIP-Myc-Cse4 plasmid (RC192). The primers used amplify a +/− 2kb region around CEN3. A depiction of the features within the region is shown below each histogram. GAL2 serves as a negative control for Cse4 localization. Error bars represent +/− the average deviation of biological replicates.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.
4.
Figure 4

Figure 4. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Co-Immunoprecipitation of histones from MNase-solubilized chromatin. Nuclear lysates were made from a strain expressing both 3FLAG-H2A and Cse4-12Myc (RC188). The chromatin fraction was pelleted and treated with MNase. Western blots were probed with each antibody listed. For each antibody, the lanes are from a single exposure of the same gel. Vertical lines indicate that intervening lanes were cropped. A. DNA was isolated from the MNase solubilized chromatin and was visualized with ethidium bromide on an agarose gel to ensure a majority of mononucleosomes. B. Total nuclear lysate, the MNase-solubilized chromatin fraction, and the insoluble chromatin pellet after MNase treatment were loaded for Western blot analysis. C. Co-immunoprecipitation was performed using the MNase-solubilized chromatin from strain RC188. αFLAG-conjugated beads were used to pulldown 3FLAG-H2A, and the pulldown was probed using the antibodies listed. The negative control pulldown was performed using chromatin from a strain lacking 3FLAG-H2A (SBY617). D. αMyc-conjugated beads were used to pulldown Cse4-12Myc from chromatin made from RC188, and the pulldown was probed using the antibodies listed. The negative control pulldown was performed using chromatin from a strain lacking Cse4-12Myc (RC177). E. DNA was isolated from both the αFLAG and αMyc pulldowns (NChIP). qPCR was then performed to look for enrichment of DNA at either CEN3 or at the site of a well-positioned canonical nucleosome at the PHO5 promoter. Cse4 is significantly enriched at CEN3 when compared to the PHO5 promoter. Levels of 3FLAG-H2A are similar at both sites. F. qPCR signal from immunoprecipitated Cse4-12Myc and 3FLAG-H2A compared to an untagged control strain at CEN3, the rDNA, Ty elements and the PHO5 promoter. Fold enrichment was calculated by dividing IP/input ratios from the untagged control by the IP/input ratios of the epitope-tagged samples.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.
5.
Figure 3

Figure 3. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Cse4 and Scm3 location at high resolution in vivo. XChIP/qPCR analysis was performed on MNase treated chromatin from strain RC82 using overlapping primers that span ~ 400 bp across the centromere on Chromosome 3. GAL2 is a gene located on the arm of Chromosome XII and is a negative control for Cse4 localization. A. MNase-treated chromatin was visualized with ethidium bromide on an agarose gel. B. Quantitative PCR results of the Cse4-12Myc XChIP for the region surrounding CEN3. Only three primer pairs contained sequences from CDEI-II-III, as indicated. The size of each primer pair product is indicated below its respective bar on the histogram. Without antibody, the XChIP/qPCR signal was <10% of the total signal; this has been subtracted from the values presented. The signal from each XChIP has been divided by the signal obtained with total chromatin. Error bars represent +/− the average deviation of biological replicates. C. Quantitative PCR results of the Scm3-3HA XChIP for the region surrounding CEN3. Primers and error bars are the same as in 3B. The Scm3-3HA localization pattern differs from the Cse4-12Myc localization pattern, exhibiting the strongest XChIP signal at the primer which amplifies CDE III and the sequence directly downstream of the centromeric DNA elements.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.
6.
Figure 6

