EMBO/soda-nel huggingface dataset:
Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse hemophilia A (2022)
Figure 6

Figure 6. AAV-N6 intein administration results in therapeutic F8 levels without eliciting anti-F8 antibodies.

A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.

B Bethesda assay performed on plasma samples mice injected with AAV-CodopV3; on the plasma of animals injected with AAV-N6 intein n=3 and the plasma of animals injected with AAV-CodopN6 intein n=2; plasma of a wild-type animal was used as a control. The dotted line indicates the threshold above which anti F8-antibodies are considered inhibitors.

C Pro-inflammatory cytokines levels followed over-time and reported as percentage of the baseline levels (time point before the injection). Data are represented as mean±SEM. Five animals belonging to the CodopV3 group were analysed. Significant differences were assessed using the Kruskal−Wallis test; for IP-10: * indicates the p value ≤ 0.05 between 0 and 4 wpi; * indicates the p value ≤ 0.05 between 0 and 8 wpi; * indicates the p value ≤ 0.05 between 0 and 16 wpi. (n=20); for eotaxin: * indicates the p value ≤ 0.05. All p values are reported in Appendix Table S1.

EXPANDED VIEW FIGURE LEGENDS
Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers (2020)
Figure 1

Figure 1: Genetic incorporation of Bpa into β-arrestins.

A Ribbon representation of bovine βarr1 (PDB 1G4M)(Han et al., 2001). The finger loop is highlighted in blue and β-strand VI in pale green

B Sequence alignment of the finger loop and N-terminal part of β-strand VI in bovine β-arrestins, color coded as in A. Divergent positions are shown in bold.

C Western blots of the lysates of HEK293T cells, resolved on SDS-PAGE and stained with a pan-arrestin antibody. mt = mock transfected. Bpa: Benzoyl-Phenylalanine. Arrestins were equipped with a 3xHA tag at the C-terminus, which increases their size of about 3 kDa compared to the endogenous proteins (βarr1 47 kDa, βarr2 46 kDa). βarr1 and βarr2 were transfected at 1/3 DNA compared to the corresponding TAG-mutants.
Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers (2020)
Figure 2

Figure 2. Photo-crosslinking of Bpa-arrestins with three different GPCRs with and without agonist stimulation.

Each panel represents immunoblots of whole cell lysates stained with an anti-HA antibody detecting arrestin. In the left panels GPCRs were combined with βarr1 variants, in the right panels with βarr2 variants. Residues replaced by Bpa are indicated above each lane. Covalent arrestin-receptor complexes are expected between 100-200 kDa, considering the size of the receptors (V2R 41.5 kDa, PTH1R 63.8 kDa, CRF1R, 46.3 kDa) and their glycosylation.

A PTH1R, agonist PTH(1-34)

B CRF1R, agonist Urocortin 1 (Ucn1)

C V2R, agonist arginine-vasopressin (AVP)

D No receptor.
Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers (2020)
Figure 3

Figure 3. Response of photo-crosslinked samples to deglycosylation.

Aliquots from agonist-treated samples shown in Figure 2 were deglycosylated with PNGaseF, run on SDS-PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope).
Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers (2020)
Figure 4

Figure 4: Analysis of activation-independent crosslinking band at D78Bpa-βarr1.

A SDS-PAGE and immunoblots of immunoprecipitated samples. Sample 1 was immunoprecipitated without previous UV treatment. Sample 2 is the product of irradiation of sample 1. Sample 3 was treated with UV light before immunoprecipitation. All samples were run in parallel on the same gel.

B Size exclusion chromatography (SEC) of Sample 3.

C Western blot of the fractions eluted by SEC.

C MALDI-TOF analysis of the SEC fractions VR2 and VR3. The spectrum of sample VR2 shows a [M+H]+ peak featuring twice the mass (~108 000 Da) of the [M+H]+ peak of the βarr1 monomer (sample VR3, ~50 400 Da).
Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers (2020)
Figure 5

Figure 5: Proximity-induced pair-wise crosslinking.

A Mechanism of the nucleophilic substitution reaction between BrEtY and Cys. The product is a thioether, which is stable in reducing SDS-PAGE.

