EMBO/soda-nel huggingface dataset:
Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation (2020)
Figure 3

Figure 3: Class switch recombination and proliferative potential of Dot1L-deficient B cells in response to T-cell dependent and -independent stimuli in vitro

A. Statistical analysis of IgG1 switching of Dot1L-proficient (WT) and -deficient (KO) B cells after four days of activation with anti-CD40 + IL-4. Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4).

B. Number of cell divisions traced by CTV dilution of B cells stimulated for four days with anti-CD40 + IL-4. Data represent three biological replicates for each genotype.

C. Statistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT and KO B cells after four days of stimulation with anti-CD40 + IL-4. Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4).

D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13).

F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).

G. Number of cell divisions traced by CTV dilution of B cells stimulated for four days with anti-CD40 + IL-4 either in the presence of DOT1L inhibitor Pinometostat or DMSO as a control. Data represents three biological replicates for each treatment.

H. Statistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT B cells after four days of stimulation with anti-CD40 + IL-4 either in the presence of DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 3).

I. Statistical analysis of IgG3 switching of naïve B cells after four days of activation with LPS alone in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).

Data information: Statistical analyses were performed using the Student's two-tailed unpaired t-test. Statistical significance was determined by calculating the p-value. A p-value of less than 0.05 was considered as significant. Bars and error bars indicate mean ± SD.
Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation (2020)
Figure 4

Figure 4: DOT1L-deficient B cells fail to mount an efficient immune response

A. Representative flow cytometry plots showing gating strategy to identify splenic GC B cells 14 days after LCMV Armstrong infection in WT and Dot1L-KO mice (left panel) and statistical analysis of their relative number. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).

B. Representative flow cytometry plots showing gating strategy to identify plasma cells from the spleen 14 days after LCMV Armstrong infection (left panel) and statistical analysis of their relative number (right panel). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 3, KO; n = 4).

C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).

D. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.

Data information: Except for Figure 4D, statistical analyses were performed using the Student's two-tailed unpaired t-test. Statistical significance was determined by calculating the p-value. A p-value of less than 0.05 was considered as significant. Bars and error bars except Figure 4D indicate mean ± SD.
Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation (2020)
Figure 5

Figure 5: Chemical inhibition of DOT1L results in aberrant plasma cell differentiation in response to in vitro stimulation.

A. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).

B. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).

C. Representative flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).

D. Representative histograms showing the relative surface density for CD19 on total live cells after four days of stimulation with LPS (left panel) and statistical analysis of MFI of CD19 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).

Data information: Statistical analyses were performed using the Student's two-tailed unpaired t-test. Statistical significance was determined by calculating the p-value. A p-value less than 0.05 was considered as significant. Bars and error bars indicate mean ± SD.
Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation (2020)
Figure 6

Figure 6: Transcriptome analysis of in vitro activated B cells shows accelerated plasma-cell differentiation and compromised activation of MYC-target genes in the absence of DOT1L.

A. MA-Plot of normalized RNA-Seq data generated from three independent biological replicates for each genotype showing differential (false discovery rate (FDR) < 0.05) expression of genes between Dot1L-KO and WT B cells after two days of in-vitro activation with LPS+IL-4.

B. Differential (FDR < 0.05) expression of Bach2, Prdm1 (encoding BLIMP-1), Cd138 (a plasma-cell marker) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language.

C-D. Enrichment of plasma-cell signature genes in KO as compared to WT activated B cells (C) and Enrichment of MYC-target genes in WT as compared to KO activated B cells (D). Enrichment of gene sets is depicted by a BARCODE plot; P-value calculated via FRY test show the statistical significance of enrichment of each gene set. A p-value of less than 0.05 was considered as significant.

Source data are available online for this figure.
Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation (2020)
Figure 7

Figure 7: DOT1L-mediated H3K79 methylation is associated with gene activity in B cells and indirectly promotes repression of PRC2 target genes.