Figure 6. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Reconstitution of Cse4 nucleosomes. A. Cse4 and Xenopus canonical octamers (XCO) were combined with DNA containing the 601-216bp nucleosome positioning sequence and nucleosomes were assembled by salt dilution. The resulting products were resolved on a 5% native poly-acrylamide gel. B. The shifted native gel band from the XCO reconstitution (6A,1) and the bands from the Cse4 reconstitution (6A, 2-5, asterisk) were excised and placed directly into the wells of a denaturing SDS-PAGE gel. This gel was subsequently silver stained and ethidium bromide stained. The top two bands (2-3) from the Cse4 reconstitution contain a full complement of histones and also DNA. C. Both Cse4 and yeast canonical octamers (YCO) were used in a DNA supercoiling assay with recombinant yNap1 as the histone chaperone. Addition of Nap1 and Cse4 octamers to fully relaxed plasmid DNA (R) leads to reversion to a fully supercoiled state (S), an indication of nucleosome deposition onto the plasmid template. D. A supercoiling assay was performed as in 6C, both with and without Cse4 octamers. The reaction was then treated with MNase and loaded onto a Superdex 200 gel filtration column. Fractions were collected and analyzed for DNA content by agarose gel - electrophoresis and Sybr Green I staining. E. Superdex 200 fractions containing ~150bp DNA (6D, asterisks) were treated with Benzonase to digest DNA. These fractions were then analyzed by SDS-PAGE followed by silver staining to examine protein content.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.
7.
Figure 7

Figure 7. From: Cse4 is Part of an Octameric Nucleosome in Budding Yeast.

Dimerization of Cse4. Cse4 was cloned into pRS413 and subjected to site directed mutagenesis which mutated each individual amino acid to an alanine. Each mutated plasmid in the collection was transformed into a haploid cse4Δ strain containing another plasmid with a wild type copy of Cse4, and a plasmid-shuffle assay was performed. A. Growth on 5-FOA identified six single alanine substitutions that do not support growth in the cse4Δ background. B. Using the modeled Cse4 crystal structure as a guide (Bloom et al., 2006), the location of each of the 6 lethal point mutants was mapped. One molecule of Cse4 histone fold domain from the predicted Cse4 octamer crystal structure is shown in color and the essential residues are indicated. The second molecule of Cse4 is shown in grey. Five of six of the lethal point mutations lie in close proximity in either Loop II or Helix III. C. Each of the Cse4 mutants identified in our alanine scanning mutagenesis (7A) was purified from E. coli inclusion bodies. We then used these recombinant Cse4 point mutants to reconstitute octamers in vitro. Octamers were subjected to gel filtration chromatography. The efficiency of octamer formation was calculated by dividing the amount (mg) of octamer recovered by the amount of input histones (Cse4, H2A, H2B, H4). D. Co-immunoprecipitation was performed using whole cell extracts (WCE) isolated from a strain which expresses both Cse4-12Myc and 2FLAG-Cse4 (MM118). αFLAG-conjugated beads were used to pulldown 2FLAG-Cse4 from WCE, and the pulldown was probed by Western blotting with the αMyc antibody. The negative control pulldown was performed using WCE from a strain lacking the 2FLAG tag on Cse4 (MM117). E. Co-immunoprecipitation was performed from WCE isolated from a strain which expresses both Cse4-12Myc and a 2FLAG-Cse4 point mutant identified in the alanine scanning mutagenesis (MM111-116), WT 2FLAG-Cse4 (MM118), or Cse4 without a FLAG tag (MM117). αFLAG-conjugated beads were used to pulldown 2FLAG-Cse4 from WCE, and the pulldown was probed by Western blotting with the αMyc antibody. The values below each lane of the blot represent quantification of the IP band over the lysate band, with the WT sample set to 1. E. SeqXChIP was performed using sheared chromatin isolated from MM118. αMyc antibody was used for the 1st round of XChIP, followed by XChIP using either the αFLAG antibody or no antibody. The signal from each XChIP has been divided by the signal obtained with total chromatin. The centromeric primer pair spans ~ 350bp across CEN3. The GAL2 gene serves as a negative control for Cse4 localization. Error bars represent +/− the average deviation of biological replicates.

Raymond Camahort, et al. Mol Cell. ;35(6):794-805.

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