Histone H3 lysine 4 methyltransferase is required for facultative heterochromatin at specific loci

Qiaoqiao Zhu,Jinhee Park,Mukund Ramakrishnan,William J Belden

doi:10.1186/s12864-019-5729-7

Abstract

BackgroundHistone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 9 tri-methylation (H3K9me3) are widely perceived to be opposing and often mutually exclusive chromatin modifications. However, both are needed for certain light-activated genes in Neurospora crassa (Neurospora), including frequency (frq) and vivid (vvd). Except for these 2 loci, little is known about how H3K4me3 and H3K9me3 impact and contribute to light-regulated gene expression.ResultsIn this report, we performed a multi-dimensional genomic analysis to understand the role of H3K4me3 and H3K9me3 using the Neurospora light response as the system. RNA-seq on strains lacking H3 lysine 4 methyltransferase (KMT2/SET-1) and histone H3 lysine 9 methyltransferase (KMT1/DIM-5) revealed some light-activated genes had altered expression, but the light response was largely intact. Comparing these 2 mutants to wild-type (WT), we found that roughly equal numbers of genes showed elevated and reduced expression in the dark and the light making the environmental stimulus somewhat ancillary to the genome-wide effects. ChIP-seq experiments revealed H3K4me3 and H3K9me3 had only minor changes in response to light in WT, but there were notable alterations in H3K4me3 in Δkmt1/Δdim-5 and H3K9me3 in Δkmt2/Δset-1 indicating crosstalk and redistribution between the modifications. Integrated analysis of the RNA-seq and ChIP-seq highlighted context-dependent roles for KMT2/SET1 and KMT1/DIM-5 as either co-activators or co-repressors with some overlap as co-regulators. At a small subset of loci, H3K4 methylation is required for H3K9me3-mediated facultative heterochromatin including, the central clock gene frequency (frq). Finally, we used sequential ChIP (re-ChIP) experiment to confirm Neurospora contains K4/K9 bivalent domains.ConclusionsCollectively, these data indicate there are obfuscated regulatory roles for H3K4 methylation and H3K9 methylation depending on genome location with some minor overlap and co-dependency.

Highlights

Histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 9 tri-methylation (H3K9me3) are widely perceived to be opposing and often mutually exclusive chromatin modifications
Role of KMT1/DIM-5 and KMT2/SET-1 in transcriptional regulation In order to understand the role of KMT1 and KMT2 in the Neurospora light response, we performed a comprehensive RNA-sequencing (RNA-seq) on Δkmt1/Δdim-5 and Δkmt2/Δset-1 compared to WT with RNA isolated from mycelia grown in the dark for 24 h (DD24) and after a 30-min light exposure (LP30)
We performed multidimensional scaling (MDS) on the replicates to gage the variance among the strains and conditions and found the deletions contributed more to the changes in gene expression compared to environmental stimulus (Additional file 1b) we examined the expression level distribution among the different transcript types in WT under DD and LP30 and found that natural antisense transcript (NAT) and lincRNA are typically expressed at a much lower level than annotated protein-coding genes (Fig. 1b)

Summary

Introduction

Histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 9 tri-methylation (H3K9me3) are widely perceived to be opposing and often mutually exclusive chromatin modifications Both are needed for certain light-activated genes in Neurospora crassa (Neurospora), including frequency (frq) and vivid (vvd). In Neurospora, it is widely believed there is little, if any cross talk between KMT2/ SET-1 and KMT1/DIM-5 because H3K9me and H3K4me appear to be mutually exclusive [33] and Neurospora centromeres lack H3K4 methylation [34]. Both KMT2/SET-1 and KMT1/DIM-5 assist circadian negative feedback of the clock frequency (frq), and both are required for DNA methylation (5mC) at frq. The loss of DNA methylation at frq in Δkmt2/Δset-1 implicates H3K4me in KMT2/ DIM-5-dependent facultative heterochromatin and counters prevailing views on H3K4 methylation

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