EMBO/soda-nel huggingface dataset:
Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma (2019)
Figure 1

Figure 1 - Rpd3 and other members of the Rpd3L complex negatively regulate H3K79 methylation.

A) Schematic overview of the Epi-ID strategy.

B) Epi-ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes, calculated as described in Appendix Supplementary Methods, where 0 means a wild-type H3K79me level (log2 scale). The gray dots indicate accessory subunits. UpTag and DownTag are barcode reporters in a promoter and terminator context, respectively. Data was obtained on all Rpd3L/Rpd3S subunits except Sds3.

C) Mass spectrometry analysis of H3K79 methylation in wild-type and mutant strains. Mean and individual data points of two biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.

D) Heatmaps of H3K79me1, H3K79me3, H2Bub and H3 in wild-type cells, aligned on the TSS. Genes were sorted based on the average H3K79me3/H3K79me1 ratio in the first 500 bp.

E) Snapshot of depth-normalized ChIP-seq data tracks from wild-type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF, which is the top gene in the heatmap in panel (F). All tracks have the same y axis (0-20 RPM). A snapshot of another top-regulated gene is shown in Fig EV1D.

F) Heatmap of the H3K79me3/H3 change in rpd3Δ versus wild-type cells, aligned on the TSS. Genes were sorted based on the average ratio in the first 500 bp.
Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma (2019)
Figure 2

Figure 2 - Rpd3 represses transcription, H2B ubiquitination and H3K79 methylation at its target sites.

A) ChIP-seq and RNA-seq data for genes ranked on H3K79me3/H3 in rpd3Δ/WT, smoothed using locally weighed regression. The gray band around the line shows the 95% confidence interval. Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion. ChIP-seq data of H3K79me1, H3K79me3 and H2Bub were generated in this study (plotted is the average coverage in reads per genomic content, RPGC), Rpd3 binding, H4ac and WT gene expression data were from (McKnight et al, 2015), and the relative expression in rpd3Δ/WT from Kemmeren et al (2014).

B) H3K79me3/H3 ChIP-qPCR efficiencies (relative to a non-transcribed region, which was unaffected by RPD3 deletion) in wild-type and in rpd3Δ cells harboring empty or RPD3-encoding CEN plasmids. The H188A and H150A-H151A mutations have previously been shown to abrogate catalytic activity (Kadosh & Struhl, 1998). Error bars indicate standard deviation of three biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.

C-E) Gene set enrichment analysis on genes ranked on H3K79me3/H3 in rpd3Δ/WT; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines. Meiotic (C) and Ume6-bound (D) genes are enriched among the genes at which Rpd3 represses H3K79 methylation, and subtelomeric genes (<30 kb of telomere) (E) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells.
Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma (2019)
Figure 3

Figure 3 - Hdac1 deletion increases H3K79 methylation in thymocytes in vivo, and simultaneous heterozygous Dot1L deletion prolongs tumor-free survival.

A) Mass spectrometry analysis of H3K79 methylation in thymuses from 3-week-old mice, either wild-type (Cre-) or with deleted Hdac1 or Dot1L alleles, as indicated. Mean and individual data points of biological replicates; H3K79me0 is the predominant state, the y axis is truncated at 70% for readability. The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed (yet). p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.

B) Representative H&E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes. A picture with lower magnification of independent samples is included in Fig EV3B.

C) Kaplan-Meier curves of mice harboring thymocytes with indicated genotypes. An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma. Mice that died due to other causes or were still alive and event-free at the end of the experiment were censored. Mice for which the cause of death could not be determined were removed from the data. Wild-type mice were the Cre- littermates of the mice that were used for the other curves.

D) Summary of the data presented in panel C. A median latency could only be calculated when the tumor incidence was >50%. The p value was determined by comparing to the Lck-Cre;Hdac1f/f curve with a Peto test, but a logrank test yielded the same conclusions.
Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma (2019)
Figure 4

Figure 4 - Hdac1-deleted thymic lymphoma cell lines depend on DOT1L activity.

A) Immunoblots showing HDAC1 status and H3K79me/H3K9ac levels in nuclear lysates of Hdac1-proficient (p53-null) and Hdac1-deficient thymic lymphoma cell lines. The top four and bottom two panels are from separate lysates of the same cell lines.

