Lrs⁻ alterations do not show a Sin⁻ phenotype in vivo. (A) Lrs⁻ alterations do not suppress swi/snf defects in HO-lacZ transcription. Strains CY232 (SWI) and CY240 (swi1), both containing an HO-lacZ reporter gene, were transformed with plasmids expressing either wild-type or Lrs⁻ histone H3 or the Sin⁻ histone H4-R45C. Four clones of each were grown on SC-Trp plates, and HO-lacZ reporter gene expression was analyzed using a β-galactosidase filter assay. (B) Relative levels of HO-lacZ and ACT1 gene expression in panel A were determined by RT-PCR for an swi1Δ strain (CY240) transformed with plasmids expressing either wild-type histone H4, histone H3-R83A, or histone H4-R45C. Similar results were observed for strains expressing other Lrs⁻ histones. (C) Lrs⁻ alterations do not lead to derepression of PHO5 gene expression in high-phosphate media. JPY12 cells expressing either wild-type, Lrs⁻, or Sin⁻ histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and harvested for RNA. The level of PHO5 and ACT1 gene expression was analyzed by RT-PCR. PHO5 expression was normalized to ACT1 expression in each strain, with expression levels in the wild-type strain set to 1.0. The bottom panels show raw data from a representative experiment. Quantified data are graphed above and reflect the average of four independent experiments with standard deviations. WT, wild type.

Sin⁻ alterations do not disrupt ribosomal DNA silencing. Silencing was measured by assaying for expression of two RNA polymerase II-transcribed reporter genes: (A) the MET15 reporter inserted in the NTS2 region and (B) the mURA3 reporter in the 5′ region of the 35S rRNA gene. Mutant with wild-type designates the JPY12 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys⁺ Trp⁺ cells). Mutant alone designates the JPY12 strain containing only the mutant histone pDM18 plasmid (Lys⁻ Trp⁺ cells). Control strains include the wild type (WT) containing one or two wild-type histone plasmids and the histone H3 Lrs⁻ mutant A75V.

Sin⁻ and Lrs⁻ alterations do not generally disrupt H3-K79 methylation. (A) JPY12 cells expressing either wild-type, Lrs⁻, or Sin⁻ histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium. Cells were lysed in Laemmli buffer and lysates were analyzed by SDS-PAGE, followed by Western blot analysis with antibodies raised against histone H3 dimethyl K79 and TBP (loading control; upper panel) or histone H3 trimethyl K79 and total histone H3 (loading control; lower panel). (B) Soluble chromatin was isolated from wild-type (JPY12), dot1Δ (EMHY234), and a series of dot1Δ Lrs⁻ strains and incubated with recombinant Dot1p and [³H]S-adenosyl-methionine. Reaction products were separated by SDS-PAGE, and methylated histone H3 was detected by fluorography (upper panel). Histone H3 was detected by immunoblot analysis to normalize input levels of histone proteins in each reaction (lower panel).

Sin⁻ and Lrs⁻ histones do not disrupt Sir2p and Sir4p binding to silent HML and HMR chromatin. Binding of Sir2p and Sir4p to the silent mating-type loci HML and HMR and the nonspecific PHO5 promoter in JPY12 cells harboring either wild-type, Lrs⁻ (A to C), or Sin⁻ (D to F) histones as the sole source of histone H3 or H4 was examined using ChIP analysis, as described in the legend to Fig. 7. An asterisk designates mutations that have previously been shown to disrupt mating (24, 35).

Sir2 overexpression does not rescue the ribosomal DNA-silencing phenotype of lrs or sin mutant alleles. JPY12 strains harboring both sin and lrs mutant alleles were transformed with a 2-μm vector containing SIR2 or an empty vector, and silencing of the mURA3 reporter gene inserted in the 5′ region of the 35S rRNA gene was assayed by measuring growth on SC-Ura and SC-His plus 0.1% 5-FOA. Mutant with wild type designates the ANY34 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys⁺ Trp⁺ cells). Mutant alone designates the ANY34 strain containing only the mutant histone pDM18 plasmid (Lys⁻ Trp⁺ cells).

Mapping of Sin and Lrs alterations onto the surface of the yeast nucleosome core particle. (A) Surface representation of the yeast nucleosome core particle (Brookhaven PDB 1ID3) viewed down the DNA superhelix axis (disk surface). Sin alleles map to SHL ±0.5, and the LRS alleles map to SHL ±2.5. (B) A 90° rotation about the x axis of the image shown in panel A shows the relationship of LRS alleles to the DNA. (C) A 90° rotation about the y axis of the image shown in panel A shows the relationship of the Sin alleles to the DNA. (D) An overlay of the Sin and Lrs histone fold domains demonstrates their structural similarities.

