Histone-histone interactions and centromere function - PubMed (original) (raw)

Histone-histone interactions and centromere function

L Glowczewski et al. Mol Cell Biol. 2000 Aug.

Abstract

Cse4p is a structural component of the core centromere of Saccharomyces cerevisiae and is a member of the conserved CENP-A family of specialized histone H3 variants. The histone H4 allele hhf1-20 confers defects in core centromere chromatin structure and mitotic chromosome transmission. We have proposed that Cse4p and histone H4 interact through their respective histone fold domains to assemble a nucleosome-like structure at centromeric DNA. To test this model, we targeted random mutations to the Cse4p histone fold domain and isolated three temperature-sensitive cse4 alleles in an unbiased genetic screen. Two of the cse4 alleles contain mutations at the Cse4p-H4 interface. One of these requires two widely separated mutations demonstrating long-range cooperative interactions in the structure. The third cse4 allele is mutated at its helix 2-helix 3 interface, a region required for homotypic H3 fold dimerization. Overexpression of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression of cse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants, suggesting that histone H3 competes with Cse4p for histone H4 binding. However, the relative resistance of the Cse4p-H4 pathway to H3 interference argues that centromere chromatin assembly must be highly regulated.

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Figures

FIG. 1

FIG. 1

Conditional growth and protein levels of Cse4p point mutants. (A) The growth phenotypes of the wild-type plasmid shuffle strain and the histone fold Cse4p mutants at the permissive (28°C) and restrictive (37°C) temperature are shown. Tenfold serial dilutions of each strain were spotted onto synthetic complete medium minus tryptophan and grown for 2 days at the indicated temperatures. The location of the point changes in the mutants can be found in Table 1. (B) Mutant Cse4 proteins are stable at the restrictive temperature. Crude lysates were made from the indicated strains grown at 28°C and then shifted to 37°C for 4 h. Equal amounts of protein were run on a sodium dodecyl sulfate–12.5% polyacrylamide gel and electroblotted to nitrocellulose. Western blot analyses were performed using and anti-HA antibody (HA.11).

FIG. 2

FIG. 2

G2/M arrest in cse4 mutants. (A) Flow cytometry of cse4 strains in exponential cultures grown at 28°C. Wild-type and cse4 mutant strains were stained for DNA content with propidium idodide and analyzed by flow cytometry. Representative histograms of cell number versus relative fluorescence are shown. In each panel the circles show the channel data points and the solid line shows the overall fit of the data to a model of G1, S, and G2/M phase cell populations. Below each plot the individual G1, S, and G2/M model fits are shown at half scale. (B) Cell division cycle profiles. The relative lengths of the cell cycle phases were approximated using culture doubling times and the percentage of cells in each phase as estimated from the flow cytometry histograms. The estimates are the mean of six independent measurements for each mutant. The profiles are aligned at the S/G2/M boundary for comparison of the relative lengths of the G2/M cell cycle phase. (C) Morphology of cse4 cells accumulated at restrictive temperature. The nuclear and spindle morphology are shown for the class of cse4-102, cse4-110, and cse4-111 mutant cells that accumulate following arrest at the restrictive temperature (Table 2). Each pair of micrographs illustrates the nuclear staining (DAPI) and the spindle staining (tubulin) visualized with an antitubulin antibody.

FIG. 3

FIG. 3

hhf1-20 and cse4 mutants activate the spindle-kinetochore checkpoint pathway. (A) Checkpoint control in hhf1-20 mutants. Cultures were grown to mid-log phase at 28°C and then shifted to 37°C, the restrictive temperature for hhf1-20 cells. The kinetics of cell viability are shown following the shift to the restrictive temperature. Open circle, MSY559 (HHF1); open square, MSY554 (hhf1-20); open diamond, MSY556 (hhf1-20 rad9::URA3); filled circle, MSY713 (hhf1-20 bub2::URA3). (B) Checkpoint control in GAL1::CSE4 conditional null mutants. Cultures were grown to mid-log phase in galactose medium and then shifted to raffinose medium to turn off CSE4 expression. The kinetics of cell viability are shown following the shift to raffinose medium. Open square, MSY753 (GAL1::CSE4); open diamond, MSY824 (GAL1::CSE4 rad9::URA3); filled diamond, MSY819 (GAL1::CSE4 mad1).

