EMBO/soda-nel huggingface dataset:
Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator (2018)
Figure 6

Figure 6. H2Bac levels are severely decreased in tauopathic mice and increased by CSP-TTK21 treatment.

A Pie charts showing the genomic distribution of H2Bac and H3K27ac relative to whole genome, obtained from CHIP-sequencing experiments performed in WT dorsal hippocampus. The percentage of peaks in promoters, Upstream Regulatory Regions (Upstream; -1 kb to -20 kb relative to TSS), introns, exons, and intergenic regions is shown.

B Mean H2Bac profiles at TSS relative to gene expression obtained from RNA-seq data. Expressed genes were separated into 4 groups (Q1 to Q4, 6483 genes/group), Q1 representing the less expressed genes (0-25%) and Q4, the most expressed genes (75-100%). Profiles were established with SeqMiner centered on TSS ±2Kb.

C Mean H2Bac profiles established with SeqMiner for all genes at TSS (top) and gene bodies (bottom) for the 4th quartile (most expressed genes) in the two replicates (rep1 and rep2). Significance (Benjamini, p<0.05) of the annotations associated with both TSS (green) or Gene profiles (red) are shown (DAVID, KEGG Pathways).

D Differentially regulated H2Bac peaks were analyzed with SICER and significant Fold Changes (FC>1; FDR<0.001) between comparisons are shown for the pathology (TAU VEH vs. WT VEH, blue) and the effect of the molecule (TAU MOL vs. TAU VEH, red). Fold Change is shown as log2 (Fold Change) (X-axis) and significance, as -log10(FDR) (Y-axis).

E Mean H2Bac and H3K27ac profiles established with SeqMiner for the 1624 decreased peaks obtained in the comparison TAU VEH vs. WT VEH. H2Bac but not H3K27ac peaks show differential regulation.

F Time series Like plot. For every significant H2Bac decreased regions (1624) in WT VEH vs TAU VEH, normalized read counts were computed for each of the different conditions (WT VEH, TAU VEH, TAU MOL). Fold changes are all referring to WT VEH. Y-axis: Fold change. X-axis: Conditions.

G Functional annotation charts using DAVID performed on the differentially regulated H2Bac genes corresponding to the peaks either decreased in TAU VEH vs. WT VEH (blue, 1624) or increased in TAU MOL vs. TAU VEH (red, 2617). Significance is indicated as -log10(Benjamini p_value).

H Venn diagram showing the rescue of H2Bac levels by comparing differentially regulated peaks before (TAU VEH vs. WT VEH) and after (TAU MOL vs. TAU VEH) the treatment for all peaks with an FDR <0.001. About 95% of the peaks were not deregulated after CSP-TTK21 treatment.
Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator (2018)
Figure 7

Figure 7. CSP-TTK21 treatment of THY-Tau22 mice affects the regulations of H2Bac enrichment at different genomic loci.

A Mean enrichment profiles performed with SeqMiner presenting the genomic distribution of H2Bac and H3K27ac reads obtained in ChIP-seq experiments in the different experimental groups, established along putative neuronal CBP enhancers (± 4Kb) (Kim et al., 2010). In the dorsal hippocampus, H2Bac levels are enriched at CBP enhancers in WT WEH (blue), decreased in tauopathic mice (TAU VEH, pink) and induced by CSP-TTK21 treatment (TAU MOL, green).

B Mean profiles were separated in two sets of loci, including 17817 peaks highly enriched in H2Bac and showing a differential regulation in the experimental groups and 23331 peaks that were poorly enriched in H2Bac and not regulated.

C Functional annotation of the 17817 peaks highly enriched in H2Bac performed with GREAT, GOTERM Cellular Component and Biological Process.

D Mean profiles established with SeqMiner for all genes at TSS for H2Bac (Left) and H3K27ac (Right). H2Bac but not H3K24ac presents a differential regulation at TSS.

E Mean H2Bac profiles established with SeqMiner on the differentially regulated genes obtained in transcriptomic experiments (RNA-seq), either decreased (left) or increased (right). The "basal" H2Bac TSS enrichment state of these genes is differentially regulated whether the genes are decreased or increased in response to learning.

