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A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich's ataxia - PubMed

  • ️Fri Jan 01 2021

A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich's ataxia

Oscar A Campos et al. Sci Adv. 2021.

Abstract

Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. A source of damage to Fe-S clusters is cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ for copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3–mediated Cu1+ toxicity is a major determinant of cellular functional pool of Fe-S clusters. Inadequate Fe-S cluster supply, due to diminished assembly as occurs in Friedreich’s ataxia or defective distribution, causes severe metabolic and growth defects in Saccharomyces cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster–dependent metabolism and growth. Our findings reveal an interplay between chromatin and mitochondria in Fe-S cluster homeostasis and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.

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Figures

Fig. 1.
Fig. 1.. H3H113N rescues the growth defects caused by dysfunctional Fe-S cluster assembly or distribution.

(A) Spot test assays in glucose-containing minimal media with or without additional CuSO4. Copper concentration in minimal medium at baseline is ~0.25 μM. (B) Spot test assay in fermentative medium with or without YFH1 deleted. (C to E) Spot tests with strains in which the YFH1, GRX5, or ATM1 genes were placed under the control of the GAL1 promoter, which is repressed in glucose-containing SC and minimal media. Genes were transcriptionally suppressed for 1, 2, or 3 days (d) before spot testing.

Fig. 2.
Fig. 2.. H3H113N restores Fe-S cluster–dependent cellular processes.

(A) Spot test assays in glucose-containing media with gradually decreasing amounts of all 20 amino acids. Baseline (i.e., 1×) concentration of each amino acid is 85.6 mg/liter with some exceptions (see Materials and Methods). (B) Spot test assay with amino acid–depleted media ± 80 mg/liter of each of the amino acids that are either not dependent [non–Fe-S amino acids (AAs)] or dependent (Fe-S amino acids) on Fe-S cluster–containing enzymes. (C) Spot test assays with SC medium lacking lysine or glutamate. (D) 55Fe content of immunoprecipitated (IP) Aco1-myc, with representative Western blot shown above. Bars show mean scintillation counts per minute (cpm), and each dot is an independent experiment (n = 3). (E) Aconitase activity assay using whole-cell extracts, with representative loading control Western blot shown above. Bars show mean activity relative to the matched WT or H3H113N, and each dot is an independent experiment (n = 3). (F) Spot test assays in rich medium containing either glucose (SC) or ethanol and glycerol (SCEG). (G) Spot test assays in SC with or without MMS.

Fig. 3.
Fig. 3.. H3H113N prevents global transcriptional rewiring in YFH1-off.

(A) Principal components (PC) analysis of global gene expression values from cells in SC medium from four independent experiments. YFH1-off strains were grown in SC medium for 44 hours before RNA sequencing. (B) Volcano plots of average log2 fold changes of YFH1-off compared to WT (left) or H3H113N YFH1-off compared to H3H113N (right). A subset of iron regulon genes (ARN1 and FIT2) or Fe-S cluster–binding genes (ACO1, ISU1, and LYS4) are indicated. (C and D) Average mRNA expression levels for either (C) genes up-regulated by at least twofold (n = 280) or (D) genes down-regulated by at least twofold (n = 214) in YFH1-off compared to WT. The white box and whisker plots overlaid on the violin plots are the median and interquartile ranges. Dots are outlier data points. The gray and blue dashed lines are the median expression levels in WT and YFH1-off, respectively. (E) Significantly enriched (Bonferroni-Šidák P value of <0.05) Gene Ontology (GO) terms among genes that were significantly differentially expressed in YFH1-off compared to WT and H3H113N YFH1-off.

Fig. 4.
Fig. 4.. Differences in cellular iron content do not account for the protective effect of H3H113N when YFH1 is shut off.

(A) Spot test assays in SC medium or SC without lysine and glutamate. (B) Growth after 44 hours (see Materials and Methods for growth procedure) in liquid SC without lysine and glutamate. Bars show means, and each dot is an independent experiment (n = 5). (C) Intracellular iron content for strains grown in liquid SC without lysine and glutamate. Bars show means, and each dot is an independent experiment (n = 4 to 5). ***P < 0.001; n.s., not significant.

Fig. 5.
Fig. 5.. H3H113N rescues YFH1-off by diminishing copper toxicity.

(A) Intracellular copper content for strains grown in liquid SC medium. YFH1-off strains were grown in SC for 44 hours. Bars show means, and each dot is an independent experiment (n = 4 to 5). (B to D) Spot test assays in SC medium or SC without lysine and glutamate and with or without the indicated amount of (B) BCS or (D) additional CuSO4.

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