Roles for H2A.Z and its acetylation in GAL1 transcription and gene induction, but not GAL1-transcriptional memory

Abstract

H2A.Z is a histone H2A variant conserved from yeast to humans, and is found at 63% of promoters in Saccharomyces cerevisiae. This pattern of localization suggests that H2A.Z is somehow important for gene expression or regulation. H2A.Z can be acetylated at up to four lysine residues on its amino-terminal tail, and acetylated-H2A.Z is enriched in chromatin containing promoters of active genes. We investigated whether H2A.Z's role in GAL1 gene regulation and gene expression depends on H2A.Z acetylation. Our findings suggested that H2A.Z functioned both in gene regulation and in gene expression and that only its role in gene regulation depended upon its acetylation. Our findings provided an alternate explanation for results that were previously interpreted as evidence that H2A.Z plays a role in GAL1 transcriptional memory. Additionally, our findings provided new insights into the phenotypes of htz1Delta mutants: in the absence of H2A.Z, the SWR1 complex, which deposits H2A.Z into chromatin, was deleterious to the cell, and many of the phenotypes of cells lacking H2A.Z were due to the SWR1 complex's activity rather than to the absence of H2A.Z per se. These results highlight the need to reevaluate all studies on the phenotypes of cells lacking H2A.Z.

Figure 1. Acetylated H2A.Z was important for GAL1 induction.

Q-RT PCR of GAL1 mRNA performed on HTZ1 (JRY7971), htz1Δ (JRY7754), and htz1-K3,8,10,14R (JRY7983) cultures that were grown long-term in YP-glucose (2%) prior to being transferred into YP-galactose (2%). Open circles represent the average of three biological replicates. Bars represent standard deviations of values from these replicates. Solid lines represent the best-fit curve for the measured data. See text for details. (B) ChIP analysis of H2A.Z-FLAG at the GAL1 promoter in cells grown long-term in YP-glucose (2%). (C) Q-RT PCR of GAL1 mRNA performed on HTZ1 (JRY7971), htz1Δ (JRY7754), and htz1-K3,8,10,14R (JRY7983) cultures that were grown for 20 h in YP-galactose (2%) prior to 12 h of growth in YP-glucose (2%) prior to being transferred into YP-galactose (2%). Open circles represent the average of three biological replicates. Bars represent standard deviations of values from these replicates. Solid lines represent the best-fit curve for the measured data. See text for details.

Figure 2. H2A.Z was not required for GAL1 transcriptional memory.

(A) Q-RT PCR of GAL1 mRNA performed on RNA from HTZ1 (CRY1) and htz1 (DBY50) grown long term in CSM-Glucose (2%) prior to being transferred into CSM galactose (2%). Open circles represent the average of three biological replicates. Bars represent standard deviations of values from these replicates. Solid lines represent the best-fit curve for the measured data. See text for details. (B) Q-RT PCR of GAL1 mRNA performed on RNA from HTZ1 (CRY1) and htz1 (DBY50) grown in CSM-galactose (2%) for 20 h prior to being grown in CSM-Glucose (2%) for 12 h prior to being transferred into CSM galactose (2%). Open circles represent the average of two biological replicates. Bars represent standard deviations of values from these replicates. Solid lines represent the best-fit curve for the measured data. See text for details.

Figure 3. Acetylated H2A.Z was important for GAL1 gene induction.

Flow cytometry analysis was performed using Gal1-GFP on HTZ1 (JRY9002), htz1Δ (JRY9004), and htz1-K3,8,10,14R (JRY9003) cells grown long-term in YP-glucose (2%) prior to being transferred into YP-galactose (2%). The histograms in this figure represent the distribution of cells within each culture as a function of their GFP intensity.

Figure 4. htz1Δ cells' galactose induction phenotypes are more severe than those of htz1-K3,8,10,14R cells.

A threshold level of GFP-intensity was set so that between 1% and 2% of glucose-grown HTZ1 cultures were classified as GFP-positive cells. (A) The frequency of GFP-positive cells within HTZ1, htz1Δ, and htz1-K3,8,10,14R cultures. (B) The average GFP intensity of the GFP-positive populations of HTZ1, htz1Δ, and htz1-K3,8,10,14R cultures. (C) Q-RT PCR of GAL1-GFP mRNA performed on HTZ1 (JRY9002), htz1Δ (JRY9004), and htz1-K3,8,10,14R (JRY9003) cultures that were grown long-term in YP-glucose (2%) prior to being transferred into YP-galactose (2%). (D) The average GFP intensity of the entire population of cells, both GFP positive and negative, within HTZ1, htz1Δ, and htz1-K3,8,10,14R cultures. Bars in all panels represent the standard deviations of values from three biological replicates.

