EMBO/soda-nel huggingface dataset:
Pseudo-repeats in doublecortin make distinct mechanistic contributions to microtubule regulation (2020)
Figure 1

Figure 1. Each DC domain in DCX contributes a distinct function in regulation of MT assembly

A. Two routes of MT polymerization in the presence of DCX are considered: direct nucleation, involving stabilization of early tubulin oligomers by DCX; stabilization, involving DCX binding only between straight PFs of a complete MT lattice. Question marks, unknown mode of DCX binding to tubulin/MT.

B. Human DCX schematic, sequence alignment of the DC domains from human isoform 2 (Uniprot ID: O43602-2) with indication of secondary structures and 3D structure alignment, in which selected amino acid positions are indicated using single-letter codes. Sequence identity is 24.1 % by Clustal 2.1 (Larkin et al., 2007). H, α-helix; S, β-strand.

C. List of DCX constructs with their abbreviated names and a summary of their activity. Construct boundaries are indicated with residue numbers. +, WT activity; +/-, impaired activity compared to WT; ++, enhanced activity compared to WT; -, no activity; nd, not determined.

D. Plot of initial MT polymerization velocities at 37 °C in the presence of increasing concentrations of WT or NN. Tubulin concentration = 5 μM. Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength (OD500/min). Fitted are sigmoidal dose-response curves: Y = YB + ((YT - YB)/(1 + 10Xi - X)), where YB and YT are Y values (OD500/min) at the bottom and top plateaus, respectively, and Xi is the X value at the inflection point. Representative MT samples from 2:5 μM WT:tubulin or NN:tubulin incubations are shown in E and F, respectively. MT architecture distributions for WT and NN are shown with pie graphs.

E, F. Representative low and high magnification images of MTs nucleated for 10 min at 37 °C from 5 μM tubulin, in the presence of: 2 μM wild-type DCX (WT) (E) or 2 μM NDC-NDC (NN) (F).

G. Schematic of TIRF microscopy-based MT stabilization assay reported in H and I. Various concentrations of unlabelled DCX variants were added to 9 μM unlabelled tubulin mixed with 1 μM HiLyte Fluor™ 488-labeled tubulin in the presence of surface-immobilised HiLyte Fluor™ 488-labeled GMPCPP MT seeds; e-field, evanescent field.

H. Representative kymographs with arrowheads indicating MT catastrophes.

I. Quantification of MT catastrophe frequency in the presence of different DCX constructs observed with MT stabilization TIRF assay. Total free tubulin concentration was 10 μM in all experiments. The basal (tubulin alone) catastrophe frequency level at this tubulin concentration is indicated with a grey bar.

Data Information: In (D), data are presented as mean ± SD. In (I), data are presented as mean ± SD and smoothed curves connect the means. Number of MTs analyzed across 2-3 movies for each condition: 1 μM WT, n=26; 0.5 μM WT, n=27; 250 nM WT, n=26; 50 nM WT, n=13; 1 μM NN, n=27; 0.5 μM NN, n=26; 250 nM NN, n=25; 50 nM NN, n=19; N, n=18; C1, n=24; C2, n=22; C3, n=18; C4, n=20; tubulin alone, n=29. *, p = 0.03; ****, p < 0.0001 (one-way ANOVA)
Pseudo-repeats in doublecortin make distinct mechanistic contributions to microtubule regulation (2020)
Figure 2

Figure 2. NDC and CDC have distinct roles in stabilizing different stages of tubulin assembly

A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state.

B. Isosurface +end and side views of cryo-EM reconstructions of MTs decorated with different DC domains: α-tubulin, light violet or light grey; β-tubulin, dark violet or dark grey; CDC, yellow; NDC, blue.

C. Close-up views of the DCX binding site focusing on loop 3 density, which differentiates DC domains. Refined atomic models in predominantly backbone representation are fitted to their respective density maps (wireframe). Selected residues are labelled with single letter codes and selected side chains are shown as sticks. Nucleotide in tubulin subunit designated as β1 is shown as sticks in the top left corner of each panel. Colours as in (B), or oxygen atom, red; nitrogen atom, navy blue.

