Tolerance of DNA Replication Stress Is Promoted by Fumarate Through Modulation of Histone Demethylation in Saccharomyces cerevisiae Faeze Saatchi1*, Ann L. Kirchmaier1 1 Department of Biochemistry and Purdue Center for Cancer Research , Purdue University, West Lafayette, IN 47907 *Current address: Department of Internal Medicine, Kidney Cancer Research Program, UT Southwestern Medical Center, Dallas, TX 75235 Abstract Conclusions Introduction me me Ku70/80 me me FH Ku70/80 H2A H2A H2A.Z H2A.ZH3K36H3K36 FH H2A H2A.Z H2A H2A.Z Malate Fumarate KDM2B Malate Fumarate Figure 1. Upon DNA damage, fumarase (FH) is recruited to sites of double stranded break (DSB) through interaction with the histone variant H2A.Z. Fumarate generated by DSB-associated FH acts as an inhibitor of α- ketoglutarate-dependent histone demethylase KDM2B causing an increase of H3 K36 methylation at DSBs. This, in turn, promotes binding of Ku70/80 to DSB ends and enhances DSB repair by NHEJ. Sensitivity of htz1Δ mutants to DNA replication stress caused by HU is suppressed by high cellular levels of the metabolite fumarate (achieved by addition of exogenous fumarate or deletion of Fumarase, Fum1p) Figure 3. Cells with genotypes as indicated were grown overnight in rich (YPD) medium, then 3 ul of 10-fold serial dilutions were spotted onto YPD medium containing the indicated concentrations of fumarate and/or HU, and incubated at 30°C for 2 days prior to imaging. Loss of JmjC domain-containing histone demethylase Jhd2p suppresses the sensitivity to DNA replication stress of htz1Δ mutants. Results Our study uncovers links between the metabolic enzyme fumarase plus the metabolite fumarate and chromatin modifications during DNA damage response in Saccharomyces cerevisiae. In mammalian cells, fumarase becomes enriched at sites of double stranded break (DSB) through interaction with the histone variant H2A.Z. At DSBs, fumarase promotes DNA repair by nonhomologous end joining (NHEJ) by local production of fumarate which in turn acts as an inhibitor of KDM2B, a H3 K36-specific histone demethylase. We have demonstrated that treatment with hydroxyurea (HU), which creates stalled replication forks by depletion of nucleotides, leads to upregulation as well as nuclear enrichment of Fum1p. We have also shown that increased cellular levels of fumarate (upon deletion of FUM1 or addition of exogenous fumarate) suppresses the sensitivity to HU in htz1Δ mutants in a manner that is independent of modulating nucleotide levels. In fact, fumarate confers resistance to HU in htz1Δ mutants by inhibition of the H3 K4-specific histone demethylase Jhd2p, and increasing H3 K4 methylation levels. Sensors and mediators of the DNA replication checkpoint were required for fumarate- dependent resistance to HU in htz1Δ whereas factors involved in processing of regressed replication forks were dispensable. Together, our findings imply that high cellular levels of fumarate support processing of replicative intermediates by regulation of histone methylation, thereby promoting genome integrity. Fumarase is induced and becomes enriched in the nuclear fraction upon exposure to hydroxyurea (HU) Fumarate modulates levels of JDH2-dependent H3 K4 methylation. Figure 4. Strains with indicated genotypes were analyzed in serial dilution growth assays as described in Figure 3 YKu70p is not required for suppression of sensitivity of htz1Δ mutants to DNA replication stress by fumarate. Figure 2. Yeast expressing Fum1-GFPp were incubated in the absence or presence of 200 mM HU at 30°C 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. Figure 5. (A) Enhancing histone methyl