A. Integrative analyses of differentially (FDR < 0.05) expressed transcripts from naïve and activated Dot1L-deficient B cells (left panel) with H3K79me2 ChIP values from WT naïve and activated B cells (right panel).

B. The distribution of mean H3K79me2 amongst different gene sets (6A, left panel) from activated and naïve B cells depicted by box plots. Boxes in Box plot indicate Inter quartile range (IQR) and whiskers show 1.5 IQR of highest and lowest quartile. Central horizontal line within the bars represent median of the TMM normalized H3K79me2 counts + 1 values of the respective genes for each condition. Results represent the data generated from three biological replicates for each group.

C. Differential (FDR < 0.05) expression of Ezh2 and Cdkn1a as indicated by counts per million after TMM normalization from WT and Dot1L-KO activated B cells. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language.

D. H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells, as determined by reads per genomic content (RPGC). Data represent three independent biological replicates.

E. Correlation between expression of Ezh2 and Dot1L as depicted by TPM in different mature B-cell subsets; B1: B1 cells, MZB: marginal Zone B, FOB: follicular B, GCB: germinal Center B, SPLPB: spleen plasma blast, SPLPC: spleen plasma cells) and Bone Marrow (BMPC: Bone marrow plasma cells) A20: Germinal Center like cell lymphoma cell line. Correlation shown by scatter plot.

F. Coverage plot of H3K27me3 from naïve B cells (Frangini et al., 2013) and H3K79me2 (from WT activated and naïve B cells) flanking 4 kb around transcriptional start sites (TSS) for genes upregulated in KO (KO Gain) or non-differential Expression Matched genes. Coverage was calculated as reads per genomic content cutoff at the 0.995th quantile and rescaled to a maximum of 1. Black boxes indicate the relavant B cell population for comparing the distribution of H3K27me3 and H3K79me2 for the indicated gene sets obtained from the differential gene expression analysis of Dot1L-proficient and deficient B cells.

Source data are available online for this figure.
Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway (2013)
figf1

(a) Left, the structure of THPN. Right, THPN specifically induces the cell death in several melanoma cell lines but not in any other types of cancer cell lines. The cells were treated with THPN for 48 h, as indicated; cell survival was determined by flow cytometry. '–' represents vehicle treatment only. (b) THPN-induced cell death is prevented by the autophagic inhibitors wortmannin (100 nM) and chloroquine (20 μM) but not by the apoptotic inhibitor zVAD (20 μM) or the necrotic inhibitors butylated hydroxyanisole (100 μM) and necrostatin-1 (30 μM). The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment (20 μM, 48 h). Ctrl, control. (c) THPN induces autophagy in melanoma cells. Top, THPN (20 μM, 24 h) induces a change in the localization of GFP-LC3 fusion proteins from a ubiquitous, diffuse pattern to cytoplasmic puncta. Serum starvation (SS, 24 h) was used as a positive control (scale bars, 10 μm). Middle, the GFP-LC3–positive cells with puncta were calculated from five randomly picked regions. Bottom, the conversion of LC3-I to LC3-II induced by THPN was determined by western blotting. Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.
Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway (2013)
figf2

(a) THPN induces TR3 translocation from the cytoplasm to the mitochondria, detected by western blotting for the various cell fractionations (top) and by confocal microscopy (bottom). The cells were treated with THPN at the times indicated and detected by TR3 (red) and Hsp60 (green). The nuclei were stained with DAPI (blue) (scale bars, 10 μm). PARP, Tubulin and Hsp60 were used as loading controls for nuclear extracts (NE), cytoplasmic extracts (CE) and mitochondrial fractions (Mito), respectively. IB, immunoblotting. (b) TR3 mutant Flag-ΔNLS but not Flag-NLS promotes the induction of autophagy in response to THPN treatment. The siRNA-resistant versions of Flag-ΔNLS and Flag-NLS were transfected into TR3-knockdown A375 cells. Top, the cells cotransfected with GFP-LC3 were further treated with THPN for 24 h before microscopic analysis (scale bars, 10 μm). Middle, the conversion of LC3-I to LC3-II was analyzed by western blotting. The rate of cell survival was determined after THPN treatment for 48 h (bottom). '–' represents vehicle treatment only. (c) Nix assists the TR3 mitochondrial targeting. Cells with Nix, Pink1 and Bnip3 knockdown were treated with THPN (20 μM, 6 h). Top, mitochondrial TR3 was assayed by western blotting. Nix knockdown abolishes the THPN-induced (20 μM, 24 h) formation of LC3 foci (middle and bottom; scale bars, 10 μm). Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01; NS, not significant; ctrl, control.
Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway (2013)
figf3