B) Mass spectrometry analysis of H3K79 methylation in the cell lines from panel A. Mean and individual data points of two independent cell lines; H3K79me0 is the predominant state, the y axis is truncated at 80% for readability. p<0.01 (**) and p<0.001 (***) by two-way ANOVA.

C) Immunoblot showing H2BK120 ubiquitination levels in Hdac1-proficient and -deficient cell lines (two independent lines each).

D) Growth curves of Hdac1-proficient and -deficient cell lines that were left untreated or were infected with empty virus (pLKO) or shRNAs against Dot1L and selected with puromycin. Growth curves were determined by a series of resazurin assays, which measure metabolic activity, starting four days post-infection. Error bars indicate the range of two replicates from independent cell lines.

E) Inhibitor dose-response curves of the two DOT1L inhibitors EPZ-5676 (Pinometostat) and SGC-0946 in Hdac1-proficient and -deficient cell lines. Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO-treated cells. Two independent cell lines are plotted separately; error bars indicate the range of two biological replicates.
Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma (2019)
Figure 5

Figure 5 - Hdac1-deficient thymic lymphoma lines require DOT1L activity for survival.

A) Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC-0946 for two days. Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live (Annexin V low; DAPI low), apoptotic (Annexin V high; DAPI low) and dead (Annexin V high; DAPI high) cells.

B) Quantification of several independent FACS experiments, including the experiment shown in panel (A). Mean with individual data points of 2-4 replicates each of two independent lines per genotype. p<0.001 (***) by two-way ANOVA, comparison to corresponding DMSO control.

Expanded View Figure Legends
A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells (2020)
Figure 1

Figure 1. The dynamic alternative splicing program during hESC hematopoietic differentiation.

(A) Schematic representation of the strategies for fluorescence-activated cell sorting (FACS) and transcriptomic analyses. During inducing hematopoietic differentiation, human embryonic stem cells (hESCs, H1) on day 0, FACS-purified lateral plate mesodermal APLNR+ cells on day 2, purified CD31+CD34+ endothelial progenitor cells (EPCs) on day 5, and purified CD43+ hematopoietic stem and progenitor cells (HSPCs) on day 8 were collected for RNA-Seq, respectively. STAR Cufflinks, MISO, DESeq2 and rMATS were used to analyze the expression abundance of genes and transcripts, alternative splicing events, differentially expressed genes and transcripts, and differential splicing events, respectively. n = 3 technical replicates.

(B) Cumulative distribution curves of Log2(FPKM) of splicing factors (n=235) in hESCs APLNR+, CD31+CD34+, and CD43+ cells. The upper and lower dotted lines represent cumulative scores of 1 and 0, respectively. n=3 technical replicates. The P-value was calculated by a two-tailed Wilcoxon signed-rank test.

(C) The heatmap illustrates the row normalized expression of components within the major spliceosomal machinery (n=182) using unsupervised hierarchical clustering. The heatmap was scaled with Z-Score using the log2(FPKM) expression of components within the major spliceosomal machinery. n=3 technical replicates.

(D) The boxplots depict the expression of splicing factors in cluster 1 (n=115) and cluster 2 (n=67) identified in (C) at distinct differentiation stages. The central band indicates the median level of expression. Boxes presents 25%-75% of genes expression. The whiskers indicate the lowest and highest points within 1.5× the interquartile. n=3 technical replicates. P-values were calculated by a two-tailed Wilcoxon signed-rank test.

(E-F) The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were row normalized. The heatmaps were scaled with Z-Score using the log2(FPKM) expression of indicated components. n=3 technical replicates.

(G) The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT-qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P-values were determined by an unpaired two-tailed Student's t-test. n ≥ 3 biological replicates.

(H) The number of differentially expressed transcripts at the isoform level (fold change (FC) > 2 & FDR < 0.01), but not at the gene level (FDR > 0.01), by comparing two consecutive differentiation stages (hESC vs APLNR+ cells, APLNR+ cells vs CD31+CD34+ cells, and CD31+CD34+ cells vs CD43+ cells).

Data information: * presents P < 0.05. ** presents P < 0.01. *** presents P < 0.001.
A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells (2020)
Figure 2

Figure 2. Inhibition of splicing disrupts EPC and HEP generation.

(A) The upper schematic illustrates the stage-specific supplementation of splicing inhibitor PLB and on day 8 CD43+ HSPCs were examined by flow cytometry. The bottom bar graph showing the normalized frequency of CD43+ cells to DMSO control under distinct PLB treatment windows. P-values were calculated by one-way followed by Dunnett's test.

