Yeast cells lacking histone modifying enzyme subunits are sensitive to histone overexpression. (A) Sensitivity of mutant yeast cells lacking histone modifying enzyme subunits to histone overexpression. Wild type (WT) or the indicated mutant strains were transformed with a plasmid carrying galactose inducible HA-epitope tagged H3 (pYES2-HTH-HHT2) or the empty vector (pYES2-HTH).11 The rad53Δ strain carries the crt1Δ as well to suppress the loss of viability due to the essential nature of Rad53.46 10-fold serial dilutions of the indicated strains were plated on glucose or galactose media and incubated for 3 days at 30°C prior to being photographed. (B) Histone H3 overexpression in yeast cells lacking histone modifying enzyme subunits. Four independently isolated transformants for each of the indicated mutant strains carrying the plasmid pYES2-HTH-HHT2 were grown overnight in minimal media lacking uracil and with raffinose as the carbon source. Cells were then treated with galactose for 90 minutes prior to harvesting 10 million cells per sample. Whole cell lysates were prepared by the alkaline lysis method47 and processed for Western blotting to detect both the endogenous and overexpressed epitope-tagged histone H3 using the H3-C polyclonal antibody as described previously.10,11 Asterisks indicate the transformants used in the experiment shown above in (A).

Indirect end-labeling analysis of chromatin following in vivo DNase I cleavage. Galactose inducible DNase I was expressed in wild type cells either expressing or not expressing GAL-HA-H3 as described previously.26 The DNase I cleaved genomic DNA was purified and analyzed by indirect end-labeling with multiple cycle primer extension25 using a ³²P-end-labeled primer specific to the ACT1 locus in addition to the primers for MATa and RDN loci described above in Figure 2B. The reaction products were resolved on a 5% denaturing polyacrylamide gel which was processed for autoradiography. On the right hand side of the parts, a “−” sign indicates loss of a DNase I hypersensitive site while a “+” sign indicates the gain of a site upon GAL-HTH-H3 overexpression.

Excess histones can bind to RNA. An exponentially growing yeast strain with a FLAG epitope on the chromosomal HHT2 gene (FLAG-H3) and carrying the plasmid pYES6/CT-HA3-HHT2 for galactose inducible HA3-H3 overexpression was treated with or without galactose prior to performing RNA immunoprecipitation (RIP) using FLAG or HA antibodies as described in Materials and Methods. The amount of ACT1, MATa, HHT1 and HHT2 transcripts co-immunoprecipitated using the FLAG and HA antibody beads in the absence of galactose mediated histone overexpression (i.e., the background signal) was arbitrarily set to “1” and the relative binding of HA-H3 to these RNAs upon the addition of galactose is shown here. No signal above background was obtained in control qPCR carried out following RNaseA treatment of the immunoprecipitated material, as well as when the reverse transcriptase enzyme was left out of the reverse transcription reaction used to generate the cDNA for qPCR analysis. Error bars represent standard error of the mean from three experiments.

Histone overexpression does not affect the bulk chromatin structure but alters the fine structure of chromatin. (A) Micrococcal nuclease digestion of bulk chromatin. MNase cleaved DNA isolated from cells in the presence or absence of histone overexpression was purified as described in the Materials and Methods section. This DNA was then resolved on a 2% agaorse gel and stained with ethidium bromide to visualize the nucleosomal ladder. The positions of mono-, di-, tri- and tetra-nucleosomes are indicated. (B) Analysis of chromatin structure at specific loci using MNase cleavage. The MNase digested genomic DNA purified above in (A) was used as a template for analysis by indirect end-labeling with multiple cycle primer extension25 using ³²P-end-labeled primers specific to the MATa and RDN loci to perform linear PCR. The reaction products were resolved on a 5% denaturing polyacrylamide gel which was processed for autoradiography. On the right hand side of the parts, a “-” sign indicates loss of a MNase cleavage site while a “+” sign indicates the gain of a site upon GAL-HTH-H3 overexpression.

Genome wide comparison of gene expression in wild type yeast cells upon overexpression of individual or pairs of histone genes. (A) Overexpression of histone H3 alone does not result in appreciable changes in gene expression. Microarray analysis was carried out on total RNA isolated from the wild type W303-1A strains carrying either the empty vector or a galactose inducible histone H3 gene as described in the Materials and Methods section. A scatter plot is shown and illustrates the expression level of genes along the x- and y-axes representing transcript levels reflecting gene expression values on a log₂ scale for the strains indicated. The three solid green lines represent fold-changes in transcript levels from the baseline, which is represented by the green line in the middle and indicates identical transcript levels in the two strains being compared. The upper solid green line represents a two-fold increase in transcript levels, while the lower green line represents a two-fold reduction in transcript levels. The dashed purple line is the linear regression with the coefficient of correlation R2 indicated on the bottom right corner. Each transcript is represented by a color coded data point to reflect where it is in comparison to the middle green line, with red color for upregulated transcripts and blue color for downregulated transcripts (the darker the color, the greater the fold-change in the transcript levels). Only the transcript levels of the overexpressed histone H3 (HHT2) was significantly elevated in this experiment and the corresponding data point is indicated by the arrow. (B) Changes in gene expression upon co-overexpression of the histone H3-H4 gene pair. Scatter plot for the indicated strains as in (A). (C) Changes in gene expression upon simultaneous overexpression of all four core histones. Scatter plot for the indicated strains as in (A). (D) Summary of the gene expression data derived from microarray analyses. The number of transcripts altered two-fold or more upon overexpression of different histones is summarized in a tabular form.