D. Cartoon illustration of the modes of WT or NN binding to MT lattice, based on cryo-EM results. WT decorates the GTP/GDP.Pi lattice predominantly via CDC ('C') or via both DC domains ('N-C') and the GDP lattice predominantly via NDC ('N') or via both DC domains ('N-C'). NN can decorate the GDP lattice via its N-terminal NDC ('N'), C-terminal NDC ('C') or both ('N-C'). Modes 'N' and 'C' are compatible with MT cross-linking (bundling) by NN.

E-G. Assessment of the model-map fit. Upper panels show single DC domains bound to four tubulin subunits: t, MT polymerization time; density maps, semi-transparent isosurface; models, backbone. Lower panels show close-up views of the MT-facing side of the DC domains (backbone + side chains shown as sticks). The main differentiating features between NDC and CDC are highlighted with dashed lines. NDC density in the NN reconstruction (G) results from averaging of two NDCs with varying N-terminal region, causing less defined density for that region (N-term scrambling, shown without side chains). Colouring as in (C).
Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development (2016)
Figure 1

Figure 1 – Jmjd2a and Jmjd2b are individually dispensable for ESC self-renewal and embryogenesis.

A-B. Schematic representation of the KO strategies used for the conditional deletion of Jmjd2a (A) and Jmjd2b (B). FRT: flippase recognition site, LoxP: Cre recombinase recognition site.

C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for WB (C), growth curves and RT-qPCR (D,E).

F-G. 2a and 2b ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves and RT-qPCR analyses.

H-I. Jmjd2a(+/-); Jmjd2a(+/-) (H) or Jmjd2b(+/-); Jmjd2b(+/-) (I) mice were inter-crossed and the number of pups alive at time of weaning reported. P values were calculated using a Chi-square test.

Data in panels C-G are representative of results obtained with at least 2 independently derived ESC lines of each genotype. Graphs show mean ± standard deviation (SD) for 3 cultures counted in parallel (growth curves) or 3 technical replicates (RT-qPCR).
Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development (2016)
Figure 3

Figure 3 - The combined functions of Jmjd2a and Jmjd2c are essential for early embryonic development.

A-B. 2ac ESCs expressing GFP were cultured in the absence or presence of OHT and subsequently used for morula injections. (A) Average volume of the GFP-positive area in chimeric embryos at 24 and 72 hrs after morula injection. Number of embryos used for quantification: Ctrl 24h n=18; +OHT 24h n=15; Ctrl 72h n=5; +OHT 72h n=5. Data are presented as mean ± SD. (B) Representative images of chimeric embryos. The white bars indicate a length of 32 µm. Relative intensities of the GFP signal are indicated for each image.

C-D. Jmjd2a(+/-);Jmjd2c(+/-) mice were inter-crossed. The number of (C) pups alive at weaning or (D) embryos recovered at E6.5 is presented. P values were calculated using exact binomial test with the most conservative estimate of expected (-/-;-/-) embryos (6.25 %, assuming no linkage between the Jmjd2a and Jmjd2c loci even though they are located on the same chromosome).
Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development (2016)
Figure 4

Figure 4 - Jmjd2a and Jmjd2c both localize to H3K4me3 marked TSSs.

A. Examples of ChIP-seq data obtained for Jmjd2a, Jmjd2c (Pedersen et al, 2014), and H3K4me3.

B. Diagram illustrating the distribution of Jmjd2a peaks with regard to TSSs and open reading frames. TES: transcription end site.

C. Unsupervised k-means clustering of ChIP-seq tags over all TSSs (+/- 5kb).

D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (D) an H3K4me3 or (E) a Jmjd2c peak. Jmjd2a peaks were classified as TSS-associated (‘TSS’) if overlapping a region of +/- 1kb of a TSS.

F. Venn diagram illustrating the number of TSSs containing binding sites for Jmjd2a and/or Jmjd2c within +/- 1kb. The p value was calculated using hypergeometric test.