FIG. 4

FIG. 4

Transcriptional read-through of CEN DNA in cse4 and hhf1-20 mutants. For each panel the indicated strains were grown in synthetic complete galactose medium lacking histidine, and cell extracts were prepared after 18 h of growth at the semipermissive temperature. The units for levels of β-galactosidase activity (LacZ Activity) are nanomoles of ONPG cleaved per milligram of lysate protein per minute. (A) LacZ expression was determined in wild-type, ctf13-30, and isogenic cse4 mutant strains grown at 33°C. In these strains GAL1::LacZ expression requires transcription through a CEN DNA insert located in an intron of the reporter gene. The CEN DNA insert partially blocks transcription in wild-type cells, giving low levels of expression. In ctf13-30 mutants this block is compromised, giving increased expression (14). The values represent the average of two to three independent cultures. (B) Transcription through CEN DNA was determined for isogenic HHF1 and hhf1-20 strains as described above. Cells were grown at a semipermissive temperature of 34°C, and the values represent the average of three to four independent cultures. (C) LacZ expression was determined for wild-type and two cse4 mutant strains as described above. However, in these strains the GAL1::LacZ reporter gene is carried on a 2μ plasmid and lacks the CEN DNA insert. In all panels, error bars show standard deviations.

FIG. 5

FIG. 5

Model of the locations of cse4 amino acid substitutions. The protein structures of the Cse4p-H4 dimer were modeled based on the X-ray crystal structures of histones H3 and H4 in the nucleosome (Materials and Methods). In each panel structures and labels referring to Cse4p are shown in green and those referring to histone H4 are shown in white. The wild-type residue that is mutated in each cse4 allele is shown in red. Atoms that are within 5 Å of the side chain of the mutated residue are colored yellow. (A) A partial nucleosome structure depicting a Cse4p-H4 dimer and one turn of DNA is shown. The view is down the superhelical axis of the DNA, perpendicular to the pseudodyad axis of the nucleosome which is within the plane of the illustration extending from left to right. Approximate vectors for the points of view shown in panels B, C, D, and E are indicated by the red arrows. (B) The region surrounding L175, one of the two amino acids mutated in cse4-102, is shown. The view is looking down between loop 1 of Cse4p and loop 2 of histone H4. (C) The region around M217, the second amino acid mutated in cse4-102, is shown. The view is looking down just behind loop 1 of histone H4 onto helix 3 of Cse4p. Foreground residues in helix 1 of histone H4 have been clipped to expose the interior region. (D) The region around L196, the residue mutated in cse4-110, is shown. The view is looking up towards the dyad axis along helix 3 of Cse4p. (E) The region around L193, the residue mutated in cse4-111, is shown. The view looks down through the dimer where helix 2 of Cse4p and helix 2 of histone H4 cross.

FIG. 6

FIG. 6

Chromatin immunoprecipitation of CEN3 DNA with wild-type and mutant Cse4 protein. The region of chromosome III from approximately bp 112,800 to 115,200 is illustrated in the diagram. The location of CEN3, containing the CDEI, CDEII, and CDEIII sequences, is indicated by the open box in the center of the region separating the left (L) and right (R) arms of chromosome III. Cells expressing epitope-tagged Cse4 protein from either the wild-type gene (CSE4-HA) or the mutant cse4-111 allele (cse4-111-HA) were grown at the restrictive temperature for 4 h. Formaldehyde cross-linked chromatin was prepared and immunoprecipitated with anti-HA antibody specific for Cse4-HAp (α-HA) or mock treated (No Ab). The coimmunoprecipitated DNAs and total input DNA (Total) were analyzed by PCR with primers corresponding to short, overlapping intervals flanking CEN3. A map of the amplified intervals is provided by location of the boxes shown below the chromosome III diagram. The sets of PCR products from these reactions are shown in the images located below the corresponding sequence interval. Mutant Cse4-111p remains specifically associated with CEN3 DNA at the restrictive temperature. Similar results were obtained for cse4-102 and cse4-110.