Figure 5. The distribution of GAL1-induction times and Gal-GFPp accumulation rates among cells as modeled as a Gamma distribution of values.

See text for details. (A) shows the Gamma distribution of GAL1-induction times that were used in the best-fit simulation of HTZ1's (JRY9002) GAL1-GFP expression phenotype. (B) shows the Gamma distribution of Gal1-GFP accumulation rates that were used in the best-fit simulation of HTZ1's (JRY9002) GAL1-GFP expression phenotype. (C) compares the GAL1-GFP induction phenotypes observed for HTZ1 cultures with the phenotype predicted for HTZ1 based on its best-fit simulation.

Figure 6. Role of H2A.Z acetylation in GAL1 induction.

Flow cytometry analysis was performed using Gal1-GFP on HTZ1/htz1Δ (JRY9007), htz1-K3,8,10,14R/htz1Δ (JRY9008), htz1-K3R/htz1Δ (JRY9009), htz1-K8R/htz1Δ (JRY2010), htz1-K10R/htz1Δ (JRY2011), and htz1-K14R/htz1Δ (JRY2012) cells grown long-term in YP-glucose (2%) prior to being transferred into YP-galactose (2%). A threshold level of GFP-intensity was set so that between 1% and 2% of glucose-grown HTZ1 cultures were classified as GFP-positive cells. (A) The frequency of GFP-positive cells within HTZ1, htz1-K3,8,10,14R, htz1-K3R, htz1-K8R, htz1-K10R, and htz1-K14R cultures. (B) The average GFP intensity of the GFP-positive populations of HTZ1, htz1-K3,8,10,14R, htz1-K3R, htz1-K8R, htz1-K10R, and htz1-K14R cultures. Bars in all panels represent the standard deviations of values from three biological replicates.

Figure 7. The htz1Δ mutant phenotypes were partially suppressible by mutations in genes encoding members of SWR1-Com.

The stress sensitivities of htz1Δ (MK1027), swr1Δ (MKY1028), htz1Δ/swr1Δ (MKY1029), swc2Δ (MKY1030), htz1Δ/swc2Δ (MKY1031), swc3Δ (MKY1032), htz1Δ/swc3Δ (MKY1033), swc5Δ (MKY1034), htz1Δ/swc5Δ (MKY1035), swc6Δ (MKY1036), and htz1Δ/swc6Δ (MKY1037) strains were assessed by plating ten-fold serial dilutions of these double mutant cultures onto solid YP-glucose (2%) medium with the following conditions: 2% formamide, 3 mM caffeine, 125 mM hydroxyurea, and 10 µg/ml benomyl.

Figure 8. The severity of the htz1Δ mutant GAL1 expression defect was suppressible by the swr1Δ mutation.

Flow cytometry analysis was performed using Gal1-GFP on HTZ1 (JRY9002), htz1Δ (JRY9004), htz1-K3,8,10,14R (JRY9003), swr1Δ HTZ1 (JRY9005), and swr1Δ htz1Δ (JRY9006) cultures grown long-term in YP-glucose (2%) prior to being transferred into YP-galactose (2%). The histograms represent the distribution of cells within HTZ1, htz1Δ, and htz1-K3,8,10,14R cultures as a function of their GFP intensity.

Figure 9. swr1Δ mutants' GAL1 induction phenotypes resembled those of htz1-K3,8,10,14R cultures.

A threshold level of GFP-intensity was as above. (A) The frequency of GFP positive cells within HTZ1, htz1Δ, htz1-K3,8,10,14R, swr1Δ HTZ1, and swr1Δ htz1Δ cultures. (B) The average GFP intensity of the GFP-positive populations of HTZ1, htz1Δ, htz1-K3,8,10,14R, swr1Δ HTZ1, and swr1Δ htz1Δ cultures. Bars in all panels represent the standard deviations of values from three biological replicates.