Inhibition of α-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors - PubMed (original) (raw)

. 2012 Jun 15;26(12):1326-38.

doi: 10.1101/gad.191056.112. Epub 2012 Jun 7.

Hui Yang, Wei Xu, Shenghong Ma, Huaipeng Lin, Honguang Zhu, Lixia Liu, Ying Liu, Chen Yang, Yanhui Xu, Shimin Zhao, Dan Ye, Yue Xiong, Kun-Liang Guan

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Inhibition of α-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors

Mengtao Xiao et al. Genes Dev. 2012.

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Abstract

Two Krebs cycle genes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are mutated in a subset of human cancers, leading to accumulation of their substrates, fumarate and succinate, respectively. Here we demonstrate that fumarate and succinate are competitive inhibitors of multiple α-ketoglutarate (α-KG)-dependent dioxygenases, including histone demethylases, prolyl hydroxylases, collagen prolyl-4-hydroxylases, and the TET (ten-eleven translocation) family of 5-methlycytosine (5mC) hydroxylases. Knockdown of FH and SDH results in elevated intracellular levels of fumarate and succinate, respectively, which act as competitors of α-KG to broadly inhibit the activity of α-KG-dependent dioxygenases. In addition, ectopic expression of tumor-derived FH and SDH mutants inhibits histone demethylation and hydroxylation of 5mC. Our study suggests that tumor-derived FH and SDH mutations accumulate fumarate and succinate, leading to enzymatic inhibition of multiple α-KG-dependent dioxygenases and consequent alterations of genome-wide histone and DNA methylation. These epigenetic alterations associated with mutations of FH and SDH likely contribute to tumorigenesis.

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Figures

Figure 1.

Figure 1.

Fumarate and succinate inhibit the activity of α-KG-dependent KDMs in vitro. (A) Structural comparison among fumarate, succinate, α-KG, and 2-HG. (B) Fumarate and succinate inhibit the demethylase activity of C. elegans KDM7A (CeKDM7A). CeKDM7A activity toward monmethylated H3K9 was assayed in the presence or absence of 15 μM α-KG with increasing concentrations of either fumarate or succinate as indicated. The demethylated products were analyzed by mass spectrometry (MS), and mean activity values of duplicated assays, represented by percentage of remaining methylated peptides, are shown. (C) Fumarate and succinate inhibit the demethylase activity of human KDM4A (HsKDM4A) toward trimethylated H3K36, determined by mass spectrometry (MS) assay as in B. Error bars represent standard deviation (SD) for triplicate experiments. (D) Succinate is more potent to inhibit HsKDM4A than fumarate. The half maximal inhibitory concentration (IC50) of fumarate and succinate on HsKDM4A was determined by MS assay as in C. Error bars represent standard deviation (SD) for triplicate experiments. Additional MS results are shown in Supplemental Figure S1.

Figure 2.

Figure 2.

Fumarate and succinate increase genome-wide histone methylations, accumulate HIF1α, and reduce endostatin in cultured cells. (A) Cell-permeable fumarate (2.5 mM) or succinate (5 mM) increases histone methylation and HIF1α and reduces endostatin in HEK293T cells (left) and HeLa cells (right), as determined by Western-blot. (B) Cell-permeable octyl-α-KG (5mM) diminishes the effect of FH or SDH suppression on increasing histone methylations, accumulating HIF1α, and decreasing endostatin in HEK293T cells with knockdown of FH (left panel), SDHA (middle panel), and SDHB (right panel), as determined by Western blot. See also Supplemental Figure S3.

Figure 3.

Figure 3.

Knocking down FH or SDH reduces the TET-catalyzed 5hmC production in cultured cells. (A) HEK293T cells with stable knockdown of FH or SDHA/B were transiently transfected with plasmids expressing the indicated proteins. Thirty-six hours to 40 h after the transfection, cells were fixed and stained with antibodies specific to Flag to determine the expression of TET protein, with antibodies specific to 5hmC to determine the levels of 5hmC, and with antibodies specific to DAPI to view the cell nuclei. Bars, 251 μm. See also Supplemental Figure S4. (B_–_D) HEK293T cells were transiently transfected as described in A. Genomic DNAs were isolated from cells and immunoblotted with an antibody specific to 5hmC. Quantification of 5hmC was calculated from three independent assays. The expression of individual proteins was determined by immunoblotting as shown at the right. One representative quantification of the 5hmC level determined from the assays using 100 ng of genomic DNA is included. Error bars represent standard deviation (SD) for triplicate experiments.

Figure 4.

Figure 4.

Knocking down Fh or Sdha inhibits multiple α-KG-dependent dioxygenases and regulates gene expression in vivo. (A) Mice with transient knockdown of Fh or Sdha in the liver were generated by using the hydrodynamic tail vein-based delivery technique (n = 3 per group). The Fh or Sdha knockdown efficiency and levels of histone methylation, HIF1α, and endostatin were determined by Western blot. (B) The ratios of succinate/α-KG and fumarate/α-KG in mouse livers after suppression of Fh or Sdha were determined by GC-MS. Error bars represent standard deviation (SD) for triplicate experiments. (**) P < 0.01 versus Scramble; (n.s.) not significant. See also Supplemental Figure S5. (C,D) The mRNA expression of Hoxa genes in mouse livers after suppression of Fh or Sdha was determined by quantitative RT–PCR. Error bars represent standard deviation (SD) for triplicate experiments. (E,F) The 5hmC levels in mouse livers after suppression of Fh or Sdha were determined by immunohistochemistry (E) and dot blot assay (F). Bars, 334 μm. Error bars represent standard deviation (SD) for triplicate experiments. See also Supplemental Figure S6.

Figure 5.

Figure 5.

Ectopic expression of tumor-derived FH or SDH mutants inhibits α-KG-dependent dioxygenases in cultured cells. (A) HEK293T cells with stable FH or SDHA/B knockdown were transiently transfected with plasmids expressing the indicated proteins, and the enzyme activity of wild-type and mutant FH or SDH was measured as described in the Material and Methods. Error bars represent standard deviation (SD) for triplicate experiments. (**) P < 0.01 versus wild type; (##) P < 0.01 versus vector. (B) Overexpression of tumor-derived FH or SDHA/B mutants inhibits histone demethylation and hydroxylation of prolyl residues. Levels of histone methylation, HIF1α, and endostatin were determined by Western blot. (C) Overexpression of tumor-derived FH or SDHA/B mutants inhibits the TET-catalyzed 5mC oxidation. 5hmC levels were determined by dot blot. Error bars represent standard deviation (SD) for triplicate experiments.

Figure 6.

Figure 6.

Tumor-derived FH or SDH mutants are not functional in fumarate or succinate metabolism. (A) HEK293T cells with stable FH or SDHA/B knockdown were transfected with the indicated plasmids. Quantitative RT–PCR was performed to determine mRNA expression of HOXA genes in these cells. Error bars represent standard deviation (SD) for triplicate experiments. (B) The ratios of succinate/α-KG and fumarate/α-KG were determined by GC-MS. Error bars represent standard deviation (SD) for triplicate experiments. (*) P < 0.05 versus Scramble;; (**) P < 0.01 versus Scramble; (n.s.) not significant. See also Supplemental Figure S9.

Figure 7.

Figure 7.

Fumarate and succinate can function as α-KG antagonists to broadly inhibit α-KG-dependent dioxygenases, including the JMJD family KDMs and the TET family of 5mC hydroxylases.

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