Lrs⁻ alterations do not disrupt nucleosomal array folding in vitro. Histone octamers reconstituted from recombinant histones H2A, H2B, H4, and either wild-type H3 or H3-R83A were deposited onto 208-11 DNA templates by salt dialysis. (A) R83A Lrs⁻ nucleosomes are indistinguishable by native PAGE. Arrays harboring either the wild type or the Lrs⁻ R83A version of histone H3 were cleaved with EcoRI, electrophoretically separated on a native 4% PAGE, and stained with ethidium bromide. The mononucleosome (Nuc) and naked DNA (Naked) bands are indicated. (B) R83A Lrs⁻ nucleosomal arrays show normal intramolecular, salt-dependent folding as shown by sedimentation velocity analysis of 208-11 arrays in the presence or absence of Mg²⁺. The G(s) distributions are depicted for the indicated arrays sedimented in either TE (10 mM Tris [pH 8.0], 0.25 mM EDTA) or TE with 1.75 mM MgCl₂. S_20,w is the sedimentation coefficient corrected to water at 20°C. (C) Intermolecular oligomerization is not altered by the histone H3-R83A mutation. Nucleosomal arrays were incubated in TE with varying concentrations of MgCl₂ at room temperature for 15 min, followed by centrifugation in a microcentrifuge at 14,000 × g for 10 min. The percentage of array remaining in the supernatant is plotted as a function of MgCl₂ concentration. (D) rDNA silencing of the lrs mutant allele H3-R83A was determined by assaying for growth on SC-Ura to measure expression of the mURA/His reporter and plating on Pb²⁺-containing media to assay expression of MET15 reporter, both integrated into the rDNA locus. WT, wild type. R, ratio.

Both Lrs⁻ and Sin⁻ alterations disrupt telomeric silencing. Silencing was measured in strain ANY34 by assaying for expression of two reporter genes: (A) the ADE2 reporter gene integrated at the V-R telomere and (B) the URA3 reporter gene integrated at the VII-L telomere. Mutant with wild type designates the ANY34 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys⁺ Trp⁺ cells). Mutant alone designates the ANY34 strain containing only the mutant histone pDM18 plasmid (Lys⁻ Trp⁺ cells). Control strains include the wild type (WT) containing one or two wild-type histone plasmids and the histone H3 Lrs⁻ mutant A75V, which displays loss of telomeric silencing.

Sin⁻ and Lrs⁻ histones disrupt Sir2p and Sir4p binding to telomeric chromatin. JPY12 cells expressing either wild-type, Lrs⁻, or Sin⁻ histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and then processed for Western immunoblot analysis (A) or chromatin immunoprecipitation (B to E). (A) Western blot analysis shows that Lrs⁻ and Sin⁻ histone alterations do not affect Sir2p protein levels. (B to D) ChIP analysis of Sir2p or Sir4p recruitment to the telomere, 500 bp (B) and 70 bp (C) from the end of the right arm of chromosome VI and to the nonspecific PHO5 promoter (D). An additional JPY12 strain containing an SIR4 deletion (Sir4Δ), which abolishes both Sir2p and Sir4p telomeric binding (21), was used to determine background signals in this assay. Quantification shown below the panels indicates the percent immunoprecipitated telomeric DNA (IP/Input) normalized to the percent IP from the nonspecific PHO5 locus (D) to normalize for IP efficiency. The normalized value for the wild-type strain was set at 1.0. Quantification below panel D indicates the percent immunoprecipitated PHO5 DNA (IP/Input), with the value for wild-type cells set at 1.0. (E) Rap1p binding 70 bp from the end of the right arm of chromosome VI was analyzed using antibodies to Rap1p. Similar results were observed in an independent experiment. Chr., chromosome.

In the absence of Sir1p, Sin⁻ and Lrs⁻ histones abolish Sir2p and Sir4p binding to silent HML chromatin. (A) JPY12 cells containing the indicated SIR gene deletions and harboring either wild-type, Lrs⁻, or Sin⁻ histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and processed for ChIP analysis, as described in the legend to Fig. 7. (A) Binding of Sir2p and Sir4p to HML in strains containing histone H3 Lrs⁻ alterations compared to strains containing wild-type histone H3. (B) Binding of Sir2p and Sir4p to HML in strains containing Sin⁻ histone alterations compared to wild-type histone H3 and the H3 R83A Lrs⁻ alteration. As a control for nonspecific binding, Sir2p and Sir4p binding to the PHO5 promoter is also shown. Sir2p and Sir4p binding to HML was normalized to the amount of binding observed for the nonspecific PHO5 locus as described in the legend to Fig. 7. WT, wild type.