B Ribbon representation of inactive (PDB 1G4M (Han et al., 2001)) and active (PDB 6U1N (Staus et al., 2020)) βarr1. Arrestin is in grey, M2R in pale green.

C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines. All samples were run in parallel on the same gel.

D, E Pair-wise crosslinking between BrEtY-βarr1-3xHA and wt PTH1R. (D) Ribbon representation of active PTH1R as seen from the intracellular side (PDB 6NBF) (Zhao et al, 2019). Cys residues are shown in red. ECL3 (not resolved in the structure) is represent as a yellow dashed line. The approximate position of the Cys residue in this loop is marked with a fuzzy red circle. (E) Western blot of whole cells lysates stained with an α-HA antibody. Residues exchanged with BrEtY are indicated in the upper row, mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R.

Expanded View Figure Legends
Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin (2021)
Figure 1

Fig 1 | Clinical presentation, olfaction test, immune profile in the nasal turbinated and viral load of SARS-CoV-2-infected hamsters with and without ivermectin treatment.

a, clinical signs in infected hamsters. The clinical score is based on a cumulative 0-4 scale: ruffled fur; slow movements; apathy; absence of exploration activity. Symbols indicate the median ± interquartile range.

b, olfactory performance in infected hamsters. The olfaction test is based on the hidden (buried) food finding test. Curves represent the percentage of animals that did not find the buried food. Food finding assays were performed at 3 days post-infection. Data were obtained from three independent experiments for males and two independent experiments for females.

c, viral load in the nasal turbinates and in the lungs at 4 dpi.

d, ratio between the CPD (copy per droplets, normalized to γ−actin and Hprt reference genes relative expression) of structural [N, nucleocapsid] and non-structural [IP4: RdRp, RNA-dependent RNA polymerase] viral gene expression determined by digital droplet PCR (ddPCR) in the nasal turbinates and in the lungs at 4 dpi.

e, infectious viral titer in the lung at 4 dpi expressed as Plaque Forming Units (PFU)/g of tissue.

f, cytokines and chemokines transcripts in the nasal turbinates at 4dpi in male and female SARS-CoV-2 infected hamsters, treated with saline or with 400 µg/kg ivermectin.

Data information: Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test (a, c-f) and Log-rank (Mantel-Cox) test (b). M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex. See Figs EV1-EV2 and Appendix Fig S1.
Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin (2021)
Figure 3

Fig 3 | Identification of macrophages in the lung of SARS-CoV-2-infected hamsters with and without ivermectin treatment and characterization of their transcriptomic profile related to M1/M2 polarization.

a, representative histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline and CoV_ivermectin. Top panels: whole lung sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion or edema. Hematoxylin and Eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels).

b, representative immunofluorescence photomicrographies of neutrophils (Ly-6G), monocytes/macrophages (Iba1), M2 macrophages (Arg1) and SARS-CoV-2 (NP) in the lung. Scale bars = 50 μm.

c, quantification of Iba1+ cells, Arg1+ cells and Iba1+Arg1+ cells in the lungs. mock_saline n=3 (males), mock_ivermectin n=4 (males), CoV_saline n=9 (6 males and 3 females), CoV_ivermectin n=6 (4 males and 2 females).

d, heatmaps showing the differentially-expressed genes related to the M1/M2 polarization in comparison to mock-infected hamsters. * indicates Benjamini-Hochberg adjusted p-value<0.05 in the comparison between saline and ivermectin within the same sex. Color gradient represents the transcription log2 fold change comparing infected and mock-infected. Complete analyzes are listed in Dataset EV1.

e, lung weight-to-body weight ratio in the different groups (n=4/sex/group).

Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test (c, e).
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 1

Figure 1. SAGA and USP22 inducibly bind to the promoters region of ER stress response genes.

HCT116 human colorectal adenocarcinoma cells were treated for 2 hrs with 100 nM Thapsigargin to induce ER stress. Cells were cross-linked, harvested, and subjected to high-throughput ChIP-sequencing for SAGA members USP22, GCN5, and ATXN7L3.

