EMBO/soda-nel huggingface dataset:
Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs (2019)
Figure 4

Figure 4. Gadd45a affects the expression of a particular set of memory-related genes. RNA-seq was performed on Gadd45a-WT (n=6) and Gadd45a-KO hippocampi (n=6). Samples were collected 1 hour after exposure to PA.

A MA plot of DESeq differential expression analysis with the 1% false discovery rate (FDR) hits in red (5/402; and with >2-fold change 1/65, up-/down-regulated respectively). The affected genes are described in Dataset EV1.

B Gene ontology (GO) enrichment analysis of the 402 down-regulated genes revealed gene classes related to memory formation and synaptic function. For a complete analysis of gene ontology, see Dataset EV2.

C Proportion of transcripts with extended 3´UTRs (at least 2 kb beyond the canonical polyadenylation site) among the top 100 Gadd45α hits.

D UCSC browser view of reads-per-million-normalised RNA-seq coverage for Grin2a in Gadd45a-WT (black) and Gadd45a-KO (blue) representative samples. Gene structure of Grin2a (colored in purple), and the direction of transcription (red arrow). Upper pannel covers the entire transcript while lower panel is an enlargement of exons 4-8.

E Detailed view of the extended 3´UTR of Grin2a showing a significant 5´<3´ gradient in RNA-seq coverage. Note that the normalised read coverage is similar between genotypes at the actual 3´UTR end but differs significantly upstream including the annotated exons. For subsequent experiments, position of the designed primers covering proximal and distal parts of the 3´UTR are indicated below.

F, G Validation of Gadd45α-regulated levels of Grin2a mRNA by qPCR with primers covering exons 3-4. Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT=7 and Gadd45a-KO=7) and 1 hour after PA (Gadd45a-WT=5, black bars; Gadd45a-KO=6, blue bars). Random primed cDNA levels (F), were very similar in all the experimental groups. Oligo(dT)-primed cDNA (G) revealed a significant increase of Grin2a 1h after PA, which was only observed in Gadd45a-WT mice. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01, *** = p<0.001.

H, I mRNA levels of proximal vs. distal parts of the 3´UTR analyzed in the same set of oligo(dT)-primed cDNA. (H) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a-WT. (I) Distal parts of the 3´UTR remained unchanged (n=6 for all groups). Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.
Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs (2019)
Figure 5

Figure 5. Gadd45α regulates synaptosomal NMDA receptor (NR2A) levels and specifically interacts with NR2A mRNA.

A, B NR2A protein levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT, n=13 and Gadd45a-KO, n=14) and 1 hour after PA (black bars; Gadd45a-WT=14, Gadd45a-KO=14). NR2A signal (160 kDa) was normalized to the loading control (tubulin (55 kDa) for the total fraction, and synaptotagmin (68 kDa) for the synaptosomal fraction) and calculated as percentage of control. In Gadd45a-WT samples (A), NR2A protein levels were unchanged in the total fraction during memory formation, while in the synaptosomal fraction, a significant increase was detected (t20=3.199). In Gadd45a-KO hippocampi (B), levels of NR2A protein in both fractions remained unaltered during memory consolidation. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01.

C Gadd45α pulldown experiments were carried out in wild-type hippocampal lysates (n=12 for all groups). Percentage of input recovery was unchanged between GFP-FLAG (green bars) and Gadd45α-FLAG (black bars) for genes that are not regulated by Gadd45α, such as succinate dehydrogenase complex flavoprotein subunit A (Sdha, no 3´UTR extension), and mitogen-activated protein kinase 6 (Map2K6, containing 3´UTR extension of 10 kb). On the contrary, Gadd45α-FLAG bound fraction revealed a significant increase in Grin2a (t22=4.992) and Grm5 mRNA (t22=4.939) as compared to GFP-FLAG fraction. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: *** = p<0.001.
Gadd45α modulates aversive learning through post-transcriptional regulation of memory-related mRNAs (2019)
Figure 6

Figure 6. Gadd45α requires its RNA-binding capacity to mediate its action on memory.

