Histone acetylases are required for iron homeostasis in yeast

Abstract

Iron, an ancient and essential transition metal, is involved in various biological functions, including oxygen transport, DNA synthesis, heme production, and iron-sulfur clusters, which participate in electron transport, DNA repair, and other cellular processes. However, excessive iron can lead to oxidative stress, lipid peroxidation, and cell damage. Thus, maintaining the iron content within an appropriate safe range and maintaining the balance of iron metabolism play crucial roles in both cellular function and human health [1]. An important aspect of maintaining the balance of iron homeostasis is the regulation of the iron uptake system. In Saccharomyces cerevisiae, cells can either obtain iron from the external environment via the non-reducing siderophore transport system or transport iron from the extracellular space to the intracellular space via the reducing iron transport system [2]. Iron uptake system-related genes are regulated mainly by the transcription factor Aft1p. During iron deficiency, Aft1p translocates into the nucleus, binds to genes involved in iron metabolism, and regulates the expressions of genes involved in iron uptake systems [3]. In addition, when there is a problem in the synthesis of iron-sulfur clusters in the mitochondria, such as the lack of the iron chaperone Yfh1p, which promotes the synthesis of iron-sulfur clusters, the transcription and nuclear entry of the transcription factor Aft1p are also activated, thereby regulating the expressions of iron metabolism-related genes [4]. In addition to transcription factors, gene expression is also regulated by histones and their modifications at the epigenetic level. For example, histone H3K4 methylation is related to gene activation, H3K36 methylation plays an important role in the elongation of transcription, and histone acetylation results in the loss of nucleosome structure and facilitates gene expression [5]. Therefore, histone modifications should also play important roles in the regulation of iron homeostasis. The relationship between histone modifications and iron homeostasis has been reported in the literature. For example, the DNA methylation-binding protein MBD5 can change histone acetylation in the promoter region of the ferritin gene by recruiting the histone acetylase KAT2A protein [6]. Histone acetylation has also been reported to be reduced in iron-deficient environments [7,8], and direct effects of histone acetylation on iron homeostasis gene loci have also been reported both in C. albicans [9] and mammals [10]. More recently, the histone H3-H4 tetramer was found to be a copper reductase enzyme, and H3-mediated Cu+ toxicity is a major determinant of the cellular functional pool of iron-sulfur (Fe-S) clusters [11,12]. However, information on the role of histone modifications in the regulation of iron homeostasis is limited. The mechanism by which and how histone modifications are involved in the transcriptional regulation of iron uptake-related genes or the iron deficiency response require further investigation. To determine whether histone acetylation and methylation are involved in the iron deficiency response, we first deleted the histone acetylase genes, including GCN5 (histones H2B and H3 N-terminal lysine acetylase, partial deletion of the ADA2 interaction sequence [13]), RTT109 (H3 lysine 9 and 56 acetylase), SAS2 (H4 lysine 16 acetylase), and YNG2 (subunit of the histone acetyltransferase complex NuA4 for acetylation of histone H4 or histone H2A) in the wild-type (WT) strain, as well as the histone methyltransferases SET1 (H3 lysine 4 methyltransferase), SET2 (H3 lysine 36 methyltransferase) and DOT1 (H3 lysine 79 methyltransferase). The genomic deletions were confirmed by colony PCR and genomic coverage analysis, as depicted in Supplementary Figures S1 and S2. The sensitivity of single histone modification enzyme mutants to iron deficiency induced by the iron chelator bathophenanthroline disulfonate (BPS) was tested. As shown in Supplementary Figure S3, the histone acetylation-related mutants gcn51–316, rtt109Δ, sas2Δ, and yng2Δ did not exhibit significant growth defects compared with the WT on the YPD + BPS plate. None of the histone methyltransferase knockout strains presented significant growth defects. As a positive control, the iron-responsive transcription factor gene AFT1 knockout strain grew slowly on YPD + BPS plates. It is possible that histone modifications do not have a strong effect on the equilibrium status of iron deficiency but still regulate transcription induction during the iron deficiency response. To investigate the role of histone modifications during the induction of the iron deficiency response, the expressions of iron response genes in the wild-type and mutant strains before and 4 h after BPS treatment were examined.

1. Introduction

Iron, an ancient and essential transition metal, is involved in various biological functions, including oxygen transport, DNA synthesis, heme production, and iron-sulfur clusters, which participate in electron transport, DNA repair, and other cellular processes. However, excessive iron can lead to oxidative stress, lipid peroxidation, and cell damage. Thus, maintaining the iron content within an appropriate safe range and maintaining the balance of iron metabolism play crucial roles in both cellular function and human health [1].

An important aspect of maintaining the balance of iron homeostasis is the regulation of the iron uptake system. In Saccharomyces cerevisiae, cells can either obtain iron from the external environment via the non-reducing siderophore transport system or transport iron from the extracellular space to the intracellular space via the reducing iron transport system [2]. Iron uptake system-related genes are regulated mainly by the transcription factor Aft1p. During iron deficiency, Aft1p translocates into the nucleus, binds to genes involved in iron metabolism, and regulates the expressions of genes involved in iron uptake systems [3]. In addition, when there is a problem in the synthesis of iron-sulfur clusters in the mitochondria, such as the lack of the iron chaperone Yfh1p, which promotes the synthesis of iron-sulfur clusters, the transcription and nuclear entry of the transcription factor Aft1p are also activated, thereby regulating the expressions of iron metabolism-related genes [4].

In addition to transcription factors, gene expression is also regulated by histones and their modifications at the epigenetic level. For example, histone H3K4 methylation is related to gene activation, H3K36 methylation plays an important role in the elongation of transcription, and histone acetylation results in the loss of nucleosome structure and facilitates gene expression [5]. Therefore, histone modifications should also play important roles in the regulation of iron homeostasis. The relationship between histone modifications and iron homeostasis has been reported in the literature. For example, the DNA methylation-binding protein MBD5 can change histone acetylation in the promoter region of the ferritin gene by recruiting the histone acetylase KAT2A protein [6]. Histone acetylation has also been reported to be reduced in iron-deficient environments [7,8], and direct effects of histone acetylation on iron homeostasis gene loci have also been reported both in C. albicans [9] and mammals [10]. More recently, the histone H3-H4 tetramer was found to be a copper reductase enzyme, and H3-mediated Cu+ toxicity is a major determinant of the cellular functional pool of iron-sulfur (Fe-S) clusters [11,12]. However, information on the role of histone modifications in the regulation of iron homeostasis is limited. The mechanism by which and how histone modifications are involved in the transcriptional regulation of iron uptake-related genes or the iron deficiency response require further investigation.