H4k20me3 is Important for Ash1-mediated H3k36me3 and Transcriptional Silencing in Facultative Heterochromatin in a Fungal Pathogen

Abstract

Facultative heterochromatin controls development and differentiation in many eukaryotes. In metazoans, plants, and many filamentous fungi, facultative heterochromatin is characterized by transcriptional repression and enrichment with nucleosomes that are trimethylated at histone H3 lysine 27 (H3K27me3). While loss of H3K27me3 results in derepression of transcriptional gene silencing in many species, additional up- and downstream layers of regulation are necessary to mediate control of transcription in chromosome regions enriched with H3K27me3. Here, we investigated the effects of one histone mark on histone H4, namely H4K20me3, in the fungus Zymoseptoria tritici, a globally important pathogen of wheat. Deletion of kmt5, the gene encoding the sole methyltransferase responsible for H4K20 methylation, resulted in global derepression of transcription, especially in regions of facultative heterochromatin. Derepression in the absence of H4K20me3 not only affected known genes but also a large number of novel, previously undetected transcripts generated from regions of facultative heterochromatin on accessory chromosomes. 

Introduction

Chromatin, the assembly of DNA, RNA, and proteins that constitutes chromosomes, can assume active and inactive states that are correlated with different histone and DNA modifications [1]. Transcriptionally inactive, “silent” chromatin is separated into “constitutive heterochromatin” and “facultative heterochromatin”. Constitutive heterochromatin is most often marked by H3K9me2/3 and DNA methylation and found within or near centromeres, subtelomeric regions or telomeric repeats, rDNA, and transposable elements. Facultative heterochromatin is typically enriched with H3K27me3 and found on specific genes often associated with development [2,3], in large broad local enrichments (BLOCs) in mice [4], or across long sections of chromosomes in many fungi [5,6]. Facultative heterochromatin can be more dynamic than constitutive heterochromatin and “on” or “off” states vary between cell types or individuals within a species [7]. While H3K27me3, mediated by Polycomb Repressive Complex 2 (PRC2), is considered to be a hallmark histone modification correlated with facultative heterochromatin [8], little is known about other chromatin marks that are important for formation, maintenance, and gene silencing in these regions. In animals, the PRC1 complex and H2AK119ub1 play an important role in PRC2 and H3K27me3 recruitment [9], but so far there is no evidence for a canonical PRC1-like complex in fungi [10].

Methods

Fungal and bacterial growth conditions

Zymoseptoria tritici cultures were grown on YMS (4 g yeast extract, 4 g malt, 4 g sucrose per liter, with 16 g agar per liter added for solid medium). Glycerol stocks (1:1 YMS and 50% glycerol) were maintained at -80°C. Escherichia coli strain DH10beta (NEB) was grown at 37°C in LB (10 g tryptone, 10 g NaCl, 5 g yeast extract per liter) with appropriate antibiotics (100 μg/mL kanamycin). Agrobacterium tumefaciens strain AGL1 was grown in dYT (16 g tryptone, 5 g NaCl, 10 g yeast extract per liter) or LB media with appropriate antibiotics (100 μg/mL kanamycin, 100 μg/mL carbenicillin, 50 μg/mL rifampicin).

Results

Kmt5 and Ash1 are important for normal growth but not essential in Zymoseptoria tritici
We identified kmt5 (Zt_chr_3_00475) and ash1 (Zt_chr_13_00232) in the genome of the reference isolate IPO323 by BLAST searches with S. pombe SET9 and N. crassa ASH-1 sequences as baits. We found a single H4K20 methyltransferase homolog, consistent with findings in other fungi but different from metazoans, where multiple enzymes are involved in catalyzing H4K20me1 and H4K20me2/3 (Fig 1A). Protein sequence alignments of the SET domains of H4K20 methyltransferases from Z. tritici, S. pombe, D. melanogaster, Danio rerio, and human revealed higher sequence similarity between Kmt5 in fungi and known H4K20me2/3 methyltransferases than H4K20me1 methyltransferases (S1 Fig). We showed that Kmt5 in Z. tritici is most likely responsible for all H4K20 methylation, as kmt5 deletion strains are unable to generate either H4K20me1 or H4K20me3 (Figs 1B and S2).

Discussion

We report chromatin dynamics and interactions in facultative heterochromatin that identify Kmt5 and H4K20me3 as important regulators for transcriptional repression and recruitment of Ash1-mediated H3K36me3 in Z. tritici, which itself at least partially regulates the distribution of H3K27me3. Here we investigated potential epistasis rules for repressive histone modifications and show that presence of H4K20me3 and Kmt5 are important for Ash1-mediated H3K36me3, because we observed absence of H3K36me3 in selected regions upon either deletion of Kmt5 or introduction of mutant histone alleles. Our findings suggest that H4K20 methylation needs to be established before Ash1 can catalyze H3K36me3.

Acknowledgments

We thank Eva Stukenbrock for supplying materials and strains, and for supporting this work while MM was still at CAU Kiel. We thank colleagues in the Freitag lab and Zachary Lewis (University of Georgia) for conversations and comments on the manuscript.

Citation: Möller M, Ridenour JB, Wright DF, Martin FA, Freitag M (2023) H4K20me3 is important for Ash1-mediated H3K36me3 and transcriptional silencing in facultative heterochromatin in a fungal pathogen. PLoS Genet 19(9): e1010945. https://doi.org/10.1371/journal.pgen.1010945

Editor: Alessia Buscaino, University of Kent, UNITED KINGDOM

Received: April 20, 2023; Accepted: August 30, 2023; Published: September 25, 2023

Copyright: © 2023 Möller 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: Sequencing raw reads (FASTQ files) of all ChIP-seq and RNA-seq are available online at Sequence Read Archive (SRA) under BioProject ID PRJNA902413. Normalized bigwig files for all ChIP-seq datasets, reference genome, and annotation files have been deposited at zenodo under doi: https://zenodo.org/record/8218851.

Funding: MM was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, grant number MO 3755/1-1). JBR was supported by a grant from the United States Department of Agriculture-Agriculture and Food Research Initiative (USDA-AFRI, grant number 2019-67012-29722). Chromatin research in the Freitag lab is supported by grants from the National Science Foundation (NSF, grant number MCB1818006), National Institutes of Health (NIH, grant number R01GM132644), and the United States-Israel Binational Science Foundation (BSF, grant number #2019034) to MF. 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.

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010945&rev=2#abstract0