A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).

B, Heat map of USP22, GCN5, and ATXN7L3 association at the TSS for 29 genes whose promoters undergo significant recruitment of USP22 following induction of ER stress.

C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.

D‑F, Linear regression analysis of SAGA member recruitment to target gene promoters following induction of ER stress.
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 2

Figure 2. USP22 is required for the biological consequences of ER stress.

A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm.

B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *.

C, Whole-cell lysates from the indicated time points were subjected to immunoblotting with the indicated antibodies.
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 3

Figure 3. USP22 regulates transcription of ER stress response genes.

A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.

B, Cells were treated as in A, but over an extended series of time points. RNA was isolated and subjected to qRT-PCR against the indicated transcripts. Three independent experiments are represented as mean ± SEM.

C, A Tetracycline (Tet)-inducible, shRNA-resistant USP22 transgene was stably introduced into HCT116 cells. Cells were treated as in A, with and without expression of the transgene via Tet treatment. Whole-cell lysates were subjected to immunoblotting with the indicated antibodies.

D, RNA was isolated from conditions described in C subjected to qRT-PCR analysis against the CHOP mRNA. Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test. ***p<0.005.
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 4

Figure 4. USP22 is required for recruitment of Pol II to ER stress response genes independent of H2B deubiquitylation.

HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross-linked, harvested, and subjected to high-throughput ChIP-sequencing for RNA Pol II and Ub-H2B.

A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).

B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005

C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 5

Figure 5. USP22 mediates PIC stability at ER stress response genes.

A, Schematic of the CHOP locus. Black boxes indicate amplicons for subsequent ChIP-qPCR analysis.

B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross-linked, harvested, and subjected to ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at the loci across CHOP (indicated in A). Three independent experiments are represented as mean ± SEM

C, ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.

D‑G, Cells were treated as in B and subjected to ChIP-qPCR for TBP, TFIIB, TFIIF, and TAF7 at the indicated gene promoters. Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, ***p<0.005.

H, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 6

Figure 6. Deubiquitylation of PIC components MED16 and MED24 is controlled by USP22. A, High-throughput proteomic analysis of Mediator tail subunits MED16 and MED24 following depletion of USP22, with significance measured by Student's t-test. Specific ubiquitylated lysines detected are highlighted in red text.

B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.

C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.
The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22 (2021)
Figure 7

Figure 7. USP22 is required for efficient recruitment of the Mediator tail module to ER stress response gene promoters and enhancers.

HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross-linked, harvested, and subjected to high-throughput ChIP-sequencing for Mediator subunits MED1 (middle) and MED16 (tail).

A, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).

B, H3K4me3 HiChIP signal (log2) at promoter-enhancer loops before and after induction of ER stress or USP22 depletion at indicated Bound group genes.

C,D, Mean ChIP-seq signal MED1 and MED16 surrounding gene promoters from gene groups defined in Fig 4.

E,F, Linear regression analysis of Mediator and USP22 recruitment to USP22-bound target gene promoters following induction of ER stress.

Expanded View Figure Legends
A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson’s disease (2016)
Figure 1

Figure 1 - PBX1 is present in mDAn. (A) Tru-Seq RNA sequence analysis of E12.5 midbrain floor-plate (mFP), midbrain roof-plate (mRP), anterior (A, adjacent anterior FP) and posterior (P, adjacent posterior FP). Pbx1 is enriched in the midbrain FP, together with Th and Pitx3. Lower levels of Pbx3 are also expressed in the mFP. Gdf7 and Wnt1 are restricted to the mRP at E12.5. (B) Pbx1 is expressed in the intermediate (IZ) and marginal zones (MZ), but not the ventricular zone (VZ), of the mFP at E12.5, as detected by in situ hybridization. (C) PBX1 is first detected in the ventro-lateral part of the LMX1a+ domain at E10, preceding the birth of the first (TH+) mDA neurons at E10.5. At E12.5, PBX1 is present in all mDA neurons, but not all PBX1+ cells are TH+. White boxes indicate the area showed in higher magnification (right). (D) At E11.5, PBX1 protein defines a subpopulation of NURR1+ neuroblasts and labels all NURR1+TH+ mDA neurons. (E) PBX1 co-localizes with PITX3 and is also detected in a subpopulation of NURR1+PITX3- postmitotic neuroblasts at E12.5. Higher magnification revealed three different populations of postmitotic cells: primary neuroblasts (NURR1+PBX1A-PITX3- cells, green), secondary neuroblasts (NURR1+PBX1A+PITX3- cells, yellow/orange) and tertiary neuroblasts/mDA neurons (NURR1+PBX1A+PITX3+ cells, white). Nuclear staining, Dapi (4′,6-diamidino-2-phenylindole, blue). All scale bars, 20µm.
A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson’s disease (2016)
Figure 2