FIG. 7

FIG. 7

Cse4p suppression of hhf1 mutations. (A) Cse4p is a specific suppressor of hhf1-20. The individual hhf1 mutant yeast strains indicated on the left of each row were transformed with a high-copy plasmid expressing CSE4. Tenfold serial dilutions of each strain were spotted on synthetic complete medium lacking uracil and grown at permissive (28°C) and restrictive (37°C) temperatures. (B) High-copy expression of mutant cse4 alleles does not suppress hhf1-20. hhf1-20 cells were transformed with five different plasmids containing the CSE4 alleles indicated to the left of each row: vector (pRS425), CSE4 (pLG8), cse4-102 (pLG50), cse4-110 (pLG48), or cse4-111 (pLG49). Tenfold serial dilutions were spotted onto synthetic complete medium lacking leucine and grown at permissive (28°C) and restrictive (37°C) temperatures.

FIG. 8

FIG. 8

Genetic interactions of histones H3 and H4 with cse4 alleles. (A) Overexpression of histone H4 suppresses cse4-102 and cse4-111 but not cse4-110. The histone H4 gene HHF1 was placed under control of the GAL1 promoter in plasmid pLG39. The CSE4 wild-type, cse4 mutant, and ctf13-30 strains indicated on the left of each row were transformed with either vector alone (pRS426) or pLG39 carrying the GAL1::HHF1 construct (2μ HHF1). Strains were grown on synthetic complete galactose medium at either permissive (28°C) or minimal restrictive (35, 37, or 37.5°C) temperatures. (B) Overexpression of histone H3 is dosage lethal in cse4 mutants. The histone H3 gene HHT1 was placed under control of the GAL1 promoter in plasmid pLG41. The CSE4 wild-type, cse4 mutant, and ctf13-30 strains were transformed with vector (pRS426) or pLG41 (2μ HHT1) and grown at either permissive (28°C) or semipermissive (33 or 32°C) temperatures.

FIG. 9

FIG. 9

ChIP of CEN3 DNA with Cse4 protein following overexpression of histones H3 or H4. Strains expressing epitope-tagged Cse4 protein from wild-type, cse4-102, and cse4-110 alleles were grown on galactose medium to induce overexpression of either histone H3 (top panel) or histone H4 (bottom panel) from the GAL1 promoter carried on 2μ multicopy plasmids (+). Control cells contained the 2μ vector only (−). Cultures were grown for 4 h at the temperatures indicated, and the in vivo cross-linking of Cse4 protein with the core CEN3 DNA fragment was determined by ChIP as described in the legend to Fig. 6. The cse4-102 and cse4-110 mutants are able to form colonies at 33°C on galactose medium with vector alone but are inviable at that temperature when histone H3 is overexpressed. Conversely, cse4-102 cells are inviable at 35°C on galactose medium but can form colonies at that temperature when histone H4 is overexpressed. The cse4-110 mutant is poorly suppressed by histone H4 overexpression and fails to give colonies at 37°C with either vector or histone H4 overexpression. The association of Cse4 protein with CEN3 DNA was unaffected by overexpression of either histone H3 or histone H4 in the mutant strains.

FIG. 10

FIG. 10

A model of the Cse4p-H4 assembly pathway. (A) A simple model for the interactions of histones H3 and H4 with Cse4p is shown. In this model, histones Cse4p and H3 compete for the binding of histone H4. The structural and genetic results reported here suggest that the formation of the H4-Cse4p dimer is impaired in hhf1-20, cse4-102, and cse4-111 mutants, while formation of the (H4-Cse4p)2 tetramer is impaired in the cse4-110 mutant. In those mutants, overexpression of histone H3 further antagonizes the formation of the centromere variant nucleosome, leading to dosage lethality. (B) Cooperative downstream interactions would favor the assembly of a CEN variant nucleosome in the face of histone H3 competition for histone H4. Additional known centromere components are illustrated. It is not known whether histone H2A-H2B dimers participate in the structure, or whether specialized chromatin assembly factors (CAFs) may be required.

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