EMBO/soda-nel huggingface dataset:
Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organization (2015)
Figure 1

Figure 1. Impact of Abo1 on transcriptome signatures.

A Domain architecture of S. pombe bromodomain AAA-ATPases. The D1 and D2 ATPase domains (AAA) are shaded blue and orange, respectively, and the bromodomain (BD) is grey. The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [74] using the scoring matrix BLOSUM50.

B Venn diagrams showing overlap between genes up-regulated (1.5 fold) in abo1Δ mutants with genes up-regulated under the indicated condition, along with the significance of the overlaps (based on hypergeometric distribution).
Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organization (2015)
Figure 2

Figure 2. Deletion of abo1+ results in the perturbation of nucleosomal organization at coding sequences.

A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines (“-4” nucleosome and “+4” nucleosome) are shown.

B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1∆ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.

C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells.

D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.
Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organization (2015)
Figure 3

Figure 3. Abo1 associates with FACT and suppresses cryptic transcription.

A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.

B As for A.

C RNA purified from wild type, abo1∆ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.

D RNA purified from wild type, and abo1∆ cells was analysed by strand specific RT-PCR. RNA from hip1∆ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.

E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated.

F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test.

G The indicated strains were subjected to ChIP analysis as described for E.

H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.
Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organization (2015)
Figure 4

Figure 4. Abo1 is required for heterochromatic silencing.

A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.

B Strains carrying the otr::ura4+ were analysed as described for A.

C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.

D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student’s t-test.

E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.
Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organization (2015)
Figure 5

Figure 5. Deletion of abo1+ perturbs centromeric heterochromatin.

A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.

B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.

C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.

D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.

E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.

F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.
Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organization (2015)
Figure 6

Figure 6. Abo1 represses LTR retrotransposons.

A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT-PCR, normalised to act1+ mRNA and scaled relative to the wild type level. Data are the mean of three independent biological repeats and error bars represent ±SEM.

B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β-galactosidase assays. Data are the mean of three independent biological repeats and error bars represent ±SEM.

C As for B

D ChIP DNA samples from wild type (untagged) and abo1-GFP cells were analysed by qPCR for Tf2 LTR. Data are the mean of four independent biological repeats and error bars represent ±SEM.

E Normalised cumulative nucleosome (150 ± 30 bp) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase (biorep1) and high MNase (biorep 2) datasets.

P-values were calculated using a two-tailed unpaired t-test.
Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP (2022)
Figure 2

Figure 2. Ala/Phe mutagenic scanning demonstrates the involvement of the investigated pocket in the regulation of GSEC processivity

(A) The graphs depict the Aβ profiles generated by WT or mutant PSEN1 MEF cell lines transduced with adenovirus encoding APPC99. ELISA quantified secreted Aβ37, Aβ38, Aβ40 and Aβ42 peptide levels are shown as % of total Aβ (sum of the four peptides). The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively. Data are presented as mean ± SD, N ≥ 3 independent experiments. Mutagenic primers used in the generation of mutant cell lines are listed in Appendix Table S1.

(B) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1/GSEC cell lines. The mutant Aβ(37+38)/(40+42) ratios are shown as % of the WT cell line. Data presented as mean ± SD, N ≥ 3 independent experiments. As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05); **P < 0.01, ***P < 0.001, ****P < 0.0001.
Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP (2022)
Figure 3

Figure 3. The investigated pocket plays a key role in GSM III-driven modulation

(A) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III. The chemical structure of the bicyclic heterocycle GSM III is depicted. Data are presented as mean ± SD, N ≥ 3 independent experiments. The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively.

(B) Fold change in GSEC processivity of APPC99 estimated by the Aβ(37+38)/(40+42) ratio and presented as % of the WT response. The data are presented as mean ± SD, N ≥ 3. The symbol # marks mutations that fully abolished the GSEC response towards GSM III, as indicated by data presented in Appendix Figure S3. As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). **P < 0.0001, ****P < 0.0001 (F(DFn, DFd): F (32, 101) = 112.5.
Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP (2022)
Figure 4

Figure 4. In silico and experimental data propose a binding mode for imidazole-based GSM III

(A) GSEC-APPC99 model with GSM III docked in the identified pocket, which is highlighted with a blue square. Colour scheme: PSEN1 in tan, PEN-2 in sienna brown, APH1A in gold, NCSTN in green and APPC83 substrate fragment in orange. Further information on the construction of this model is given in (Appendix Figures S4 B-D).