G. ChIP-qPCR validations. Graphs show mean ± SD for 3 technical replicates and are representative of results obtained in at least 2 independent experiments.

All data were obtained using ESCs cultured in 2i medium.
Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development (2016)
Figure 5

Figure 5 - Jmjd2a and Jmjd2c redundantly regulate histone methylation levels.

A. Density plots showing average ChIP-seq signals with 95% confidence intervals of the mean indicated in grey. y-axes show mean Tags per Million (TPM). Data were obtained using the 2ac ESC line #4. TSSs were classified as ‘Bound’ if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb (see Fig 4F), or ‘Not bound’ if neither protein binds within the same region.

B. Heat map showing H3K9me3 ChIP-seq data for Jmjd2a/c bound TSSs (+/- 5kb) sorted according to read number in 2abc #13+OHT.

C. ChIP-qPCR validations for selected Jmjd2a/c targets (see Fig 4G). Graphs show mean ± SD for technical triplicates and are representative of results obtained in at least 2 independent experiments.

All data were obtained using ESCs cultured in 2i medium.
Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development (2016)
Figure 6

Figure 6 - H3K9me3 accumulation in Jmjd2a/c KO ESCs impairs gene expression

A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESC lines of each genotype in independent experiments.

C-D. Density plots showing average ChIP-seq signals for (C) H3K9me3 or (D) H3K36me3 with 95% confidence intervals of the mean indicated in grey. Plots are shown for genes, which are down- or up-regulated according to the microarray analyses (FDR<0.05, absolute fold change log2(|FC|)>0.5) for both 2ac and 2abc KO ESCs (overlaps presented in Appendix Fig S6A), or do not show strong expression changes (log2(|FC|) < 0.2). Genes are further classified as “Bound” if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb of a TSS.

E. Heat map presenting array data in the form of log2(fold change) values comparing OHT with control treated ESCs. Genes are shown for which TSS regions (+/- 1kb) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP-seq analyses (defined in Fig EV4).

All data were obtained using ESCs cultured in 2i medium.
Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development (2016)
Figure 7

Figure 7 - The catalytic activity is essential for Jmjd2a/c functions.

A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (A) WB, (B) RT-qPCR analyses or (C) serial plating for the generation of growth curves.

All data were obtained using ESCs cultured in 2i medium. Data are representative of results obtained in at least 2 independent experiments, and graphs show mean ± SD for 3 cultures counted in parallel (growth curves) or 3 technical replicates (RT-qPCR).
Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator (2018)
Figure 1

Figure 1. Targeting the CBP/p300 pathway restores long-term spatial memory retention in THY-Tau22 mice.

A Immunohistochemistry performed on 12 month-old THY-Tau22 mice showing AT100-positive neurons (green), CBP-positive nuclei (red) and DAPI-stained nuclei (blue) in the CA1 region of the dorsal hippocampus. A representative image is shown with a focus. Scale bars: 40µm and 20µm as noted. (n=5 mice). Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining. The star depicts a ghost tangle (AT100-positive neuron and no nucleus).

B Western blot analyses perfomed in the dorsal hippocampus of 12 month-old THY-Tau22 mice compared to age-matched controls. NeuN, actin, tubulin and total H2B levels are not changed. Phosphorylated tau on serine 404 (Tau-Ph404) attest for samples from tauopathic mice. Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes, with WT arbitrarily set at 1 (fold induction). Bar graphs are mean ± SEM. n=5-7/group as noted, multiple t tests, CBP * p=0.003 and GFAP **p=0.0001 for THY-Tau vs. WT mice.

C RNA-sequencing data performed in the hippocampus of 8 month-old mice. Functional analyses (DAVID GOTERM) performed on significantly deregulated genes in THY-Tau22 mice compared to WT mice. Representative genes are presented below. |log2 Fold Change| > 0.2; * indicates FDR<0.05. IEG, Immediate Early Genes. Predicted promoter motif (right) analyses performed with GREAT. Groups: WT mice (n=3), THY-Tau22 mice (n=2).