F UCSC genome browser view of the c-fos genomic locus with H3K27ac ChIP-seq (blue), H2Bac ChIP-seq (green) and Input (black) signals. Blue shading indicates locations of the c-fos enhancers (Joo et al., 2016); gray shading indicates location of the promoter. Differential peaks were analyzed with SICER. This set of data shows that H2Bac levels were significantly decreased at e1 (FDR=1.95.10-08), e2 (FDR=0.0005) and e5 (FDR= 2.44.10-05) enhancers in THY-Tau22 compared to WT mice, and significantly enriched at e1 (FDR= 4.01.10-08) the regions encompassing e3, e4 and TSS (FDR=7.53.10-07) and e5 (FDR= 0.001) after CSP-TTK21 treatment.

Expanded View Figure Legends
Transcription factor ANAC032 modulates JA/SA signalling in response to Pseudomonas syringae infection (2016)
Figure 1

Figure 1. Enhanced expression of ANAC032 by Pst DC3000-derived signals.

(A) ANAC032 expression in WT plants sprayed with Pst DC3000, 6 and 24 hpi, compared to control (sprayed with 10 mM MgCl2 (Mock)). Error bars represent means ± SD (n = 3; ’n’ represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). ANAC032 expression in WT treated with (B) SA or MeJA or (C) COR for 3 and 6 h compared to non-treated controls. In B and C, means ± SD are given (n = 3; ’n’ represents independently performed experiments, each including at least 20 seedlings). FCh, fold change. Asterisks indicate a significant difference from their respective controls (p < 0.01; Student´s t-test).
Transcription factor ANAC032 modulates JA/SA signalling in response to Pseudomonas syringae infection (2016)
Figure 2

Figure 2. Role of ANAC032 in resistance against Pst DC3000.

(A) Plants after pressure infiltration with Pst DC3000, at 4 dpi. The experiment was repeated three times (each time 9 - 12 plants) with similar results. (B) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration. Two independent experiments were performed with three replications per experiment, each replicate consisting of three plants grown in individual pots (six measurements in total). The graph shows data points of the two individual experiments (I and II) along with their mean (Avg). (C) Disease status of plants sprayed with Pst DC3000, 5 dpi. The experiment was repeated four times with similar results. (D) Disease severity index (1, small chlorotic lesions; 5, large lesions) scored 5 dpi after spraying with Pst DC3000. Data are from three independent experiments with at least six plants per genotype in each. Means are shown ± SD. Asterisks indicate significant (*p < 0.05 and **p < 0.005) differences between transgenic and WT plants in chi-square analysis. (E) Heat map showing the fold change (log2 basis) in the expression ratio of defense-/stress-related differentially expressed genes in anac032-1 and 35S:ANAC032 compared to WT after spraying with Pst DC3000 (6 hpi) normalised to their respective controls. Blue, downregulated; red, upregulated. Data represent means of three independent experiments, each including the rosettes leaves of at least three plants grown in individual pots. Asterisks indicate significant differences from WT plants (Student’s t test, p ≤ 0.05).
Transcription factor ANAC032 modulates JA/SA signalling in response to Pseudomonas syringae infection (2016)
Figure 3

Figure 3. ANAC032 directly regulates MYC2, PDF1.2A and NIMIN1.

(A) MYC2, PDF1.2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 (24 hpi) normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; ’n’ represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). (B) EMSA showing binding of ANAC032 to MYC2, PDF1.2A and NIMIN1 promoter regions (in 5´-DY682-labelled double-stranded oligonucleotides) harbouring ANAC032 binding sites. 1, labelled probe only; 2, labelled probe plus GST protein 3, labelled probe plus ANAC032-GST protein; 4, labelled probe, ANAC032-GST protein and 100 x competitor (unlabelled oligonucleotide containing ANAC032 binding site). (C) Confocal microscope image showing nuclear localization of ANAC032-GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom:ANAC032-GFP/anac032-1 seedlings treated with Pst at 6 hpi. Left, bright field; right, chlorophyll auto-fluorescence (red) and GFP fluorescence (green) under bright field. (D) Expression of MYC2, PDF1.2A, NIMIN1 and ANAC032 in two-week-old ANAC032prom:ANAC032-GFP/anac032-1 seedlings compared to WT at 6 hpi with Pst, normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; ’n’ represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from WT, normalised to their respective controls (p < 0.05; Student´s t-test). (E) ChIP-qPCR shows enrichment of MYC2, PDF1.2A and NIMIN1 promoter regions containing ANAC032 binding site compared to a promoter region lacking the ANAC032 binding site (AT5G09810; Neg Cont). Means ± SD (n = 3; ’n’ represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from negative control (p < 0.01; Student´s t-test).
Transcription factor ANAC032 modulates JA/SA signalling in response to Pseudomonas syringae infection (2016)
Figure 4

