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Cited by 156Adrenal and Paraganglionic TumorsEpigenetics and DNA MethylationChromatin Remodeling and Cancer

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Inhibition of succinate dehydrogenase dysregulates histone modification in mammalian cells

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Cervera, Ana M, Bayley, Jean-Pierre, Devilee, Peter, McCreath, Kenneth J·2009·Molecular Cancer
DOI 10.1186/1476-4598-8-89PMC PMC2770992
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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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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.
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.CC BY · PubMed Central
Figure 2 Succinate dehydrogenase gene inhibition dysregulates histone methylation. (A) HEK293 cells were transfected in duplicate with ON-TARGETplus SMARTpool siRNAs targeting SDHD or with a non-targeting control (100 nM) for 72 h. One half of the sample was used to prepare RNA and the other half was processed for histone extraction. The silencing efficiency of SDHD was analysed by RT-PCR (top panel). Gel lanes were loaded with 5 μg histone extract (bottom panel), and immuno blotting carried out with the antibodies indicated. Histone H3 was used as a loading control. Graph shows densitometric analysis of two independent experiments. (B) Hep3B cells were transfected in duplicate with ON-TARGETplus SMARTpool siRNAs targeting SDHB or with a non-targeting control (100 nM) for 72 and 96 h. One half of the sample was used to prepare whole cell extracts and the other half was processed for histone extraction. Gel lanes were loaded with 20 μg total cell extract (top panel) or 5 μg histone extract (bottom panel), and immuno blotting carried out with the antibodies indicated. Tubulin and histone H3 were analysed as loading controls for whole cell and histone extracts, respectively. Graph shows densitometric analysis of three independent experiments. (C) HEK293 cells were transfected as in (B) and processed for analysis after 96 h. Graph shows densitometric analysis of two independent experiments. (D) Immunoblot analysis of wild-type pU6, and SDHB-silenced D11 and D20 cell lines. Samples were processed as described above. Graph shows densitometric analysis of three independent experiments.
Figure 2 Succinate dehydrogenase gene inhibition dysregulates histone methylation. (A) HEK293 cells were transfected in duplicate with ON-TARGETplus SMARTpool siRNAs targeting SDHD or with a non-targeting control (100 nM) for 72 h. One half of the sample was used to prepare RNA and the other half was processed for histone extraction. The silencing efficiency of SDHD was analysed by RT-PCR (top panel). Gel lanes were loaded with 5 μg histone extract (bottom panel), and immuno blotting carried out with the antibodies indicated. Histone H3 was used as a loading control. Graph shows densitometric analysis of two independent experiments. (B) Hep3B cells were transfected in duplicate with ON-TARGETplus SMARTpool siRNAs targeting SDHB or with a non-targeting control (100 nM) for 72 and 96 h. One half of the sample was used to prepare whole cell extracts and the other half was processed for histone extraction. Gel lanes were loaded with 20 μg total cell extract (top panel) or 5 μg histone extract (bottom panel), and immuno blotting carried out with the antibodies indicated. Tubulin and histone H3 were analysed as loading controls for whole cell and histone extracts, respectively. Graph shows densitometric analysis of three independent experiments. (C) HEK293 cells were transfected as in (B) and processed for analysis after 96 h. Graph shows densitometric analysis of two independent experiments. (D) Immunoblot analysis of wild-type pU6, and SDHB-silenced D11 and D20 cell lines. Samples were processed as described above. Graph shows densitometric analysis of three independent experiments.CC BY · PubMed Central
Figure 3 Functional consequences of increased histone methylation in SDHB-silenced cells. (A) Confocal images of SDHB-silenced D11 cells transiently transfected with the overexpression plasmids JMJD3 (wildtype) or JMJD3 mutated (mutant). At 72 h post transfection, cells were fixed and stained with anti-HA and anti-H3K27me3 antibodies; nuclei were stained with DAPI. Arrows indicate transfected cells. (B) Immunoblot analysis of cell lines pU6 and D11, transiently transfected for 72 h with pcDNA control (vec), JMJD3 wild-type (wt) or JMJD3 mutated (mut). Results are representative from two independent experiments. (C) ChIP analysis of H3K27me3 occupancy of the core promoter regions of IGFBP7, SNCA, PTGER, and KRT17 in pU6 and D11 cells. Gels show representative RT-PCR analysis of H3K27me3 and control IgG immunoprecipitates.
Figure 3 Functional consequences of increased histone methylation in SDHB-silenced cells. (A) Confocal images of SDHB-silenced D11 cells transiently transfected with the overexpression plasmids JMJD3 (wildtype) or JMJD3 mutated (mutant). At 72 h post transfection, cells were fixed and stained with anti-HA and anti-H3K27me3 antibodies; nuclei were stained with DAPI. Arrows indicate transfected cells. (B) Immunoblot analysis of cell lines pU6 and D11, transiently transfected for 72 h with pcDNA control (vec), JMJD3 wild-type (wt) or JMJD3 mutated (mut). Results are representative from two independent experiments. (C) ChIP analysis of H3K27me3 occupancy of the core promoter regions of IGFBP7, SNCA, PTGER, and KRT17 in pU6 and D11 cells. Gels show representative RT-PCR analysis of H3K27me3 and control IgG immunoprecipitates.CC BY · PubMed Central
Figure 4 Immunohistochemical staining for methylated histones in a sporadic carotid body paraganglioma. (A) Haematoxylin-eosin staining of a sporadic paraganglioma (100× magnification) showing typical cell nest structure. (B) Nuclear and cytoplasmic staining of sustentacular cells using an antibody directed to S100 (400×). Brown staining highlights the typical zellballen structures. (C) Nuclear staining of chief (type I) cells with the tyrosine hydroxylase marker antibody (400×). (D) Anti-H3K27me3 staining (400× with digital enlargement). A clear zellballen structure is visible, showing exclusively nuclear staining of chief cells (dark arrow) but no staining of sustentacular cells (red arrow). (E) High-magnification image of anti-H3K27me3 staining showing heterogeneous nuclear staining of chief cells (arrow). (F) Anti-H3K36me2 staining (400×). There is nuclear and some cytoplasmic staining of chief cells (dark arrow) but no nuclear staining of sustentacular cells (red arrow) and only light (background) staining of the cytoplasm. This specific staining pattern again highlights the zellballen structures. In all cases a sporadic tumour is illustrated, chosen for clarity. SDHD tumours show similar patterns of staining.
Figure 4 Immunohistochemical staining for methylated histones in a sporadic carotid body paraganglioma. (A) Haematoxylin-eosin staining of a sporadic paraganglioma (100× magnification) showing typical cell nest structure. (B) Nuclear and cytoplasmic staining of sustentacular cells using an antibody directed to S100 (400×). Brown staining highlights the typical zellballen structures. (C) Nuclear staining of chief (type I) cells with the tyrosine hydroxylase marker antibody (400×). (D) Anti-H3K27me3 staining (400× with digital enlargement). A clear zellballen structure is visible, showing exclusively nuclear staining of chief cells (dark arrow) but no staining of sustentacular cells (red arrow). (E) High-magnification image of anti-H3K27me3 staining showing heterogeneous nuclear staining of chief cells (arrow). (F) Anti-H3K36me2 staining (400×). There is nuclear and some cytoplasmic staining of chief cells (dark arrow) but no nuclear staining of sustentacular cells (red arrow) and only light (background) staining of the cytoplasm. This specific staining pattern again highlights the zellballen structures. In all cases a sporadic tumour is illustrated, chosen for clarity. SDHD tumours show similar patterns of staining.CC BY · PubMed Central

