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Inhibition of succinate dehydrogenase dysregulates histone modification in mammalian cells.

Cervera AM, Bayley JP, Devilee P, McCreath KJ - Mol. Cancer (2009)

Bottom Line: Recently, the jumonji-domain histone demethylases have emerged as a novel family of 2-oxoglutarate-dependent chromatin modifiers with credible functions in tumourigenesis.Using pharmacological and siRNA methodologies we show that increased methylation of histone H3 is a general consequence of SDH loss-of-function in cultured mammalian cells and can be reversed by overexpression of the JMJD3 histone demethylase.ChIP analysis revealed that the core promoter of IGFBP7, which encodes a secreted protein upregulated after loss of SDHB, showed decreased occupancy by H3K27me3 in the absence of SDH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Regenerative Cardiology, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain. acervera@cnic.es

ABSTRACT
Remodelling of mitochondrial metabolism is a hallmark of cancer. Mutations in the genes encoding succinate dehydrogenase (SDH), a key Krebs cycle component, are associated with hereditary predisposition to pheochromocytoma and paraganglioma, through mechanisms which are largely unknown. Recently, the jumonji-domain histone demethylases have emerged as a novel family of 2-oxoglutarate-dependent chromatin modifiers with credible functions in tumourigenesis. Using pharmacological and siRNA methodologies we show that increased methylation of histone H3 is a general consequence of SDH loss-of-function in cultured mammalian cells and can be reversed by overexpression of the JMJD3 histone demethylase. ChIP analysis revealed that the core promoter of IGFBP7, which encodes a secreted protein upregulated after loss of SDHB, showed decreased occupancy by H3K27me3 in the absence of SDH. Finally, we provide the first evidence that the chief (type I) cell is the major methylated histone-immunoreactive constituent of paraganglioma. These results support the notion that loss of mitochondrial function alters epigenetic processes and might provide a signature methylation mark for paraganglioma.

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Pharmacological SDH inhibition increases histone methylation. (A). Immunoblot analysis of histone methylation in Hep3B, HT1080 and PC12 cells treated for 24 h with 500 μM 2-thenoyltrifluoroacetone (TTFA). In all cases lanes were loaded with 5 μg histone extract, and blots were analysed with the indicated antibodies. Histone H3 total expression was analysed as a loading control. (B) Densitometric analysis of three independent experiments. Fold increase was calculated as the ratio of methylated lysine to H3 control, between treatments.
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Figure 1: Pharmacological SDH inhibition increases histone methylation. (A). Immunoblot analysis of histone methylation in Hep3B, HT1080 and PC12 cells treated for 24 h with 500 μM 2-thenoyltrifluoroacetone (TTFA). In all cases lanes were loaded with 5 μg histone extract, and blots were analysed with the indicated antibodies. Histone H3 total expression was analysed as a loading control. (B) Densitometric analysis of three independent experiments. Fold increase was calculated as the ratio of methylated lysine to H3 control, between treatments.

Mentions: Cultured cells were exposed to pharmacological suppression of SDH activity with 2-thenoyltrifluoroacetone (TTFA). Using Western blot analysis with methylation-state-specific antibodies, we determined the steady-state levels of histone 3 methylated on residues K9, K27, and K36. Addition of TTFA resulted in a reproducible increase in global histone 3 methylation in Hep3B and HT1080 human cell lines and also in rat PC12 phaeochromocytoma cells, although the lysine affected and the degree of increase was cell line-dependent (Figure 1A and 1B). We next silenced expression of the endogenous SDHD gene in cultured cells. Transient silencing of SDHD in HEK293 cells resulted in a significant reduction of SDHD mRNA in whole cells (Figure 2A). At the same time, analysis of nuclear histones revealed an increase in steady-state levels of both H3K27me3 and H3K36me2 upon SDHD silencing, with H3K36me2 presenting the greatest increase (Figure 2A). To further validate this response we silenced a second SDH gene, SDHB. Transient silencing of SDHB in Hep3B cells resulted in a robust reduction of SDHB protein as measured by Western blot, and analysis of nuclear histones showed increased steady-state levels of both H3K27me3 and H3K36me2 (Figure 2B). Similar results were obtained after transient silencing of SDHB in the HEK293 cell line (Figure 2C), confirming the generality of this response. Moreover, analysis of cells in which SDHB was chronically silenced by integrated siRNA (cell lines D11 and D20) [9] revealed a consistent increase in methylated histone residues (Figure 2D). Given that histone methylation is a dynamic phenomenon, we wanted to ensure that the SDH-dependent methylation could be reversed by increasing demethylase activity. We therefore forced overexpression of the H3K27me3-specific Jmjd3 histone demethylase [17] in cells. Transfection of an HA-tagged C-terminal region of Jmjd3, containing the JmjC domain, but not a mutated (non-active) C-terminal region was sufficient to downregulate H3K27me3 levels in Hep3B cells, as shown by double staining with an anti-HA antibody and the methylation-specific anti-H3K27me3 antibody (Figure 3A). Consistently, when overexpressed in the D11 (SDHB-deficient) cell line, wild-type but not mutated Jmjd3 downregulated H3K27me3 levels (Figure 3B). Together, these data strongly suggest that SDH gene inactivation leads to a reversible dysregulation of chromatin remodelling by increasing the global level of histone H3 methylation.


