Extended Data Fig. 4: Valine deprivation increases the activity of TET2 dependent on the nuclear translocation of HDAC6.
From: Human HDAC6 senses valine abundancy to regulate DNA damage
a, Schematic representation of experimental workflow to identify potential HDAC6 binding proteins under valine deprivation for 6 h by the SILAC quantitative proteomics. b, Identification of differential TET2-binding proteins in valine deprivation for 12 h via data-independent acquisition-based mass spectrometry. c, Effects of valine restriction on the endogenous interaction between HDAC6 and TET2 detected by co-immunoprecipitation. Valine was deprived for 6 h and then HCT116 cells subjected to add valine in a concentration gradient. d, Addition of valine (10 mM) to the cell extract expressing Flag-HDAC6 and Myc-TET2 negatively affects the interaction between HDAC6 and TET2 upon valine deprivation for 12 h via co-immunoprecipitation. e, Ectopically expressed HDAC6 binds to the TET2 CD ___domain via co-immunoprecipitation assay. HDAC6 binds to the CD ___domain of TET2 upon valine deprivation for 12 h. f, TET2 interacts with the exogenous overexpressed DAC ___domain of HDAC6 upon valine deprivation for 24 h or not. g, h, Effects of valine deprivation on 5hmC levels in HCT116 cells upon valine deprivation for different time. Quantification of dot blots in g. i, Effect of valine deprivation on 5hmC levels in Rhinopithecus bieti-fibroblast cells upon valine deprivation for different times via Immunofluorescence. j, UPLC–MS/MS results showing the effects of valine deprivation in HCT116 cells. k, Effects of valine deprivation on 5hmC levels. HCT116 HDAC6 WT or KO cells were exposed to valine deprivation at different time points and the cells were fixed and immunostained with anti-5hmC (red) antibodies. Scale bar, 10 μm. l, m, Effects of valine deprivation on 5hmC levels in WT or HDAC6 knockout HCT116 cells examined by flow cytometry (l). n. Quantification of dot blots in Fig. 3b. o, p, Bar plot showing the fraction of 5hmCG against covered CG sites (o) and the fraction of 5mCG against covered CG sites (p) in HDAC6 knockdown, TET2 knockdown and WT HCT116 cell lines upon valine deprivation for 24 h or not. q, Principal component analysis (PCA) of TET2 ChIP-Seq data. “-Val”, valine deprivation for 24 hrs. r, Clustering of TET2 binding peaks mainly contributing to the separation of the four groups in PCA. Three replicates for each sample. s, Violin plots showing the 5hmC level (q) and 5fC/5caC level (r) in WT, HDAC6 knockdown, TET2 knockdown HCT116 cell lines upon valine deprivation at the valine deprivation-specific enhanced TET2 binding regions. The red point indicates the mean value of the given data. t, u, In vitro TET2 catalytic activity assay. Synthesized methylated dsDNA or genomic DNA from HCT116 cells were incubated with purified proteins of TET2 and HDAC6, TET2-catalyzed oxidation was measured by 5mC decrease and 5hmC accumulation as determined by dot-blot assay (t). Quantification of dot blots in u. v, Analysis the deacetylase of HDAC6 and various truncated proteins eluted from HCT116 cells expressing Flag-HDAC6 or its various truncations via immunoprecipitation. w, Effects of valine deprivation on the acetylation of TET2 in WT, HDAC6 knockdown or SIRT1 knockdown HCT116 cells upon valine deprivation for 6 hrs. x, y, Analysis of 5hmC level of HDAC6 or SIRT1 knockdown HCT116 cells upon valine deprivation at different time (x). Quantification of dot blots in y. z. Schematic of HDAC6 promoting TET2 activity in response to valine deprivation. For h, j, m, n, u, v and y, data are presented as mean ± s.d. (n = 3 independent experiments for h, m, n, u, v, y; n = 6 independent experiments for j). Statistical analysis was performed using one-way ANOVA (h, j, n, v, y) and two-way ANOVA (m, u); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. Schematic in z was created using BioRender (https://BioRender.com). For gel source data, see Supplementary Fig. 1.