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Metadata

Authors
Cervera, A. M., Bayley, J.-P., Devilee, P., & McCreath, K. J.
Affiliations
  • Spanish National Centre for Cardiovascular Research (ES)
  • Leiden University Medical Center (NL)
Corresponding author
McCreath, Kenneth J
ORCID
Venue
Molecular Cancer
Publisher
Springer Science and Business Media LLC
Published
2009-10-22
Volume
8
Issue
1
Pages
89-89
ISSN
1476-4598 (ISSN-L)
DOI
10.1186/1476-4598-8-89
PMC
PMC2770992
OpenAlex
W2166443483
Cited by
156
References
24
Citations by year
2026: 12 · 2025: 4 · 2024: 7 · 2023: 8 · 2022: 13 · 2021: 9 · 2020: 7 · 2019: 5 · 2018: 5 · 2017: 9 · 2016: 9 · 2015: 14 · 2014: 18 · 2013: 12 · 2012: 16
FWCI
8.11
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Molecular Cancer (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • European Commission
  • Ministerio de Ciencia e Innovación
  • Centro Nacional de Investigaciones Cardiovasculares
  • Instituto de Salud Carlos III (PI0600299)
Topics
  • Health Sciences › Medicine › Surgery › Adrenal and Paraganglionic Tumors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
Keywords
succinate dehydrogenase, histone H3 methylation, JMJD3, H3K27me3, IGFBP7, paraganglioma, pheochromocytoma, mitochondrial metabolism, epigenetic dysregulation, chief cells, cancer epigenetics
MeSH terms
Animals, Carotid Body Tumor, Histones, Humans, Immunohistochemistry, Methylation, Protein Processing, Post-Translational, Staining and Labeling, Succinate Dehydrogenase, Thenoyltrifluoroacetone, Gene Silencing, Cell Line, Tumor, Rats

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