Inhibition of succinate dehydrogenase dysregulates histone modification in mammalian cells.

Cervera AM, Bayley JP, Devilee P, McCreath KJ - Mol. Cancer (2009)

Pharmacological SDH inhibition increases histone methylation. (A). Immunoblot analysis of histone methylation in Hep3B, HT1080 and PC12 cells treated for 24 h with 500 μM 2-thenoyltrifluoroacetone (TTFA). In all cases lanes were loaded with 5 μg histone extract, and blots were analysed with the indicated antibodies. Histone H3 total expression was analysed as a loading control. (B) Densitometric analysis of three independent experiments. Fold increase was calculated as the ratio of methylated lysine to H3 control, between treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2770992&req=5

Figure 1: Pharmacological SDH inhibition increases histone methylation. (A). Immunoblot analysis of histone methylation in Hep3B, HT1080 and PC12 cells treated for 24 h with 500 μM 2-thenoyltrifluoroacetone (TTFA). In all cases lanes were loaded with 5 μg histone extract, and blots were analysed with the indicated antibodies. Histone H3 total expression was analysed as a loading control. (B) Densitometric analysis of three independent experiments. Fold increase was calculated as the ratio of methylated lysine to H3 control, between treatments.
Mentions: Cultured cells were exposed to pharmacological suppression of SDH activity with 2-thenoyltrifluoroacetone (TTFA). Using Western blot analysis with methylation-state-specific antibodies, we determined the steady-state levels of histone 3 methylated on residues K9, K27, and K36. Addition of TTFA resulted in a reproducible increase in global histone 3 methylation in Hep3B and HT1080 human cell lines and also in rat PC12 phaeochromocytoma cells, although the lysine affected and the degree of increase was cell line-dependent (Figure 1A and 1B). We next silenced expression of the endogenous SDHD gene in cultured cells. Transient silencing of SDHD in HEK293 cells resulted in a significant reduction of SDHD mRNA in whole cells (Figure 2A). At the same time, analysis of nuclear histones revealed an increase in steady-state levels of both H3K27me3 and H3K36me2 upon SDHD silencing, with H3K36me2 presenting the greatest increase (Figure 2A). To further validate this response we silenced a second SDH gene, SDHB. Transient silencing of SDHB in Hep3B cells resulted in a robust reduction of SDHB protein as measured by Western blot, and analysis of nuclear histones showed increased steady-state levels of both H3K27me3 and H3K36me2 (Figure 2B). Similar results were obtained after transient silencing of SDHB in the HEK293 cell line (Figure 2C), confirming the generality of this response. Moreover, analysis of cells in which SDHB was chronically silenced by integrated siRNA (cell lines D11 and D20) [9] revealed a consistent increase in methylated histone residues (Figure 2D). Given that histone methylation is a dynamic phenomenon, we wanted to ensure that the SDH-dependent methylation could be reversed by increasing demethylase activity. We therefore forced overexpression of the H3K27me3-specific Jmjd3 histone demethylase [17] in cells. Transfection of an HA-tagged C-terminal region of Jmjd3, containing the JmjC domain, but not a mutated (non-active) C-terminal region was sufficient to downregulate H3K27me3 levels in Hep3B cells, as shown by double staining with an anti-HA antibody and the methylation-specific anti-H3K27me3 antibody (Figure 3A). Consistently, when overexpressed in the D11 (SDHB-deficient) cell line, wild-type but not mutated Jmjd3 downregulated H3K27me3 levels (Figure 3B). Together, these data strongly suggest that SDH gene inactivation leads to a reversible dysregulation of chromatin remodelling by increasing the global level of histone H3 methylation.

Bottom Line: Recently, the jumonji-domain histone demethylases have emerged as a novel family of 2-oxoglutarate-dependent chromatin modifiers with credible functions in tumourigenesis.Using pharmacological and siRNA methodologies we show that increased methylation of histone H3 is a general consequence of SDH loss-of-function in cultured mammalian cells and can be reversed by overexpression of the JMJD3 histone demethylase.ChIP analysis revealed that the core promoter of IGFBP7, which encodes a secreted protein upregulated after loss of SDHB, showed decreased occupancy by H3K27me3 in the absence of SDH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Regenerative Cardiology, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain. acervera@cnic.es

ABSTRACT
Remodelling of mitochondrial metabolism is a hallmark of cancer. Mutations in the genes encoding succinate dehydrogenase (SDH), a key Krebs cycle component, are associated with hereditary predisposition to pheochromocytoma and paraganglioma, through mechanisms which are largely unknown. Recently, the jumonji-domain histone demethylases have emerged as a novel family of 2-oxoglutarate-dependent chromatin modifiers with credible functions in tumourigenesis. Using pharmacological and siRNA methodologies we show that increased methylation of histone H3 is a general consequence of SDH loss-of-function in cultured mammalian cells and can be reversed by overexpression of the JMJD3 histone demethylase. ChIP analysis revealed that the core promoter of IGFBP7, which encodes a secreted protein upregulated after loss of SDHB, showed decreased occupancy by H3K27me3 in the absence of SDH. Finally, we provide the first evidence that the chief (type I) cell is the major methylated histone-immunoreactive constituent of paraganglioma. These results support the notion that loss of mitochondrial function alters epigenetic processes and might provide a signature methylation mark for paraganglioma.

Show MeSH
Related in: MedlinePlus