Tolerance of DNA Replication Stress Is Promoted by Fumarate Through Modulation of Histone Demethylation and Enhancement of Replicative Intermediate Processing in Saccharomyces cerevisiae

Abstract

Fumarase is a well-characterized TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate. In mammals, fumarase acts as a tumor suppressor, and loss-of-function mutations in the FH gene in hereditary leiomyomatosis and renal cell cancer result in the accumulation of intracellular fumarate-an inhibitor of α-ketoglutarate-dependent dioxygenases. Fumarase promotes DNA repair by nonhomologous end joining in mammalian cells through interaction with the histone variant H2A.Z, and inhibition of KDM2B, a H3 K36-specific histone demethylase. Here, we report that Saccharomyces cerevisiae fumarase, Fum1p, acts as a response factor during DNA replication stress, and fumarate enhances survival of yeast lacking Htz1p (H2A.Z in mammals). We observed that exposure to DNA replication stress led to upregulation as well as nuclear enrichment of Fum1p, and raising levels of fumarate in cells via deletion of FUM1 or addition of exogenous fumarate suppressed the sensitivity to DNA replication stress of htz1Δ mutants. This suppression was independent of modulating nucleotide pool levels. Rather, our results are consistent with fumarate conferring resistance to DNA replication stress in htz1Δ mutants by inhibiting the H3 K4-specific histone demethylase Jhd2p, and increasing H3 K4 methylation. Although the timing of checkpoint activation and deactivation remained largely unaffected by fumarate, sensors and mediators of the DNA replication checkpoint were required for fumarate-dependent resistance to replication stress in the htz1Δ mutants. Together, our findings imply metabolic enzymes and metabolites aid in processing replicative intermediates by affecting chromatin modification states, thereby promoting genome integrity.

Keywords: DNA replication stress; HTZ1; JHD2; fumarate; histone methylation.

Figure 1

Fumarate can complement sensitivity of htz1Δ mutants to DNA replication stress. (A and B) Expression of Fum1p is induced, and Fum1p becomes enriched in the nuclear fraction upon exposure to hydroxyurea, HU. Yeast expressing Fum1-GFPp were incubated in the absence or presence of 200 mM HU at 30° for 3 hr. Whole cell extracts (A), or nuclear fractions (B) were analyzed by immunoblotting using anti-GFP, and anti-PCNA antibodies. A representative immunoblot and fold enrichment of Fum1p from two independent experiments is shown. Levels of Fum1-GFPp were normalized to levels of PCNA (loading control), then expressed relative to signal that was observed in the absence of HU, which was set to 1. $FoldenrichmentofFum1p=\frac{\left(Fum1p/PCNA\right)indicatedsample}{\left(Fum1p/PCNA\right)noHU}$. (C) Genetic interaction between fum1Δ and htz1Δ mutants. (D) The effect of exogenous fumarate on DNA replication stress in mre11Δ, rad50Δ and xrs2Δ mutants. (C and D) Cells with genotypes as indicated were grown overnight in rich (YPD) medium, then 3 μl of 10-fold serial dilutions were spotted onto YPD medium containing the indicated concentrations of fumarate and/or HU, and incubated at 30° for 2 days prior to imaging.

Figure 2

Fumarate-mediated suppression of sensitivity to DNA replication stress of htz1Δ mutants is independent of modulation of nucleotide pools. (A) Fumarate suppresses the sensitivity to DNA replication stress of htz1Δ mutants in the absence of an inhibitor of ribonucleotide reductase. (B) Fumarate is a product in the purine nucleotide synthesis cycle. In the purine nucleotide cycle, aspartate becomes converted to fumarate in a two-stage reaction, which is facilitated by hydrolysis of GTP. This two-stage reaction involves generation of adenylosuccinate from inosine monophosphate (IMP) and aspartate, which is then converted to fumarate and adenosine monophosphate (AMP). This reaction is followed by deamination of AMP to IMP by AMP deaminase. (C) Fumarate suppresses the sensitivity to DNA replication stress of htz1Δ mutants in the absence of adenylosuccinate synthase (ADE12). Strains with genotypes as indicated in (A and C) were analyzed in serial dilution growth assays as described in Figure 1.

Figure 3

Loss of JmjC domain-containing histone demethylase Jhd2p suppresses the sensitivity to DNA replication stress of htz1Δ mutants. (A) Enhancing histone methylation by deletion of histone demethylase(s) or enzyme inhibition by fumarate. (B) Genetic interaction analyses between htz1Δ mutants and histone demethylase mutants. Strains with indicated genotypes were analyzed in serial dilution growth assays as described in Figure 1.

Figure 4

Fumarate-dependent suppression of sensitivity to DNA replication stress of strains expressing H3 mutants with lysine to arginine mutations at H3 K4, K36, or K79. Strains with genotypes as indicated were analyzed in serial dilution growth assays as described in Figure 1.

Figure 5

Impact of histone methylation, loss of JHD2 and exogenous fumarate on sensitivity to DNA replication stress of htz1Δ mutants. Strains with indicated genotypes were analyzed in serial dilution growth assays as described in Figure 1.

Figure 6

Fumarate modulates levels of JDH2-dependent H3 K4 methylation. Wild-type yeast and htz1Δ mutants carrying an empty vector or a plasmid for overexpression of FLAG-Jhd2p were grown logarithmically in selective medium with or without 5 mM fumarate. (A and C) Whole cell extracts of strains with indicated genotypes were analyzed in at least three independent experiments by immunoblotting against H3 K4me3 (A), or H3 K4me2, H3K36me3, H3 K79me3 (C) and H3 (loading control). (B and D) Levels of H3 K4me3 (B) or H3 K4me2 (D) were normalized to H3, and expressed relative to that observed in wild-type with vector (vec), which was set to 1 (Avg. ± STD, n = 3; representative independent experiments shown in (A and C). The level of H3 K4me3 or H3 K4me2 relative to H3 was calculated as $\frac{\left(H3 K4meX/H3\right)sample}{\left(H3 K4meX/H3\right)WT + vec}$. The statistical analysis was performed using Wilcoxon Rank Sum test and P-value ≤ 0.05 is shown by *.

Figure 7

Fumarate-dependent suppression of sensitivity to DNA replication stress of htz1Δ mutants requires components of the intra-S phase checkpoint. (A) The 9-1-1 complex and the 9-1-1 loader Rad24p are required for fumarate to suppress the sensitivity to DNA replication stress of htz1Δ mutants. (A and B) htz1Δ mutants require the DRC mediator Mrc1p during DNA replication stress. Strains with genotypes as indicated were analyzed as described in Figure 1.

Figure 8

htz1Δ mutants do not require the DDC mediator Rad9p during DNA replication stress. Strains with genotypes as indicated were analyzed as described in Figure 1.

Figure 9

Impact of loss of HTZ1 and exogenous fumarate on sensitivity to DNA replication stress of mutants with defects in DRC and processing and restart of aberrant replication forks. (A) Fumarate suppresses the sensitivity to DNA replication stress in cells lacking EXO1, or SAE2 in the presence or absence of HTZ1. (B) Loss of HTZ1 confers resistance to DNA replication stress of cells lacking subunits of the MRX complex, and fumarate enhances this effect. Strains with genotypes as indicated were analyzed as described in Figure 1.

Figure 10

Model: Fumarate promotes cell survival in htz1Δ mutants upon DNA replication stress by inhibition of the JmjC domain-containing histone demethylase Jhd2p.