(B) Binding mode of GSM III at the GSEC-APPC99 interface. Colour scheme: PSEN1 in tan, and APPC83 substrate fragment in orange. Residues involved in drug-target interactions are coloured according to their mode of interaction with the drug (H-bonding: salmon pink, π-stacking: green, general hydrophobic: violet)

(C) Schematic interaction diagram of the GSM III within the GSEC-APPC99 complex: PSEN1, NCSTN and APP residues are shown in black, green and orange. The hydrogen bond between PSEN1 Y106 and GSM III is depicted in green and van der Waals contacts in red. The plot has been generated with LigPlot+ (Wallace et al., 1995).

(D) Aβ profiles generated by WT or mutant PSEN1 cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III. Data are presented as mean ± SD, N = 2 independent experiments.

(E) GSEC processivity estimated by the Aβ(37+38)/(40+42) ratio was determined for the dose-response analysis (presented in panel D) and shown as % of DMSO. Data are presented as mean ± SD.
Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP (2022)
Figure 5

Figure 5. Experimental and in silico analyses implicate GSEC and APP in drug-target interactions

(A-C) (A) Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III or (C) 1 µM GSM II. Data are presented as mean ± SD, N ≥ 3 independent experiments. (B) WT and mutant GSEC processivity estimated by the Aβ (37+38)/(40+42) ratio and presented as % of WT. Data presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.

(D-E) Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by ELISA or mass spectrometry (MS). Mutagenic primers used in the generation of mutant APPC99 substrates are listed in Appendix Table S1. (D) ELISA-based Aβ profiles are presented as % of total Aβ (Aβ37+ Aβ38+ Aβ40+ Aβ42) (mean ± SD, N ≥ 3); (E) APP mutation-driven effects on GSEC processivity according to the Aβ(37+38)/(40+42) ratio are presented as % of the WT condition (mean ± SD). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68. (F) MS-based profiles show the proportion of individual Aβ peptides on the total immunoprecipitated pool. Data shown as mean of 3 independent experiments. See also (Appendix Figure S6B).
Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP (2022)
Figure 6

Figure 6. The pocket filling PSEN 1-V236W mutation mimics key GSM-GSEC interactions and allosterically activates GSEC

(A) In silico data shows that the Trp236 side chain mimics the interactions of the GSM methylimidazole ring with the protease. The position of GSM III in absence of substrate (identical with the position of GSM E2012 in the cryo-EM structure PDB: 7D8X, Data ref: Yang et al, 2021) is shown in purple, while the binding mode of GSM III suggested by our work is illustrated in grey. The side chain of W236 (indicated in orange) occupies the same space as the imidazole moieties in both GSM binding modes.

(B) Aβ profiles generated by WT or mutant PSEN1 cell lines transduced with APPC99. Dotted lines indicate the levels of Aβ peptides (%) in WT profiles. Data presented as mean ± SD, N ≥ 3 independent experiments.

(C) Pocket filling PSEN1 V236W and Y106W-V236W substitutions lead to increased Aβ(37+38)/(40+42) ratio, indicating the activation of the sequential GSEC-mediated cleavage of APPC99. Data presented as mean ± SD, N ≥ 3 independent experiments.

(D) Aβ profile analysis in dose-response experiments for GSM III revealed no modulation of the PSEN1 V236W mutant cell line. Data are presented as mean ± SD, N = 2 independent experiments. For comparison purposes, WT and V236W mutant Aβ profiles are presented in panel 6B. We note that dose response analyses for GSM II showed similar results (Appendix Figure S8B)

(E) GSEC activity assay using purified GSEC and an APP-based fluorogenic peptide as substrate (Farmery et al., 2003). Upper panel: In the non-cleaved state the Dpn (2,4-dinitrophenyl) quencher group suppresses fluorescent emission from the Nma (N-methyl-o-aminobenzoic acid) fluorophore through Förster resonance energy transfer (FRET). Upon GSEC cleavage, the Nma group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm. Lower panel: Specific activities for the purified WT or mutant GSECs (bearing the indicated substitutions in PSEN1) treated with DMSO (vehicle), 10 µM GSMIII or GSM II. Data are normalized to the WT GSEC activity in DMSO (vehicle). Data are shown as mean ± SD, N ≥ 3 independent experiments. Significance is shown for meaningful comparisons; two-way ANOVA with comparison to WT was used to determine statistical significance; **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

(F) Transition state analogue inhibitor L-685,458 (Inhibitor X) treatment of WT, V236W or Y240W mutant PSEN1 cell lines. Inhibitory profiles and derived IC50 values (table) indicate that the PSEN1-V236W significantly increases the affinity for the inhibitor. Dotted line indicates 50% inhibition. Data presented as mean ± SD, N = 3 independent experiments.
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 1

Figure 1. evi1 is expressed in emerging HSPCs and critically regulates definitive hematopoiesis.