D Long-term spatial memory testing: Mice were injected 3 times (1 per week) with Vehicle (WT mice saline, WT VEH, n=17), Vehicle (THY-Tau22 mice CSP, 500 μg /mouse, TAU VEH, n=10) or Molecule (THY-Tau22 mice CSP-TTK21, 500 μg/mice TAU MOL, n=13) before training of spatial memory in the Morris water maze (MWM); retention (Probe test) was tested 10 days after the last training session. Acquisition (Escape latencies, seconds) and retention performances (Time in target quadrant, seconds) are shown for the 3 groups of mice. All groups of mice displayed significant acquisition of the platform location (Day effect, F(4,148)= 26.45, p<0.001; Group and Group X Day effects, ns), but only TAU VEH exhibited impaired retention. CSP-TTK21 treatment fully restored the ability of THY-Tau22 mice to remember the platform location. Bar graphs are mean ± SEM. Student's t test to a constant value, # when compared to random (dotted line, 15sec) WT VEH, t(16)=4.6323, #p=0.0002; TAU VEH, t(9)=0.3606, p=0.7267; TAU MOL, t(12)=3.945, #p=0.0019. One-way ANOVA; F(2,37)= 3.55; p= 0.03; * in the different comparisons: TAU MOL vs. TAU VEH, *p=0.0166; WT VEH vs. TAU VEH, *p= 0.040; TAU MOL vs WT VEH, non significant p=0.437). TQ, Target Quadrant; O, Other, corresponds to the mean of the 3 other quadrants.
Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator (2018)
Figure 3

Figure 3. CSP-TTK21 injections of Tauopathic mice prior to learning and memory re-established part of the deregulated transcriptome.

A Time line of the experiment and experimental groups: 8 month-old mice were injected 3 times (1 per week) with Vehicle (WT mice, WT VEH, saline), Vehicle (THY-Tau22 mice (TAU VEH), CSP 500 μg /mouse) or Molecule (THY-Tau22 mice (TAU MOL), CSP-TTK21 500 μg/mice). One sub-group of WT mice was left in their Home cage (WT VEH_HC) and the other groups of mice (WT and THY-Tau22) were subjected to a 3-day spatial training (Learning). RNA extracts were isolated from the dorsal hippocampus, one hour after the last training (n=5/group).

B Volcano plot showing that 2756 genes are differentially regulated between THY-Tau22 and WT mice during learning. The log2(Fold-Change) was estimated by DESeq2. FDR<0.05 and |log2 Fold Change| > 0.2. Red dots correspond to genes with adjusted p-value <0.05. Numbers in the corners represent the number of down-regulated (left) and up-regulated (right) genes.

C Heatmap representing expression of z-score of the 2756 deregulated genes in all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson's distance. Groups: WT VEH_HC (n=3), WT VEH (n=2); TAU VEH (n=2); TAU MOL (n=2).

D Volcano plot showing that 1756 genes are differentially regulated between treated-THY-Tau22 and WT mice during learning. Same parameters as in B.

E Venn diagram showing the rescue of the transcriptome in learning mice, by comparing differentially regulated genes before (TAU VEH vs. WT VEH) and after (TAU MOL vs. TAU VEH) the treatment for all genes with an adjusted p-value <0.05.

F, G Volcano plots showing differentially regulated genes by the molecule in THY-Tau22 mice (F) and by learning in WT mice (G). Same parameters as in B.

H Fold changes for every significant pathology-regulated gene (TAU VEH vs. WT VEH) condition are plotted for down-regulations (top) and up-regulations (bottom) separately (Adjusted p-value <0.05). Functional annotation performed with DAVID for GOTERM_Biological Process and their significance are shown on the right before (green) and after (red) treatment. Significantly regulated pathways with -log10(Benjamini p_value) <0.05 are given.
Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator (2018)
Figure 4

Figure 4. CSP-TTK21 injections induced the expression of 98 genes in tauopathic mice during learning.