A Diagram of the virally-mediated approach to induce Gadd45a-overexpression in hippocampal glutamatergic neurons of Nex-Cre(+/-) mice. On the one hand, Nex-Cre(-/-) (n=12) and Nex-Cre(+/-) (n=11) were injected with the AAV-Gadd45a-HA used in Fig 2. On the other hand, a group of Nex-Cre(+/-) (n=11) was injected with a mutated form of Gadd45α (K45E) that lacks the RNA-binding capacity.

B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a-overexpression (q24 = 4.094) and Gadd45aK45E-overexpression groups (q24 = 4.726) (n=9 for all groups). Values shown are mean ± SEM; Tukey´s multiple comparison test: * = p<0.05, ** = p<0.01.

C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (left), Gadd45a-overexpression (middle) and Gadd45aK45E-overexpression (right) samples 8 weeks after AAV injection (n=3 for all groups). Upper row depicts the entire hippocampus, while center and lower rows show magnifications of the CA3 area in which the HA signal (red) is not restricted to the nuclei (blue and white). Scale bar: 250 µm.

D Gadd45α-overexpression was confirmed at the protein level by HA-western blot on hippocampal samples from Gadd45a-WT, Gadd45a-overexpression and Gadd45aK45E-overexpression mice (n=3 for all groups). Hippocampus scale bars: 250 µm; CA3 scale bars: 50µm.

E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=12), Gadd45a-overexpression (red bars, n=11) and Gadd45aK45E-overexpression mice (grey bars, n=11) in the PA test. Note that, unlike Gadd45a-overexpression mice, those expressing the mutated form of Gadd45α (no RNA-binding capacity) did not show an increase in memory retention. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05, *** = p<0.001.

Expanded View (EV) Figure Legends
LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells (2020)
Figure 4

Figure 4: Dynamics of kinase activities.

a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1).

b t-SNE plot displaying local enrichment of phospho-sites corresponding to known substrates associated with the specified kinase. Dot transparency is scaled according to the P-value corresponding to the local enrichment of the annotation term (hypergeometric test, see methods for more detailed information).

c Illustration of the kinase-substrate interconnections characterizing the AMPK, AKT and ERK signaling pathways. Substrate phospho-sites dynamics are displayed and color-coded according to their clusters, and the corresponding scaled kinetics are presented on the right.

d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control.

e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.

Data information: In the phospho-site labels, "+" and "/" separate the phospho-localisations of doubly phosphorylated peptides or peptides having two proteins identifications with different phosphorylation localizations, respectively. Note that although TBC1D1−pS231 site falls into C1, its t-SNE coordinates are close to the clusters associated with dephosphorylation, a result consistent with its late dephosphorylation state.
LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells (2020)
Figure 5

Figure 5: ITSN2 deficiency lowers TCR activation threshold.

a t-SNE plot highlighting regulated pY sites (for sake of space, only the gene names are reported). Phospho-sites of STAM2 and ITSN2 are displayed in red.

b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots.

c Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments.

d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation.

e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.

Data information: For panels c and e: Data are representative of at least three independent experiments, Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; NS, non significant).
LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells (2020)
Figure 6

Figure 6: Reduced TCR down-modulation in ITSN2-targeted cells.

a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control).

b Heat map showing normalized levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 left unstimulated (-) or stimulated for 1, 3 or 10 min with N4 tetramers (1 or 10 nM) and analyzed by mass cytometry. Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Results obtained from independent transfections with sgITSN2-1 and sgITSN2-2 are compiled in Figure EV5a.

c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points (left panel) or stimulated with increased doses of N4 peptides for 4 h (right panel), were analyzed by FACS for surface TCR expression. Data are presented as mean ± SEM and comparisons were performed using a paired t-test (*, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001). A representative analysis of three independent experiments is shown.

d Heatmap showing scaled expression levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 and stimulated for 6 h with increasing doses of N4 peptides or PMA/Ionomycin (PI) analyzed by single-cell mass cytometry (left). Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Signal difference (mean arcsinh difference) between sgEGFP and sgITSN2-1 transfected cells is depicted on the right panel.