Figure 4. ANAC032 facilitates the SA-mediated transcriptional repression of NIMIN1 and PDF1.2A.

Transcript levels of (A) NIMIN1 and (B) PR1 in WT, anac032-1 and 35S:ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls. (C), Expression of PDF1.2A in WT and anac032-1 plants after 3 and 6 h of treatment with SA compared to their respective controls. Transcript levels were measured using qRT-PCR and numbers on the y-axis indicate fold change (FCh; log2 basis). Means are shown ± SD (n = 3; ’n’ represents independently performed experiments). Asterisks indicate a significant difference from wild type (p < 0.05; Student’s t test).
Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells (2021)
Figure 1

Figure 1 - Naïve Hdac3-/- mESCs differentiate into PrE.

(A) Nanog>GFP intensity of WT and Hdac3-/- TNG-A cells in 2iL and at d2 and d5 of SL exposure.

(B) Pairwise Pearson correlations of in vitro TNG-A and in vivo embryo (Boroviak et al, 2015) RNAseq samples.

(C) k-means clustering of gene expression changes relative to naïve WT TNG-A mESCs.

(D) Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.
Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells (2021)
Figure 2

Figure 2 - Hdac3 and Dax1 are required for PrE lineage restriction in response to FGF, LIF, and RA.

(A-E) Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- (A) and WT and Dax1-/- (D) cells after 3d in indicated conditions. Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 3d (B,E) and 4d (C). Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones.

(F,G) Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes (F). Z-score-normalized fluorescence distributions in mutants compared to WT spheroids (G). Positive Z-scores indicate peripheral, outside, and negative Z-scores central, inside, enrichment. Scale bar: 25 μm.
Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells (2021)
Figure 3

Figure 3 - Transcriptional and functional characterization of in vitro PrE cells.

(A) Principal component analysis of indicated samples. GFP sorted cells express the Nanog>GFP reporter and mCherry sorted cells the Gata6::mCherry reporter. Morula (MOR).

(B) Scatter plots of log2 fold change (FC) gene expression changes (∆mRNA) between naïve Hdac3-/- or Dax1-/- mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3-/- or Dax1-/- mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3-/- or Dax1-/- mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled.

(C) Representative control embryo, and embryos that were injected with labeled SLRA-differentiated WT cells purified for Nanog>GFP-expression and mutant cells purified for Gata6::mCherry-expression. Embryos are outlined with continuous line. Dashed lines indicate embryonic EPI and PrE compartments. Arrowheads point to injected cells. Scale bar is 25 µm.

(D) Quantification of the cell fate (based on expression of Nanog and Sox17) and the localization of injected cells. Average and SD of two independent experiments with at least 5 embryos per condition and experiment.

(E) tSNE maps of scRNAseq of WT, Dax1 and Hdac3 mutants after 3.5d in SLRA. Cells are color-coded by k-means clusters (top left), genotype (top right), and expression of indicated genesets (bottom).

(F) Expression distribution of Gata6 and cluster 5 genes in single cells of indicated scRNAseq clusters, and E4.5_EPI and E4.5_PrE (Mohammed et al, 2017).
Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells (2021)
Figure 4

Figure 4 - Hdac3 and Nr5a2/Dax1 are genetically and biochemically distinct pathways repressing PrE differentiation.

(A) Z-scores of high-confidence interactors coIPed by Hdac3, Dax1, Nr5a2, and Esrrb (indicated in red) in 2iL mESCs.

(B,C) Expression of PrE markers relative to Dax1-/- cells (B), and fraction of Gata6::mCherry positive cells after 3d in SLRA (C) in indicated genotypes. Average and SD of three independent clones.

(D) Gene expression changes in Dax1-/-, Nr5a2-/- and Dax1-/-;Nr5a2-/- cells compared to WT cells in 2iL (upper) and after 2d in SLRA (lower).
Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells (2021)
Figure 5

Figure 5 - Hdac3 and Dax1 co-repress Gata6-bound CREs, including the essential Gata6 enh-45.

(A) k-means clustered heatmap showing enrichment of TFs, and changes of chromatin marks or accessibility in indicated mutants relative to WT at Hdac3- and/or Dax1-bound regions. Repressed and induced clusters 2 and 4 are marked.

(B) Scatterplot of log2 fold accessibility changes in d2_SLRA Dax1-/- cells and of log2 fold H3K27ac ChIP signal changes in d2_SL Hdac3-/- cells relative to WT controls at Hdac3- and/or Dax1-bound regions. Regions colored in red are associated with Gata6.

(C) Genome-browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants, and of ChIPseq signals of indicated TFs and H3K27ac in E6.5 VE and EPI. The grey area indicates enh-45.

(D,E) FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA (D) and in 2iL (E). Average and SD of at least three independent clones.

EXPANDED VIEW FILES
Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions (2018)
Figure 1

Figure 1. Plasma membrane phosphoinositides and PS are essential for the polarized targeting of Cdc42 regulators in vivo.

A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display.

B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed.

C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted.

D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed.

E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud.

F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks.

G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.

H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks.

I Maximum intensity projections of z-stacks displaying Bem1-GFP in the mutants indicated. Bem1-GFP was imaged in mss4-102 cho1∆ cells after shifting to the restrictive temperature for 30 minutes.

J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.

Data information: **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 2 µm.
Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions (2018)
Figure 2

Figure 2. Identification of basic cluster motifs in Bem1 and their effect on the ordering of lipid acyl chains.

A Schematic of the liposome floatation assay. In the assay, liposomes of defined lipid composition were floated through a dense sucrose gradient by ultracentrifugation. Protein that associates with the liposomes are enriched in the supernatant.

B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5)P2 (PI(4,5)P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet.

C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% PI4P ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B).

D Scheme of full-length Bem1 with its domains and the Bem1 deletion constructs used to identify the anionic lipid interacting sequences.

E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P.

F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.

G As in panel (F), containing POPC-d31/PI4P (19:1 molar ratio).

H As in panel (F), containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).

I As in panel (F), containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).

Data information: Values are means ± SD, Floatation assays were performed in triplicate in at least 3 independent experiments. Paired Student's t-tests were performed. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions (2018)
Figure 3

Figure 3. The Bem1 BC sequence can act as a heterologous plasma membrane targeting signal in vivo.

A Schematic showing Bem1 domains with the lipid interacting motifs including the BC motifs and the PX domain. The Bem1 N-terminus contains 3 BC motifs. The first cluster (BC-1) is composed of 6 K/R residues, the second cluster (BC-2) of 3 K/R residues and the third cluster (BC-3) of 5 K/R residues. The scheme shows the full-length bem1 constructs where none, one or all BCs were mutated.

B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.

C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.

D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).

E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.

Data information: Values are means ± SD, Floatation assays were performed in triplicate in at least 3 independent experiments. Paired Student's t-tests were performed. **P < 0.01, ****P < 0.0001. Scale bars = 2 µm.
Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions (2018)
Figure 4

Figure 4. The Bem1 BC sequence is required for Bem1 targeting to the cell pole in vivo.

A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature.

B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel).

C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections.

D Frequency of cells with polarized Bem1-GFP.

E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.

Data information: Values are means ± SD. In panel (D), n > 100 cells counted in each of 3 independent experiments. Comparisons were made using paired Student's t-tests. In panel (E), n > 100 cells, counted in each of 3 independent experiments and the data were compared using a Mann-Whitney test. In panels (D and E): **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar = 2 µm.