A. Whole mount in situ hybridization (WISH) of evi1 at 20 (left) and 32 (middle) hpf. evi1 expression is visible in various structures of the brain, neuronal structures, the posterior pronephric duct (ppnd) and the branchial arches (ba), as well as in the VDA (ventral dorsal aorta) region. Additionally, evi1 co-localizes with the endothelial marker flk1 (right).

B, C. WISH of runx1/c-myb in HSPCs (B), mpo in neutrophils (left) and l-plastin (right) in monocytes/macrophages (C) at 36 hpf in control (upper) and evi1 MO (lower) injected embryos.

D-G. WISH of globin in erythrocytes of 6 dpf old embryos (D), of rag-1 in the thymus of 5 dpf embryos (E; red asterisk), of cd41 at 52 hpf (F), and gata2b at 32 hpf (G) for both control (upper) and evi1 morpholino (lower) injected embryos.

H. Quantitation of results is shown for each gene, displaying the percentages of embryos with normal vs. changed expression in each condition. A Fisher´s exact test was applied to calculate statistical significance. P < 0.001 (***).

I. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 plasmid DNA injected Tg(-1.5hsp70l:Gal4) embryos with heat-shock induction performed at 14 hpf.

J. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 mRNA in Tg(fli.1:Gal4FF;UAS:RFP) embryos, leading to endothelial-specific evi1 overexpression.

K. Graph displays quantitation of results from K and M using the same method as above.

Lateral views are shown, with anterior to the left, dorsal is up. Numbers indicate the amount of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Arrows indicate up- or down-regulation for each gene. A minimum of two biological replicates was performed for each marker with at least n=5 embryos per experiment.
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 2

Figure 2. Endothelial Notch signaling is dependent on evi1 expression levels

A. WISH of notch1b (upper) and dll4 (lower) in both control (left) and evi1 MO injected embryos (right).

B. WISH of notch1b in uninjected (left) and UAS:mEvi1 plasmid DNA injected (right) Tg(fli.1:Gal4FF;UAS:RFP) embryos resulting in endothelial-specific evi1 induction.

Lateral views are shown, with anterior to the left, dorsal is up. Squares represent enlargements of the region of interest. Numbers indicate the amount of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Arrows indicate up- or down-regulation for each gene. A minimum of two biological replicates was performed for each marker with at least n=5 embryos per experiment. For each analyzed gene, quantitation of results is shown, displaying the percentages of embryos with normal vs. changed gene expression for each condition. A Fisher´s exact test was applied to calculate statistical significance. n.s., not significant; p<0.01 (**), p < 0.001 (***).
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 3

Figure 3. evi1 regulates HSC emergence from the VDA.

A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos from 28 to 42 hpf (Movie EV 1). Shown are three representative time-points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible, indicated by the white arrowheads. For each time-point merged images are shown. White arrowheads denote double-positive cells.

B. Cell counts of total numbers of kdrl+cmyb1+ cells “in view”, dividing and respectively emerging HSPCs. n=10 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d.

C. Gene expression analysis of endothelial (kdrl, flt1, dab2, cdh5) and blood specific genes (c-myb, CD45, CD41, runx1) in sorted kdrl+c-myb+ cells from 34 hpf Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos. Cells were isolated from 15-25 pooled embryos per sample. Three biological experiments were performed with one representative shown. Error bars indicate s.d. of three technical replicates for each representative experiment.
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 4

Figure 4. Global and tissue-specific activation of Notch or upstream Vegf signaling is sufficient to rescue HSPCs in evi1 morphants.

A. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right) with global heat-shock (HS) induction at 20 hpf.

B. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;cdh5:gal4ff) embryos with endothelial-specific NICD induction (right).

C. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos (right) with global heat-shock induction at 20 hpf.

D. WISH for runx1/c-myb in 32 hpf evi1 MO injected embryos (left) and embryos injected with both evi1 MO and capped gata2 mRNA (right).

E. Quantitation of all rescue experiments. Shown are the percentages of embryos displaying normal or changed runx1/c-myb expression. A Fisher´s exact test was applied to calculate statistical significance. (n.s., not significant; p < 0.001 (***); p<0.01 (**)).

Data from three biological replicates are summarized for each experiment, with exception of (D) where data from two experiments are shown. Lateral views are shown, anterior to the left, dorsal is up. Numbers indicate the amount of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Arrows indicate up- or down-regulation of runx1/c-myb in each condition. Arrowheads denote the VDA region.