Supplementary information
Unravelling the features of somatic transposition in the Drosophila intestine (2021)
Figure 1

Figure 1 - Somatic TE insertions in Notch in spontaneous male neoplasia

A The fly intestine is maintained by the Intestinal Stem Cells (ISCs). In male flies, carrying one X chromosome, the tumor suppressor gene Notch is present in a single copy. Inactivation of Notch in a stem cell (in green) leads to a clonal expansion of the mutant cell and neoplasia. The neoplastic gut region was microdissected together with the head of the same fly. DNA isolated from both tissues was subjected to whole-genome paired-end sequencing.

B An Integrative Genomics Viewer (IGV) screenshot of the Notch de novo TE insertion site from sample P47 (clonal neoplasia) and its head control, sample P48. Bars represent sequencing reads. Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence. Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family. Two types of supporting reads can be seen: soft-clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE, and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates (not seen) mapping to the TE.

C The Notch locus and the identified somatic TE insertion sites indicated with vertical arrows. Black bars represent exons. Insertions in Notch were identified in 3 out of 33 clonal samples from the Pros>2xGFP genetic background and in 1 out of 4 Dl>nslGFP samples.

D PCR validation of 4 somatic, neoplasia-specific TE insertions. Primers were designed to target regions flanking the insertion sites. Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue (non-clonal), head or thorax for the same fly. Short wild-type amplicon was detectable in all samples. Thorax DNA sample was not available for sample P15.

E Sanger sequencing of the TE insertion breakpoints in the 3'UTR of Notch from sample P47. The rover LTR element was inserted in a reverse orientation to Notch. The 5' LTR sequence was truncated by 13 bp. Vertical dashed lines indicate insertion breakpoints. LTR - long terminal repeat
Unravelling the features of somatic transposition in the Drosophila intestine (2021)
Figure 3

Figure 3 - Somatic retrotransposition occurs before and after the clonal expansion

A A somatic insertion may arise in a normal tissue and be present in a fraction of cells of the tissue (red star). If a second somatic event (yellow) inactivates the Notch pathway, the mutant cell (indicated in green) will initiate the clonal expansion amplifying somatic variants already present in its genome. Finally, any insertion that occurs after the clonal expansion (blue triangle) will be present in a subset of neoplastic cells. The graph represents theoretical allele frequencies of somatic variants obtained from the sequencing of clonal tissue samples. The allele frequency of a Notch inactivating event, marking the onset of neoplasia is represented with a green horizontal line. A somatic insertion with allele frequency higher than the Notch inactivating event was likely present in the tissue before the clonal expansion. In contrast, an insertion with allele frequency lower than the Notch inactivating event, likely occurred after the initiation of neoplasia and is thus subclonal.

B The estimated fractions of cells containing an insertion for all somatic TE insertions identified in neoplastic samples. The onset of neoplasia is represented with a green horizontal line. Three samples (P45B, P51 and P21), where timing of the neoplasia onset could not be unambiguously estimated, were excluded from this analysis.

C The distribution of somatic insertions with estimated cell fraction higher or lower than Notch-inactivating mutations for the most represented LTR-elements (rover and copia) and LINE-like I-elements. (p-values were calculated with Fisher's exact test, two-tailed, n= number of insertions)
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 1

Figure 1. C/EBP β /δ-secretase (AEP) is age-dependently activated in the gut, mediating AD pathologies

(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse colon.

(B) Immunofluorescent staining of C/EBP β (red) and AEP (green), cleaved APP C586 (red) and AEP (green), cleaved Tau N368 (red) and AT8 (green), cleaved Tau N368 (red) and Aβ (green), cleaved APP C856 (red) and cleaved Tau N368 (green), cleaved APP C586 (red) and MAP2 (green), T22 (red) and AT8 (green), Aβ (red) and Thioflavin S (green) in colons of 3xTg mice. Scale bar: 20μm.