A Functional annotation charts (Benjamini, p<0.05) using GREAT perfomed on the 98 significantly over-expressed genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH). Significance is indicated as -log10(Benjamini p_value).

B Heatmap representing expression z-score of the 98 significantly over-expressed genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson's distance.

C Venn diagram showing that most of the genes up-regulated by CSP-TTK21 treatment were also down-regulated in the pathology (81/98, p=7.1.10-88, Khi2 test), as well as a significant overlap (p=2.10-29, Khi2 test) genes up-regulated by learning in WT.

D RNA-seq data showing expression of several immediate early genes (IEGs) : Nr4a1, Arc, Egr1, Dusp1, Fos and Junb. Most of the down-regulated IEGs in tauopathic compared to WT mice present a significant induction in THY-Tau22 mice after CSP-TTK21 injection. Adjusted p-values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH.

E RNA-seq data showing that CSP-TTK21 treatment of tauopathic mice restored the expression of plasticity/memory-relevant target genes : Klotho (Kl), and Neurotensin (Nts). Adjusted p values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH.

F RT-qPCR validations performed in a different cohort of mice from the RNA-seq study (n=4-5/group). One-way ANOVA with uncorrected Fisher's test. Klotho, Kl: F(3,16)=2.949, p=0.0643; * learning p=0.0145 for WT VEH vs. WT VEH_HC, $ pathology p=0.0395 for WT VEH vs. TAU VEH. Nts: F(3,14)=4.290, p=0.0241; $ pathology p=0.0036 for WT VEH vs. TAU VEH, # molecule p=0.0081 for TAU MOL vs. TAU VEH.

G Western blot for Klotho and Neurotensin expression in the different experimental conditions. Immunoblot results are shown (n=5/group). Bar graphs represent the quantification of the protein levels as percentage of the control group WT VEH_HC, arbitrarily set at 100%. Each detected protein was normalized to the corresponding amount of total proteins in the gels or the nitrocellulose membrane. Klotho and Neurotensin were further normalized to the level of actin. One-way ANOVA with uncorrected Fisher's test. Klotho: F(3,16)=5.192, p=0.0107, * learning p=0.0117 for WT VEH vs. WT VEH_HC, $ pathology p=0.0027 for WT VEH vs. TAU VEH, # molecule p=0.0282 for TAU MOL vs. TAU VEH. Nts: F(3,16)=3.748, p=0.0326, $ pathology p=0.0200 for WT VEH vs. TAU VEH, # molecule p=0.0079 for TAU MOL vs. TAU VEH.
Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator (2018)
Figure 5

Figure 5. CSP-TTK21 injections represses the expression of 82 genes in tauopathic mice during learning.

A Functional annotation charts (Benjamini, p<0.05) using using DAVID (GOTERM_Cellular Component) perfomed on the 82 significantly down-regulated genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH), showing a significant association with neuronal terms. Significance is indicated as -log10(Benjamini p_value).

B Heatmap representing expression z-score of the 82 significantly down-regulated genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson's distance.

C RNA-seq data showing expression of several genes belonging to « synapse » annotation (DAVID): Grik4, Homer3 and Sv2b, which are significantly down-regulated by CSPTTK21 treatment in THY-Tau22 mice. Adjusted p values corresponding to p<0.05 are indicated by * for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH.

D Venn diagram showing that a significant number of genes down-regulated by CSP-TTK21 treatment overlapped with genes up-regulated in the pathology (p=2.1.10-72. Khi2 test), as well as with some genes down-regulated by learning in WT (p=2.3.10-3, Khi2 test).

E, F Functional annotation charts using DAVID (GOTERM_Cellular Component) performed on the differentially regulated genes in Learning in the WT mice condition (WT VEH vs. WT VEH_HC). The down-regulated genes by learning (E) are associated with a neuronal terms, and up-regulated ones (F), with transcriptional and translational functions. Significance is indicated as -log10(Benjamini p_value).