Characterizing the regulatory effects of H2A.Z and SWR1-C on gene expression during hydroxyurea exposure in Saccharomyces cerevisiae
Hilary T. Brewis, Peter C. Stirling, Michael S. Kobor
Abstract
Chromatin structure and DNA accessibility are partly modulated by the incorporation of histone variants. H2A.Z, encoded by the non-essential HTZ1 gene in S. cerevisiae, is an evolutionarily conserved H2A histone variant that is predominantly incorporated at transcription start sites by the SWR1-complex (SWR1-C). While H2A.Z has often been implicated in transcription regulation, htz1Δ mutants exhibit minimal changes in gene expression compared to wild-type. However, given that growth defects of htz1Δ mutants are alleviated by simultaneous deletion of SWR1-C subunits, previous work examining the role of H2A.Z in gene expression regulation may be confounded by deleterious activity caused by SWR1-C when missing its H2A.Z substrate (apo-SWR1-C).
Introduction
Incorporation of histone variants is one of the principal mechanisms that cells use to create structurally and functionally distinct regions of chromatin. H2A.Z is an evolutionarily conserved histone variant in the H2A histone family [1]. Unlike H2A, which is produced in equal amounts to other core histones during S-phase, H2A.Z is encoded by replication-independent genes allowing for variant specific expression and deposition throughout the cell cycle [2]. Essential in higher eukaryotes, H2A.Z has been implicated in a large variety of biological functions including nucleosome turnover, maintenance of heterochromatin and euchromatin boundaries, DNA repair, resistance to genotoxic stress, and transcription regulation [1,3].
Materials and method
Yeast strains
Yeast strains used in this study, described in Table 1, were constructed in the S. cerevisiae W303 background using standard genetic techniques. Gene deletions were accomplished using the one-step gene replacement method to integrate PCR-amplified segments [71]. Double mutant strains were generated by standard genetic manipulation via mating and tetrad dissection.
Results
Apo-SWR1-C contributed to differences in gene expression profiles between wild-type and the htz1Δ mutant
In order to assess the impact H2A.Z incorporation has on gene expression in S. cerevisiae during HU exposure, we pursued three primary objectives: 1) characterizing how wild-type gene expression changes in response to HU exposure, 2) determining the influence of H2A.Z incorporation and the presence of apo-SWR1-C on gene expression profiles in both untreated and HU-treated conditions, and finally 3) comparing the patterns of HU-induced gene activation and repression between wild-type and mutants lacking H2A.Z incorporation. To achieve these objectives we sequenced poly(A)-enriched libraries from six biological replicates of wild-type, htz1Δ, swr1Δ, and htz1Δswr1Δ mutants before and after a 90 minute treatment with HU (200 mM) (Fig 1A).
Discussion
In this study, we generated a robust gene expression dataset to explore the complex relationship between H2A.Z and gene expression during HU exposure in S. cerevisiae. Our findings revealed that H2A.Z incorporation was more prominently involved in gene activation than repression, and that while relatively few genes were differentially expressed in the absence of H2A.Z, its incorporation was necessary for proper wild-type expression of several HU-induced genes.
Acknowledgments
We thank Eully Ao for her technical assistance while conducting the MNase-NChIP-seq experiment, Aaron Bogutz for his invaluable help and insights on the bioinformatic analysis pipeline, as well as Meingold Chan, Kristy Dever, and Mandy Meijer, for their critical reading of the manuscript.
Citation: Brewis HT, Stirling PC, Kobor MS (2025) Characterizing the regulatory effects of H2A.Z and SWR1-C on gene expression during hydroxyurea exposure in Saccharomyces cerevisiae . PLoS Genet 21(1): e1011566. https://doi.org/10.1371/journal.pgen.1011566
Editor: Robert Schneider, Institute of Functional Epigenetics, GERMANY
Received: August 7, 2024; Accepted: January 6, 2025; Published: January 21, 2025
Copyright: © 2025 Brewis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The genomic datasets from this study have been deposited in NCBI’s Gene Expression Omnibus (GEO) database under the accession number GSE261121.
Funding: This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC: https://www.nserc-crsng.gc.ca/index_eng.asp) through discovery grants to MSK (RGPIN-2016-04297 and RGPIN-2023-0501). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
