Oncometabolite D-2-Hydroxyglutarate enhances gene silencing through inhibition of specific H3K36 histone demethylases
- University of California, Berkeley, United States
Figures
Figure 1 with 2 supplements
Yeast versions of tumor-associated isocitrate dehydrogenase mutation caused increased stabilization of heterochromatin.
(A) Illustration of the CRASH (Cre reported altered states of heterochromatin) assay used to measure the strength of heterochromatic gene silencing at the HML locus. (B) Images of colonies with the …
Figure 1—figure supplement 1
Multiple peptide sequence alignment of human and Saccharomyces cerevisiae NADP+-dependent isocitrate dehydrogenases.
Alignment of S. cerevisiae Idp1 (CAA98631.1), Idp2 (NP_013275.1), Idp3 (NP_014389.3) and H. sapiens IDH1 (CAG38738.1) and IDH2 (AAH71828.1) was generated using T-Coffee Multiple Sequence Alignment …
Figure 1—figure supplement 2
Images of colonies with the CRASH reporter assay and idp2∆ or IDP2-R132H mutations grown on glucose.
Cells were plated on CSM-Trp glucose agar plates and grown for 7 days at 30°C. Tops of colonies were imaged in GFP and RFP channels. The RFP and GFP channels for a single representative image of …
Figure 2
The silencing phenotype of IDP2-R132H was dominant.
(A) Images of diploid colonies with the CRASH reporter assay. The diploid strains have CRE inserted at both copies of HML and two copies of the CRASH RFP-GFP reporter cassette and were wild type …
Figure 3 with 1 supplement
D2-HG levels increase in dld- and idp mutants.
(A) Plot of the average fold change from wild type (JRY10790) in levels of D-2-hydroxyglutarate from metabolite extracts of dld2Δ (JRY10733), idp2Δ (JRY10732), IDP2-R132H (JRY10731), and dld2Δ …
Figure 3—figure supplement 1
Increased D2-HG levels correspond to an increase in silencing.
(A) Images of colonies with the CRASH reporter assay from wild type (JRY10790), dld2Δ (JRY10733) and dld2Δ IDP2-R132H (JRY10734) cells transformed with the empty vector pRS315 (denoted as ‘-') or …
Figure 4
Treatment with cell-permeable octyl-D2-HG increased heterochromatin silencing.
(A) Images of wild type (JRY10790) CRASH assay reporter colonies that were grown on CSM-Trp 3% Glycerol (untreated) or with 100 µM octyl-D2-HG added to the medium (Left Panel). Plots of the …
Figure 5
D2HG did not enhance a locus-specific gene repression mechanism.
(A) Images of colonies of CRASH reporter strains with wild-type HML (JRY10757) and either a pRS413 empty vector (left panels) or a vector with cre expressed from the GAL1 promoter (pSH62) (right …
Figure 6 with 1 supplement
Bulk histone methylation increased in strains with high levels of D2-HG.
(A) Immuno-blot analysis of H3 histone methylation states from wild type (JRY10790), set2∆ (JRY10746), and dld2∆ IDP2-R132H (JRY10734) mutants. The upper panel of each set shows immuno-blot signals …
Figure 6—figure supplement 1
Immuno-blot analysis of H3 histone methyl states from wild type (JRY10790) and dld2∆ IDP2-R132H mutants (JRY10734).
The upper panel of each set shows immunoblot signals generated from antibodies against trimethyl H3K4, H3K79, and total H3, as imaged on a LI-COR Odyssey. The lower panel of each set shows a loading …
Figure 7 with 1 supplement
Loss of H3K36 demethylases Gis1 and Rph1 increased heterochromatin stability.
(A) Images of CRASH assay reporter colonies that are wild type (JRY10790) or contain mutations in H3K36 demethylases. (B) Plots of the frequency of loss-of-silencing events from mutants in panel A …
Figure 7—figure supplement 1
Images of colonies with the CRASH reporter assay and emc5Δ, jhd2Δ, and set1Δ mutations grown on glycerol.
Wild-type strains (JRY10790) and strains deleted for ecm5∆ (JRY10754) (A), jhd2∆ (JRY10755) (B), or set1Δ (JRY10756) (C) were grown on CSM-Trp-glycerol agar plates for 7 days at 30°C. Representative …
Figure 8
IDP2-R132H and dld2Δ silencing phenotypes depends on H3K36 hypermethylation.
(A) Images of CRASH assay reporter colonies that are wild type (JRY10790) or mutant dld2Δ (JRY10733), dld2Δ IDP2-R132H (JRY10734), set2∆ (JRY10746), dld2Δ set2∆ (JRY10794), or dld2Δ IDP2-R132H set2∆ …
Additional files
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Supplementary file 1
Saccharomyces cerevisiae strains used in this study.
All strains were derived from W303 with the following genotype: can1-100 his3-11 leu2-3,11 lys2 TRP1 ADE2 ura3-1.
- https://doi.org/10.7554/eLife.22451.015
