What makes a histone variant a variant: Changing H2A to become H2A.Z

Abstract

Chromatin structure and underlying DNA accessibility is modulated by the incorporation of histone variants. H2A.Z, a variant of the H2A core histone family, plays a distinct and essential role in a diverse set of biological functions including gene regulation and maintenance of heterochromatin-euchromatin boundaries. Although it is currently unclear how the replacement of H2A with H2A.Z can regulate gene expression, the variance in their amino acid sequence likely contributes to their functional differences. To tease apart regions of H2A.Z that confer its unique identity, a set of plasmids expressing H2A-H2A.Z hybrids from the native H2A.Z promoter were examined for their ability to recapitulate H2A.Z function. First, we found that the H2A.Z M6 region was necessary and sufficient for interaction with the SWR1-C chromatin remodeler. Remarkably, the combination of only 9 amino acid changes, the H2A.Z M6 region, K79 and L81 (two amino acids in the α2-helix), were sufficient to fully rescue growth phenotypes of the htz1Δ mutant. Furthermore, combining three unique H2A.Z regions (K79 and L81, M6, C-terminal tail) was sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 derepression. Surprisingly, hybrid constructs that restored the transcription of H2A.Z-dependent genes, did not fully recapitulate patterns of H2A.Z-specific enrichment at the tested loci. This suggested that H2A.Z function in transcription regulation may be at least partially independent of its specific localization in chromatin. Together, this work has identified three regions that can confer specific H2A.Z-identity to replicative H2A, furthering our understanding of what makes a histone variant a variant.

Fig 1. Amio acids that differ between H2A.Z and H2A were grouped into nine distinct regions.

Sequence alignment of the S. cerevisiae H2A.Z and H2A proteins with conserved amino acids indicated by asterisks. Regions that differ between H2A.Z and H2A were divided into nine distinct regions: N-terminal tail, C-terminal tail, M6, two histone loops (L1 and L2) and four groups in the histone fold (G1-G4). The H2A.Z secondary structure is shown above the sequence alignment with lines indicating unstructured flexible regions and boxes indicating alpha helices.

Fig 2. Systematic analysis of regions that diverge between H2A and H2A.Z revealed that the N-terminal, M6, G3, and G4 regions of H2A.Z may contribute to H2A.Z-specific function.

(A) H2A.Z (HTZ1, black) and H2A (HTA1, grey) parent constructs were used to create the hybrid H2A.Z-H2A and H2A-H2A.Z constructs. Each construct was expressed from the native H2A.Z gene promoter and C-terminally 3xFLAG tagged. (B) Growth assays of the H2A-H2A.Z mutants indicated that mutants containing either the H2A.Z N-terminus, M6, G3, or G4 region had improved growth over the H2A mutant, while (C) the H2A.Z-H2A mutants showed that of the nine regions of H2A.Z, only the M6 region could not be functionally replaced by the corresponding region from H2A. Cells expressing the indicated hybrid constructs were 10-fold serially diluted, spotted onto SC-TRP media with the indicated concentrations of formamide, caffeine, and hydroxyurea and grown for 3 days.

Fig 3. The H2A.Z M6 region was necessary and sufficient for interaction with known H2A.Z partners.

(A) Immunoprecipitation of the FLAG-tagged hybrid proteins revealed that the M6 region of H2A.Z was necessary and sufficient for co-purification with VSV-tagged SWR1-C subunits. (B) Immunoprecipitation of the FLAG-tagged hybrid constructs revealed M6 as a key region for H2A.Z interaction with the histone chaperones Nap1 and VSV-tagged Chz1. (C) The reciprocal immunoprecipitation of VSV-tagged Chz1 confirmed an increase in interaction with the H2A-H2A.Z construct in comparison to the H2A.Z construct.

Fig 4. In combination with M6, an evolutionary conserved region in the alpha 2 helix of H2A.Z distinguished the histone variant H2A.Z from replicative H2A and conferred H2A.Z-specific growth phenotypes.

(A) The H2A-H2A.Z[M6,G4] mutant had comparable growth to the H2A.Z mutant. Cells expressing the indicated hybrid constructs were 10-fold serially diluted, spotted onto SC-TRP media with the indicated concentrations of formamide, caffeine, and hydroxyurea and grown for 3 days. (B) The growth curves of the H2A.Z and H2A-H2A.Z[M6,G4] mutants were indistinguishable. Each curve was generated by taking the average OD (measured every 20 minutes) of three biological replicates grown in SC-TRP liquid media in the presence of 1.5% formamide. Asterisks indicate significant comparisons determined by comparing the area under the curve of for each mutant with unpaired one-tailed Student’s t-tests. * p-value <0.05, ** p-value <0.01, *** p-value <0.001. (C) Sequence alignment of the H2A.Z L2 loop region across species revealed that amino acids K79 and L81 (bold) are highly conserved among H2A.Z homologs and are consistently divergent from H2A. Amino acids in yellow indicate that it is found in both H2A.Z and H2A for the indicated species. Red indicates that the amino acid is unique to H2A.Z in the indicated species. (D) The growth phenotypes of the H2A-H2A.Z[M6,G4] mutant were primarily driven by L81. Growth assays were performed as previously described in panel “A”.