Figure 2 - PBX1A is present in mDA neuroblasts and in adulthood. (A) PBX1A is the isoform detected in the TH+NURR1+ mDA neurons at E12.5. (B) Scheme representing the LMX1A, NURR1, PBX1 and TH/PITX3 domains in the embryonic VM at E11.5-12.5. (C) Immunofluorescence analysis of 7-week old human VM tissue showing that all mDAn are positive for PBX1, suggesting a conserved role for this factor in the development of these neurons in mice and humans. (D) PBX1 is also present in TH+ adult mDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA). (E) Schematic representation of sequential markers expressed in the mDA lineage from progenitors (P), primary neuroblasts (Nb1) to the secondary neuroblasts (Nb2) and mDAn (N), both of which are PBX1+. Nuclear staining, Dapi (4′,6-diamidino-2-phenylindole, blue). All scale bars, 20µm.
A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson’s disease (2016)
Figure 3

Figure 3 - Pbx1 controls the specification and survival of mDAn. (A) To the left, representative immunofluorescence/confocal images showing that the number of TH+ cells is reduced in Pbx1-/- embryos. Deletion of Pbx1 decreases the proportion of NURR1+ neuroblasts that become TH+ neurons at E12.5, indicating that Pbx1 is required for the proper maturation of NURR1+ neuroblasts. To the right, quantification of these experiments. (B) Ectopic PBX3 is detected in all rostro-caudal levels of the VM in Pbx1-/- mice at E12.5, indicating a repressive function of PBX1 on Pbx3 expression. (C) Representative immunofluorescence/confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 (cKO, Pbx1flox/flox;Pbx3-/-;Th-IRES-Cre-ERT) reduces the number of PITX3+ cells in rostral VM levels, but not of NURR1+ cells, when compared to double heterozygous at E14.5 (cHET, Pbx1flox/+;Pbx3-/+;Th-IRES-Cre-ERT). White boxes are magnified to the right. (D) At E18.5, a drastic reduction of TH+, PITX3+ and NURR1+ cells is detected from anterior to posterior levels in cKO embryos, but not in cHET. Pyknotic NURR1+ nuclei are observed in cKO embryos (arrowheads). White boxes are magnified to the right. (E) The total number of TH+ mDA neurons is dramatically reduced and there is an increase in aCASP3+ cells in cKO embryos compared to cHET mice at E18.5. White boxes in C E are magnified to the right. Quantification of the results is shown below the representative images. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates per condition and p-value (T-test) are indicated in the graphs.
A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson’s disease (2016)
Figure 4

Figure 4 - PBX1 improves the differentiation of neural progenitor cells towards mDA fate. (A) Scheme of the differentiation protocol for hNES cells. The AF22 cells (NES cells derived from induced pluripotent stem cells) were infected at day 2 with TET-on PBX1 lentiviral particles, and doxycycline (DOX) treatment started the day after. (B) Representative immunofluorescence/confocal images showing that PBX1-overexpression increases the number of TH+ neurons after 8 days of differentiation of hNES cells (AF22 line derived from induced pluripotent stem cells). (C) While the number of cells (Dapi+) in both conditions did not change, the number of TH+ cells increased 2.3 fold in PBX1-overexpressing cells. (D) TH+ cells derived from the PBX1-overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates per condition and p-value (T-test) are indicated in the graphs.