(B) The top panel is a representative RT-PCR electropherogram depicting the inclusion or exclusion LAS1L exon 9 in cells at day 2 as well as cells at day 5 without or with PLB treatment with indicated concentrations, respectively. The quantification of percent spliced-in (PSI) was presented in the bottom bar graph. P-values were calculated by one-way ANOVA followed by Dunnett's test.

(C) The representative FACS plots show the generation of CD31+CD34+ EPCs at day 5 of differentiation upon PLB treatment from day 2.5 to 5.

(D) The frequency of CD31+CD34+ EPCs with PLB treatment in (C) was normalized to DMSO control. P-values were calculated by one-way ANOVA followed by Dunnett's test.

(E) Cell apoptotic level assessed using Annexin V and 7-AAD at day 5 of differentiation by flow cytometry. The cells were treated with or without PLB from day 2.5 to 5.

(F) The proportion of G0/G1, S and G2/M cells at day 5 of differentiation treated with or without PLB from day 2.5 to 5. The cell cycle was determined by flow cytometry with Propidium Iodide staining.

(G) The experimental schematic (upper panel). APLNR+ cells on day 2 were FACS-purified and treated with various dosages of PLB from day 2.5 to 5 during inducing hematopoietic differentiation. On day 5, the generation of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs were assessed. The representative FACS plots showed the frequency of APLNR+ cells, CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment. The bar graphs show the percentage of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment.

Data information: Results given are mean ± SD. P-values were determined by an unpaired two-tailed Student's t-test in (E), (F) and (G). ns represents no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were conducted for at least 3 biological replicates.
A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells (2020)
Figure 4

Figure 4. Linking alternative splicing of NUMB to EPC generation.

(A) The Venn diagram showing genes with significantly isoform changes. Pink circle represents genes with highly expressed isoform (FPKM > 5) that emerged during EPC generation. Blue circle represents genes exhibiting isoform proportion changes (delta iso > 0.15) after PLB treatment (PLB vs DMSO). Green circle represents genes with differentially expressed isoforms (FC > 10 & FDR < 0.05) after PLB treatment (PLB vs DMSO). The heatmap shows the expression of the overlapped genes ranked by FC when compared between DMSO vs PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of isoforms of indicated genes. n=2 technical replicates

(B) The schematic representation (top) indicates the isoform structure of NUMB_L and NUMB_S. The bottom curve reflects the expression of NUMB isoforms in day 2-APLNR+ cells, day 5-DMSO and day 5-PLB (2.5 nM) treated cells.

(C) A Sashimi plot to visualize the mapping of RNA-Seq reads at the exon 11-13 loci of NUMB in day 2-APLNR+ cells, day 5-DMSO-treated and day 5-PLB-treated cells.

(D) The top panel is one representative RT-PCR electropherogram showing the inclusion/exclusion of NUMB exon 9 in day 2-APLNR+, day 5-DMSO-treated and day 5-PLB treated cells. The expected isoforms are indicated on the right with exon 9 in blue and constant exons in red. At the bottom is a statistical bar graph of normalized PSI obtained from the RT-PCR electropherogram. P-values were calculated by one-way ANOVA followed by Dunnett's test.

(E) Western blotting showing NUMB_S overexpression in sorted GFP+ cells on day 5 during differentiation induced by DOX from day 2.5 to 5. GAPDH was used as the loading control. The bar graph presents the quantification of NUMB_S protein expression. The P-value was determined by an unpaired two-tailed Student's t-test.

(F) The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5.

(G) The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.

Data information: Results given are mean ± SD. ns represents no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were conducted for at least 3 biological replicates.
A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells (2020)
Figure 5

Figure 5. Splicing factor SRSF2 modulates NUMB exon 9 splicing.

(A) The putative SRSF2 binding motifs on NUMB exon 9 (labeled as red), predicted by RBPmap. M1 refers to motif 1 and M2 refers to motif 2.

(B) Western blotting showing the overexpression of SRSF2 in 293T cells, which was co-transfected with NUMB reporters of WT, ∆M1 or ∆M2. ∆M1 or ∆M2 represents plasmid deleting corresponding binding site.