Fig 5. The M6 and G4 regions were sufficient to confer H2A.Z-specific bulk chromatin association.

(A) In combination with G4 or the M6 region, the C-terminal tail conferred H2A.Z-specific growth phenotypes. However, the M6 and G4 regions were required to fully recapitulate H2A.Z identity. The majority of mutants here were previously shown in Fig 4A. Cells expressing the indicated hybrid constructs were 10-fold serially diluted, spotted onto SC-TRP media with the indicated concentrations of formamide, caffeine, and hydroxyurea and grown for 3 days. (B) All hybrid constructs associated with chromatin, however only the H2A-H2A.Z[M6,G4] and H2A-H2A.Z[M6,G4,C] constructs were present at similar levels in the soluble fraction as H2A.Z. Whole-cell extracts (W) were separated into chromatin (C) and soluble (S) (non-chromatin) fractions and analyzed by immunoblotting. FLAG antibodies detected the hybrid constructs, while H4 and Pgk1 were used as controls for the chromatin and soluble fractions, respectively.

Fig 6. H2A-H2A.Z hybrid constructs were incorporated into chromatin but did not recapitulate H2A.Z-specific patterns of enrichment at gene promoters.

(A) Unlike H2A.Z, the H2A-H2A.Z[M6], H2A-H2A.Z[M6,G4], and H2A-H2A.Z[M6,G4,C] constructs were not significantly enriched at promoters in comparison to their enrichment at respective gene ORFs. (B) At loci with relatively lower levels of H2A.Z, the H2A and H2A-H2A.Z[M6,G4,C] constructs had significantly increased enrichment in comparison to H2A.Z. FLAG-tagged hybrid enrichment levels determined by ChIP-qPCR for three replicates were normalized to their respective inputs. Asterisks indicate all the significant comparisons determined by unpaired two-tailed Student’s t-tests. * = p-value <0.05, ** = p-value <0.01, *** = p-value <0.001. All constructs were significantly enriched over the untagged control (p-value <0.05). All other unlabelled comparisons had a p-value > 0.05.

Fig 7. A combination of the three unique H2A.Z regions, M6, G4 and C-terminal tail, were sufficient to restore the mRNA levels of heterochromatin-proximal genes and GAL1 after derepression.

(A) Schematic representation of H2A.Z-dependent genes at the boundaries of the HMRa locus and the right telomere of chromosome III. Arrows represent open reading frames (ORFs) and point in the direction of transcription. (B) Expression defects observed in the htz1Δ mutant were gradually improved by the stepwise addition of the H2A.Z M6, G4, and C-terminal regions. RT-qPCR analysis of heterochromatin-proximal genes mRNA levels from three replicates were normalized to ACT1 mRNA levels. Matrices of significant comparisons for GIT1, RDS1, and YCR100c were determined by unpaired two-tailed Student’s t-tests. (C) RT-qPCR analysis of GAL1 mRNA levels performed on the indicated mutants, which were grown long-term in SC-TRP-glucose (2%) prior to being transferred to SC-TRP-galactose (2%) and collected at 2-hour intervals. During GAL1 derepression, the addition of the M6, G4, and C-terminal regions incrementally increased GAL1 expression, were the H2A-H2A.Z[M6,G4,C] mutant showed comparable levels to the H2A.Z mutant. GAL1 mRNA levels were normalized to ACT1. Error bars indicate the standard deviation between the three replicates. Significance comparisons determined by unpaired two-tailed Student’s t-tests are indicated: * = p-value <0.05, ** = p-value <0.01, *** = p-value <0.001. All unlabelled comparisons within each time point had a p-value >0.05.

Fig 8. The M6, G4, and C-terminal regions of H2A.Z contributed to its unique function and distinguished it from H2A.

Schematic of H2A.Z and H2A proteins highlighting the M6, G4, and C-terminal regions as key contributors to H2A.Z unique identity. The M6 region is necessary for H2A.Z function, while the G4 and C-terminal regions confer H2A.Z unique functions when combined with M6. Within these regions, amino acids that deviate between H2A.Z and H2A and that have been individually implicated in H2A.Z activities, by this study or previous literature, are highlighted in bold.