(a) THPN (yellow) can only be fitted in site C in structure I. Structure II has a different conformation, and site C in structure II is too narrow to accommodate THPN. (b) Site C of LBD is critical for Nix binding, as determined by the in vitro pull-down assay. The purified bead-bound GST or GST-Nix proteins were incubated with different mutants as indicated and THPN (20 μM) for 3 h; the proteins were then analyzed by western blotting. IB, immunoblotting. (c) Footprint on the surface of LBD upon which Nix binds. Left, binding of THPN to Site C forms a fitting surface for the interaction with Nix. Right, THPN binding also results in a conformational change in residues Arg563 and Ser553 as compared to the apo structure. (d) The S553A mutation creates a dent on the surface of LBD, as compared to the native structure in c. (e) DPDO cannot facilitate Nix binding to TR3. Top, the structure of DPDO. Middle, DPDO binding could not form the interacting surface for Nix, unlike THPN. The structures of Arg563 and Ser553 are the same as those in the native structure. Bottom, DPDO does not enhance the interaction between TR3 and Nix. Different plasmids were transfected into A375 cells as indicated, and cells were treated with DPDO (20 μM, 3 h). The TR3 interaction with Nix was determined by co-immunoprecipitation. Images of full blots are shown in Supplementary Figure 7. IP, immunoprecipitation; HA, hemagglutinin.
Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway (2013)
figf4

(a) THPN induces the dissipation of ΔΨm in both time- and dose-dependent manners. The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry. '–' represents vehicle treatment only. (b) Effects of different plasmids on the THPN-induced dissipation of ΔΨm. Top, endogenous TR3 was knocked down by shRNA, and the siRNA-resistant version of TR3, TR3-NLS, TR3-ΔNLS or TAD (Supplementary Fig. 2c) were separately transfected into the cells as indicated. Bottom, similarly, the siRNA-resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells. Transfected cells were treated with THPN (20 μM, 12 h), and their ΔΨm was assessed by flow cytometry. Ctrl, control. (c) ANT1 and VDAC1 are required for the THPN-induced autophagy. The ANT1 and VDAC1 shRNA-expressing plasmids, which also encoded EGFP for evaluating the transfection efficiency, were separately transfected into cells; mCherry-LC3 was cotransfected to display autophagy. Green cells are those transfected with ANT1 or VDAC1 shRNA, and the cells with red cytoplasmic puncta are autophagic cells (top; scale bars, 10 μm). (d) ANT1 and VDAC1 are required for the THPH-induced dissipation of ΔΨm and cell death. Endogenous ANT1 or VDAC1 was separately knocked down by shRNA, and the dissipation of ΔΨm was determined after THPN (20 μM, 12 h) treatment (left). ANT1 and VDAC1 knockdown abolishes the THPN-induced (20 μM, 48 h) cell death (right). All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01.
Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway (2013)
figf5