(C) AEP activity assay with colon lysates from age-dependent 3xTg mice. Data represent the mean± SEM; representative data of three samples; ***P=0.0001(***P< 0.001), ****P < 0.0001 compared with control, one-way ANOVA.

(D) C/EBP β and AEP immunofluorescent staining of colons from age-matched healthy control and AD patients. White arrows indicate that C/EBP β and AEP were co-localized in AD patient's colon.

(E) AEP and cleaved APP C586 fragments immunofluorescent staining of colons from age-matched healthy control and AD patient. White arrows indicate that AEP and cleaved APP C586 were co-localized in AD patient's colon.

(F) AEP and cleaved Tau N368 fragments immunofluorescent staining of colons from age-matched healthy control and AD patient. Scale bar: 20 μm. White arrows indicate that APP C586 and AEP were co-localized in AD patient's colon.
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 2

Figure 2. DSS induces gut inflammation and C/EBPβ/δ-secretase pathway activation in 3xTg mice.

(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse colon.

(B) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse brain.

(C) AEP activity assay with colon lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; ***P = 0.0002 (*** P ＜ 0.001), **** P ＜ 0.0001 compared with control, one-way ANOVA.

(D) AEP activity assay with brain lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; *P =0.0103 (*P< 0.05), ****P < 0.0001 compared with control, one-way ANOVA.

(E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; **P =0.0071 (**P < 0.01)，***P =0.0009 (3xTg vs 3xTg AEP KO) , ***P =0.0004 (3xTg vs WT) (***P < 0.001) compared with control, one-way ANOVA.

(F) Human Aβ1-42 concentration in brain lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; **P=0.0097(**P< 0.01), ***P =0.0006 (***P < 0.001) compared with control, one-way ANOVA.
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 3

Figure 3. DSS triggers C/EBPβ/δ-secretase activation in 3xTg mice, leading to synaptic degeneration.

(A) Immunofluorescent staining of C/EBP β (red) and AEP (green), Iba-1 (red) and AEP (green), cleaved Tau N368 (red) and AT8 (green), cleaved APP C856 (red) and Aβ (green), T22 (red) and AT8 (green) in cerebral cortex of brains from DSS-treated mice. Scale bar: 20 μm. White arrows indicate that C/EBP β and AEP were co-localized in mouse brain.

(B) Quantitative analysis of AEP positive cells and C/EBPβ positive cells, respectively. The density of both AEP positive cells and C/EBPβ positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P =0.0390 (*P < 0.05), ***P =0.0002 (***P < 0.001), ****P < 0.0001, one-way ANOVA.

(C) Quantitative analysis of AT8 positive cells (left) and TauN368 positive cells (right), respectively. The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. n=5 in each group, data are shown as mean± SEM. *P =0.0194 (*P< 0.05), ***P =0.0001 (***P < 0.001), ****P < 0.0001, one-way ANOVA.

(D) Quantitative analysis of Aβ positive cells (left) and APPC586 positive cells (right), respectively. The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P=0.0107 (*P < 0.05), **P=0.0048 (3xTg vs 3xTg AEP KO), **P=0.0044 (3xTg vs WT) (**P< 0.01), ***P=0.0006 (***P< 0.001), ****P < 0.0001, one-way ANOVA.

(E) Quantitative analysis of AT8 positive cells (upper) and T22 positive cells (lower), respectively. The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. **P=0.0014 (3xTg vs 3xTg C/EBPβ+/-), **P=0.0030 (3xTg vs 3xTg AEP KO) (**P<0.01), ***P=0.001, ****P<0.0001, one-way ANOVA.

(F) Representative electron microscopy of the synaptic structures in hippocampus of brains from DSS-treated mice. Red stars indicate the synapses. (Scale bar: 1 μm).

(G) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm).

(H) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA.

(I) Quantitative analysis of the spine density. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA.