EMBO/soda-nel huggingface dataset:
Succinylation of H3K122 destabilizes nucleosomes and enhances transcription (2020)
Figure 2

Figure 2 - H3K122succ is enriched at active TSS.

A IGV browser snapshots showing the distribution of reads around the TSS of the actively transcribed PPM1D gene (chr17:60,587,313-60,643,064; top panel) and of the inactive CD40 gene (chr20:46,114,180-46,129,721; bottom panel) for the indicated histone modifications, input and RNAseq. B Venn diagrams showing the overlap between genes with H3K122succ peaks detected at their TSS regions and genes with CpC island associated to their promoters (CGI) (upper panel), as well as housekeeping genes (HKG) (bottom panel). P-values were calculated through hypergeometric tests. C Venn diagram showing the number of H3K122succ peaks detected overlapping with H3K4me3 peaks and H3K9ac peaks. D Correlation matrix displaying the degree of overlap between H3K122succ, H3K4me3, H3K9ac and H3K27ac peaks from our ChIP experiments at TSS (peaks within ± 500 bp of TSS). Total number and fraction of overlapping peaks are displayed. E Correlation matrix displaying the overlap between our H3K122succ ChIP-seq peaks and DNase-seq and FAIRE-seq enriched regions at TSS (peaks within ± 500 bp of TSS). Total number and percentage of overlapping peak regions are displayed. F H3K122succ occupancy profile over a meta gene as a function of expression level (based on RNA-seq quartiles: Q1 - least expressed, Q4 - most expressed). All genes were normalized to the same length. G Boxplots visualizing expression values of genes (reads per kb; RPK) with peaks for indicated different combinations of histone PTMs at their TSS. Histone modifications are (from left to right): none (no PTM, black); only H3K122succ (green); H3K122succ plus one tail modification (H3K4me3, or H3K9ac, or H3K27ac, green and light orange); H3K122succ plus two tail modifications (green, yellow and light orange); H3K4me3 and H3K9ac and H3K27ac without H3K122succ (yellow, light orange and dark orange); H3K122succ and all three tail modifications (green, yellow, light orange, and dark orange). The number of genes within the groups is indicated below each boxplot. Boxes indicate the range between the first and third quartile, the central line depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Each distribution was compared to the "no PTM" distribution. P-values were calculated by Wilcoxon signed-rank test.
Succinylation of H3K122 destabilizes nucleosomes and enhances transcription (2020)
Figure 3

Figure 3 - p300/ CBP can succinylate H3K122, SIRT5 and SIRT7 desuccinylate it.

A H3K122succ levels (detected by immunoblot), relative to the scramble siRNA control, upon depletion of indicated HAT enzyme(s) (GCN5, pCAF, p300 and/or CBP) from MCF7 cells by siRNA. The bar graph shows a representative experiment (for biological replicate see Fig EV3B). B In vitro succinyltransferase assay on recombinant histone octamers as substrate. After incubation with p300 and suc-CoA, the H3K122succ levels were assessed by immunoblot. Ponceau staining of membrane is shown as a loading control. For additional controls see Fig EV3D. C In vitro succinyltransferase assay on unmodified peptides spanning H3K122. Samples were incubated with increasing amounts of p300 (0 to 160 ng) in the presence of radiolabeled suc-CoA. The radioactivity incorporated was quantified by liquid scintillation counting. The plot displays average counts per minute (CPMs), ± SD and individual datapoints of two independent replicates. Note that increasing enzyme concentrations resulted in increased succinylation D-E In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT7 (D) and SIRT5 (E). Unmodified H3K122 peptide was used as control (left dot). Peptides were probed with H3K122succ AB #2 after incubation with sirtuins. For additional controls see Fig. EV3F-G. F H3K122succ levels in Sirt5-KO cells. Histones were acid extracted from wild type (WT) and Sirt5-KO MEFs and the levels of H3K122succ determined by immunoblot. The level of H3K122succ was normalized to histones extracted from WT MEFs. Average, ± SD and individual datapoints of three biological replicates are shown. P-value was calculated by two-tailed paired t-test.
Succinylation of H3K122 destabilizes nucleosomes and enhances transcription (2020)
Figure 5

Figure 5 - H3K122succ can destabilize nucleosomes.

A Schematic representation of the smFRET assay (top), as well as merged smFRET efficiency histograms of unmodified (middle) and H3K122succ mono-nucleosomes (bottom) measured under indicated NaCl conditions (color coded). The nucleosome population is highlighted by the dashed box. B Fraction of bursts in the nucleosome population (FRET efficiency, E ≥ 0.45) for unmodified (red, open circles) and H3K122succ nucleosomes (turquoise, open circles) plotted against NaCl concentrations and normalized to the respective mean at 0 M NaCl. Mean values (filled circles) were fitted with the Boltzmann sigmoid function (solid lines) to determine the NaCl concentration where half of the nucleosomes have disassembled (x0) as 0.61 M (± 0.04) for unmodified and 0.49 M (± 0.03) for H3K122succ nucleosomes. C Model of H3K122succ function: The establishment of H3K122succ (e.g. by p300/ CBP) favors an open, accessible chromatin state through nucleosome destabilization. Higher DNA accessibility facilitates the binding of the transcriptional machinery and transcription of H3K122succ marked genes. The desuccinylation of H3K122succ, by SIRT5 or SIRT7, results in nucleosome stabilization and reduced transcriptional output.

Expanded View Figure legends
WNT inhibition creates a BRCA-like state in Wnt-addicted cancer (2021)
Figure 4

Figure 4: β-catenin regulates the expression of HR and FA pathway genes

β-catenin regulates the expression of HR and FA pathway genes in HPAF-II cells. HPAF-II cells were transfected with siRNA against β-catenin for 48 hours. The relative expression of genes as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates.

Stabilized β-catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition. Mice bearing HPAF-II xenografts without or with stabilized β-catenin were treated with ETC-159 (37.5mg/kg bid) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT-PCR. ETC-159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β-catenin. The horizontal lines represent mean of replicates.

Stabilized β-catenin reduces the sensitivity of HPAF-II cells to olaparib and ETC-159. HPAF-II cells without or with stabilized β-catenin were plated in soft agar in 48 well plates. The cells were treated with a combination of olaparib and ETC-159 as in Figure 1A and the total number of colonies were scored after 2 weeks. Data shown are average colony counts ± SD from three replicates.

Wnt inhibition reduces the expression of HR and FA pathway genes in APC-mutant COLO320HSR cells. COLO320HSR cells were seeded in 6 well plates, treated with control siRNA, siβ-catenin, DMSO or G007-LK for 72 hours and then the expression of the indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates.

E, F. G007-LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation. COLO320HSR cells were plated at a low density and treated with G007-LK, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. (E) Representative image from two independent experiments is shown. (F) The Combination Index (CI) values of olaparib and G007-LK calculated from two independent experiments using the Chou-Talalay CompuSyn software.
WNT inhibition creates a BRCA-like state in Wnt-addicted cancer (2021)
Figure 6

Figure 6: Combination of Wnt inhibition and Olaparib enhances senescence

Protein levels of HR and FA pathway genes are reduced upon Wnt inhibition in both ETC-159 treated and combination groups. Tumor lysates from mice treated with vehicle, ETC-159, olaparib or combination of ETC-159 and olaparib (as shown in Figures 1C-D) for 56 hours were resolved on a 10% SDS-PAGE gel. The indicated HR and FA pathway proteins were detected by immunoblotting. Each lane represents an individual tumor.

Wnt inhibition alone or in combination with olaparib does not induce apoptosis. Tumor lysates from mice treated as above for 56 hours or 21 days (Figures 1C-D) were resolved on 12% SDS-PAGE gel and analyzed by immunoblotting with the indicated antibodies. Each lane represents an individual tumor.

C-E. Co-treatment with olaparib and Wnt inhibitor regulates expression of senescence associated genes. (C and E) Expression of senescence-associated genes was analyzed in the tumors from all four treatment groups (treated for 56 hours) by qRT-PCR. Each data point represents an individual tumor and horizontal lines represent mean. p-values for the significant changes as calculated by Mann-Whitney U test are shown. (D) Tumor lysates from indicated groups were immunoblotted for p21. Each lane represents an individual tumor.

F, G. Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (F) Representative images of tumor sections from the four treatment groups (treated for 21 days) stained for SA-β-galactosidase, a senescence marker, and counterstained with nuclear fast red. Blue color indicates positive staining for senescent cells. (G) The percentage of SA-β-galactosidase positively stained area (blue) in the representative images from each of the groups is shown. Multiple areas were imaged from three tumor samples per group (indicated as mouse 1, 2 and 3). The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.

H, I. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (H) HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days. 53BP1 foci per cell were then assessed by immunofluorescence. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test. (I) Representative immunofluorescence images of 53BP1 foci (red) and nuclei counterstained with DAPI (blue) are shown for each group.
WNT inhibition creates a BRCA-like state in Wnt-addicted cancer (2021)
Figure 7

Figure 7: HR and FA pathway genes are expressed in the Wnt high compartment of the small intestine

Immunohistochemical staining of sections of small intestine from C57BL/6J mice for DNA repair proteins shows that the expression of BRCA1, FANCD2 and RAD51 was high in the crypts (Wnt high compartment) whereas the villi did not show any staining.

Expression of DNA repair genes correlates with the Lgr5 expression in mouse intestine: Gene expression analysis of sorted Lgr5 populations from the mouse intestine (dataset (GSE149311) shows that increased expression of known Wnt target genes Lgr5 and Axin2 correlates with Mybl2, as well as with Brca1/2, Rad51 and Fanc genes. The horizontal lines represent mean of replicates.

Small intestinal adenomas from APCmin/+ mice displayed positive nuclear immunostaining for HR and FA pathway proteins BRCA1, FANCD2 and RAD51. The bottom panels are higher magnification images of the areas marked in the top panels.

PORCN inhibition reduces the expression of HR and FA pathway genes and Mybl2 in the normal mouse intestine. Total RNA was isolated from small intestines of C57BL/6J mice treated above in Figure 7C. Expression of DNA repair genes and Mybl2 was measured by qRT-PCR. Each data point represents an individual mouse. n = 3/group. The horizontal lines represent mean of replicates.

PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine. C57BL/6J mice were treated with vehicle or ETC-159 (60 mg/kg) for 3 days. Small intestines were sectioned and analyzed by immunostaining for BRCA1, FANCD2, RAD51 and phosphorylated-BRCA1.
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 1

Figure 1. Drosophila Snap29 localizes to the mitotic KT.

(A) Immuno-precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4°C overnight. The control antibody (ab) is a rabbit anti-yeast Mad2. (B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red. (C) Single confocal section of a S2 cell. Anti-Incenp, anti-Cid mark the centromeric chromatin and the inner KT, respectively. (D-E) Maximal projections of S2 cells imaged by STED microscopy. 7 z-sections for a total of 1.5 um (D) and 4 z-sections for a total of 0.5 um (E) are shown. Anti-CenpC and anti-Spc105R mark the proximal and the distal region of different portions of the outer KT, respectively. (F-G) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29, nuclear pores (WGA), and CyclinB (F), or Snap29, Spc105R, and the nuclear lamina (LaminA) (G). Note the transient perinuclear enrichment of Snap29 in early prophase. WGA stains also cytoplasmic speckles in hemocyte-derived S2 cells (Rizki & Rizki, 1983). High magnification of the insets in C-G shows localization of Snap29 relative to the indicated markers. (H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 2

Figure 2. Snap29 localization at KTs in vivo depends on the N-terminal SNARE domain and on outer KT components.

(A) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs. A cartoon of full length Snap29 is shown above the control panel. (B) Quantification of the Snap29 signal as in panel A, considering > 30 KTs per sample. Relative mRNA expression of the downregulated genes versus control, measured by Q-PCR, is shown above the graph. Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs. (C) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over-expressed. Cartoons of the expressed Snap29 mutant forms are shown above the panels. Anti-pH3 labels mitotic cells and CenpC marks KTs. (D) Quantification of Snap29 as in panel C, considering > 28 KTs per sample. Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells. In both quantification graphs (B, D), P values are obtained by 2-way ANOVA with Dunnett’s multiple comparison test.
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 3

Figure 3. Snap29 depletion affects mitotic progression.

(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally). (B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a. (C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together. (D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads). (E) Western blot analysis of extracts relative to the experiments in panel d. (F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn’s multiple comparison analysis. OE: Over-expression.
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 4

Figure 4. SNAP29 controls MT binding to KTs in HeLa cells.

(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett’s multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate. (C) Single confocal sections of HeLa cells. CREST and CENPT were used to label the centromere region and associated KTs, respectively. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown. (D) Average of the inter KT distance based on in each of which 10 chromosomes/cell and 10 cells/sample were counted. P values are obtained by Kruskal-Wallis test with Dunn’s multiple comparison analysis. Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs. (E) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA. (F) Averaged spindle pole length based on >20 cells with bipolar spindles/sample. The P value is obtained by two-tailed paired t-test.
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 5

Figure 5. SNAP29 is required for KT integrity in HeLa cells

(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs. (D) Immunoblotting of HeLa protein extracts, treated as indicated. (D) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A-C, e relative to CREST. 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Sidak’s multiple comparison analysis relative to localization of HEC1, which is not lost on SNAP29KD cells.
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 6

Figure 6. Snap29 mutants prevent recruitment of KNL1 to KTs and cause its ectopic accumulation.

(A-C) KNL1 localization analysis in control, SNAP29 KD and SNAP29 KD expressing full length human SNAP29, CFP tagged, full length Drosophila Snap29 (CFP-Snap29), or the mutant forms schematized above the panels. Max projections of single cells in prophase are shown. (D) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST. 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Dunnett’s multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells. (E) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs. In cells expressing SNAP29 Q1 Q2, SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 (arrows).
An essential step of kinetochore formation controlled by the SNARE protein Snap29 (2016)
Figure 7

Figure 7. Drosophila Snap29 activity supports tissue formation in imaginal discs.

(A-B) Confocal sections of wild-type and Snap29 mutant eye-antennal imaginal discs (eye disc portion to the left) of L3 larvae. Discs were labeled with phalloidin and DAPI to visualize the subcortical f-actin cytoskeleton and nuclei, respectively. Arrowheads in B point to actin-rich unpolarized cells in apical, medial and basal sections of the same disc. (B) High magnification of a single confocal cross-section of wild-type and Snap29 mutant eye disc tissues. Two facing folds of the tissue are separated by the apical lumen. aPKC and Dlg mark the apical and lateral plasma membrane domains, respectively. In Snap29 mutant tissue the apical-basal polarity is disrupted. (D) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs, expressing Puckered-LacZ (Puc-lacZ), a reporter of JNK signaling activation. (E-H) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs (E-F), and of otherwise wild-type and Snap29 eye-antennal discs expressing the bacterial apoptosis inhibitor p35 (G-H). Anti-activated-Caspase 3 (Casp3) marks apoptotic cells.
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 1

Figure 1. KIF4A mediates MT-flux.

A. Representative spinning disk confocal live-cell imaging time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), treated with control and KIF4A siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.

B. Representative spinning disk confocal live-cell imaging time series images of S-trityl-L-cysteine (STLC)-treated U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red). Note that MT-flux is abrogated in presence of taxol (lower panel). Scale bars, 10 µm. Time, min:sec.

C. Illustration of 405nm laser-photoactivated regions in monopolar spindles (blue circles, left). The effect of photoactivation and region selected for kymograph generation (dashed white rectangle, right). Scale bar, 10 µm.

D. Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 30 sec.

E, F. Quantification of MT-flux in bipolar (E) and monopolar (F) spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments): - bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3); - monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3). P-values were calculated against control using one-way ANOVA and Kruskal-Wallis H test. n.s. - not significant, **** P ≤ 0.0001.