(a) Endogenous TR3 is localized in the MIM in response to THPN stimulation. Left, isolated mitochondria from cells treated with THPN (20 μM, 6 h) were incubated with proteinase K (20 μM and 40 μM) for 30 min. TR3 expression was determined. Tom20 in MOM and Tim23 in MIM were used as controls. Right, the mitochondria were further subjected to submitochondrial fractionation. VDAC1, Tim23 and Hsp60 were used to represent MOM, MIM and mitochondrial matrix protein, respectively. '–' represents vehicle treatment only. (b) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM. Endogenous Toms were separately knocked down by their corresponding shRNAs, and the TR3 expression of mitochondrial fractions was detected as described above. Ctrl, control. (c) Tom40 and Tom70 are required for the THPN-associated dissipation of ΔΨm (left), conversion of LC3-I to LC3-II (right and top) and cell death (right and bottom). Endogenous Toms were separately knocked down by the corresponding shRNAs. ΔΨm, LC3-II and cell death were detected as described above. Images of full blots are shown in Supplementary Figure 7. (d) The mitochondria were markedly eliminated after treatment with THPN for 24 h. Right, endogenous Tom40 and Tom70 were knocked down, and the amount of mitochondria was measured by the fluorescence intensity of Tom20 (green). Left, the volume of mitochondria from five randomly picked regions was quantified by ImageJ software. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.
Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway (2013)
figf6

(a) THPN inhibits the formation of tumors in the liver and lung in a TR3-dependent manner. B16 cells transfected with either control shRNA, TR3 shRNA or ATG7 shRNA (5 × 105) were separately injected into the lateral tail vein of 6-week-old C57BL/6J mice (n = 7). THPN were administered 3 d after inoculation. The tumors formed in the lung and liver are indicated (top; scale bars, 5 mm) and were counted (bottom). Ctrl, control. (b) Crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice (n = 12) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms, and mice were treated with THPN as described in Online Methods. The representative images of dorsal melanomas (top) and the tumor numbers (bottom) are presented. (c) The survival curves of crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice are shown (n > 16). (d) A signaling pathway of THPN-induced and TR3-mediated autophagic induction in the inhibition of melanoma growth. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01; NS, not significant.
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 1

Figure 1. NSUN3 localises to the mitochondrial matrix.

A. The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3-GFP (green). NSUN3-GFP and staining with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.

B. To analyse submitochondrial localization of NSUN3, human mitochondria were isolated and either left untreated, swollen in hypotonic buffer (Mitoplasts) or solubilized by sonication (Sonic.) before treatment with different amounts of Proteinase K (PK) where indicated, followed by SDS-PAGE and Western blotting using antibodies against human TIM44, TIM23, TOM70 or FLAG-tagged NSUN3. Note that TIM44 localises to the matrix, while the N-terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface. The asterisk indicates a cross-reaction of the TOM70 antibody.
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 2

Figure 2. NSUN3 crosslinks to the mitochondrial tRNAMet in vivo.

A. Structure of 5-azacytidine and formation of a covalent RNA methyltransferase adduct.

B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film.

C-E. The UV or 5-AzaC crosslinking and analysis of cDNA (CRAC) experiments with NSUN3-HisPrcFLAG (D-E) or the FLAG control (C) samples were treated as described in B. The RNA was isolated from the nitrocellulose membrane-bound protein-RNA complexes and converted into cDNA for sequence library production and Illumina deep sequencing. Pie charts present different RNA classes and the relative distribution of Illumina sequence reads that were obtained after mapping of the reads on the human genome. Bar graphs below indicate the distribution of mitochondrial (mt-)tRNA, mt-rRNA and mt-mRNA sequence reads among the reads mapped to the mitochondrial genome. Abbreviations: tRNA – transfer RNA, snRNA – small nuclear RNA, snoRNA – small nucleolar RNA, rRNA – ribosomal RNA, mtRNA – mitochondrial encoded RNA, miscRNA – miscellaneous RNA, miRNA – micro RNA, lncRNA – long non-coding RNA.

F. Relative distribution of mitochondrial tRNA sequence reads obtained from the CRAC experiments using UV or 5-AzaC crosslinking with cells expressing the NSUN3-HisPrcFLAG (NSUN3) protein or control cells (FLAG). Only mt-tRNAs that were represented by more than 5% of all mt-tRNAs reads are labelled.

G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.