(C) A minigene splicing reporter assay showing SRSF2 promoted the generation of NUMB_L isoform via M1 but not M2. The upper panel indicating the pET01 vector that contains two constitutive NUMB exons. The representative RT-PCR electropherogram showing the expression of NUMB_L and NUMB_S isoforms in WT, ∆M1 or ∆M2 transfected 293T cells with or without SRSF2 overexpression, respectively.

(D) The bar graph showing the normalized exon 9 inclusion from the RT-PCR electropherogram of (C) to the WT control cells. P-values were determined by an unpaired two-tailed Student's t-test.

(E) On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram.

(F, G) The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.

Data information: Results given are mean ± SD. P-values were calculated by one-way ANOVA followed by Dunnett's test in (E), (F) and (G). *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were conducted for at least 3 biological replicates.
A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells (2020)
Figure 6

Figure 6. NUMB alternative splicing impacts NOTCH activity during EPC generation.

(A) The top 10 Reactome terms enriched from differential splicing events regulated by PLB. To define the differential splicing events, genes with FPKM > 1 in at least one differentiation stage were selected. Among them, the differential splicing events were identified by the double cutoffs of delta PSI > 0.2 and FDR < 0.05.

(B) GSEA analysis of NOTCH signaling in isoform level between DMSO-control and 2.5 nM of PLB treated cells.

(C) The bar graph representing the percentage of CD31+CD34+ cells at day 5 of differentiation after treatment with DMSO and various concentrations of DAPT starting from day 2.5.

(D) The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicates

(E) RT-qPCR assay showing the expression of HES1 mRNA in the GFP+ cells on day 5 of differentiation with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5.

(F) RT-qPCR analysis confirmed the overexpression of HES1 mRNA after DOX induction. The ACTB gene was used as a control. The data was normalized to the mRNA level in empty vector control cells.

(G) Western blotting showing the level of HES1 overexpression after DOX induction. The GAPDH gene was used as a loading control.

(H) The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression.

(I) Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).

(J) A hypothetical model. During hematopoietic differentiation deriving from hESCs, a splicing factor switch occurs during the transition from mesoderm APLNR+ cells to EPCs. Concomitant with the switch of components in the splicing machinery, the splicing regulator SRSF2 is downregulated, leading to the enrichment of an EPC-induced NUMB_S isoform. Alternative splicing of NUMB can precisely control NOTCH activity, probably by suppressing HES1 expression and, ultimately, regulate EPC specification. The blue and red colors represent the two switching clusters within splicing factors during hematopoietic differentiation.

Data information: Results given are mean ± SD. P-values were determined by an unpaired two-tailed Student's t-test in (C), (E), (F) and (I). **P < 0.01, ***P < 0.001. All experiments were conducted for at least 3 biological replicates.

Expanded View Figure Legends
Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase (2020)
Figure 1

Figure 1. An optimal TF/SecB ratio is essential for cell viability and in vitro translocation of proOmpA.

A. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates.

B. Schematic summary of the assays used in this study. Preproteins (purified commonly in 6 M urea; except in Appendix Fig. S6B) were diluted in an aqueous buffer (final urea < 0.2 M) and their physicochemical properties and functional interactions with Sec system components analysed. Y: SecYEG; A: SecA.

C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown.

D. proOmpA translocation ATPase activity stimulation of SecA (as in C) in the absence or presence of TF2 or/and SecB4, added at the indicated molar excess over proOmpA1. The T/M ratios were calculated; the one in the absence of chaperones was considered as 100%, all other values were expressed as % of this. n= 2-4 biological replicates. Mean values (± SEM) shown. Unpaired parametric t-test, 95% confidence interval: ns: not significant (p = 0.1032); ****: p < 0.0001; **: p = 0.0037.

E. Titrated TF-mediated inhibition (0-6 µM range) of proOmpA-stimulated translocation ATPase activity (as in C). Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 1-4 biological replicates; mean values ± SEM) versus the log10[TF2]. The Ki was determined using a variable slope fit (log[inhibitor] versus normalized response; GraphPad Prism).

F. SecB-mediated relief of TF-mediated inhibition of proOmpA translocation ATPase activity(as in C). TF2 (1μM) and SecB4 (0-6 µM) were used. The T/M ratios were calculated, normalized (as in D) and plotted (n= 2-3 biological replicates; mean values ± SEM) versus the log10[SecB4]. An apparent relief constant (Krel) was determined (as in E).