EMBO/soda-nel huggingface dataset:
HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation (2022)
Figure 6

Figure 6. Allosteric ubiquitin binding to the IBR and preceding helical region are important for HOIL-1 activation.

(A) Amino acid sequence alignment of HOIL-1, HOIP and Parkin centred at the start of the IBR domain. Conserved zinc coordinating residues are highlighted in yellow, other highly conserved residues are highlighted with dark blue, and weaker conservation of property is indicated by light blue highlight. Red triangles denote the position of the mutations indicated.

(B) Time-course assay performed at 30°C featuring wild-type HOIL-1 in the absence or presence of the indicated ubiquitin oligomers, visualised by Coomassie staining.

(C) HOIL-1[I358R], assayed as in (B).

(D) HOIL-1[F381A/E383A], assayed as in (B).

(E) The truncated HOIL-1[233-510; I358R] mutant was assayed as in (B).

(F) The truncated HOIL-1[233-510; F381A/E383A] mutant was assayed as in (B).

(G) The sequence from RING1-IBR was aligned by overlaying the HOIL-1 AlphaFold prediction (residues 282-404, AlphaFold Uniprot ID: Q9BYM8) with residues 699-827 in the crystal structure of HOIP-RBR (PDB: 5EDV). The location of the allosteric ubiquitin from that HOIP structure is also shown. HOIL-1 residues I358, F381 and E383 are highlighted in red.

(H) Full-length HOIL-1 AlphaFold structural prediction (AlphaFold Uniprot ID: Q9BYM8) with likely location of allosteric ubiquitin shown. The region shown in (G) is indicated.
The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin (2019)
Figure 3

Figure 3: Structural basis of Sir4 H‑BRCT interaction with phospho-peptides

A) The electrostatic surface potential covering a range from -4 kT/e (red/acidic) to +4 kT/e (blue/basic) is mapped onto the Sir4 H‑BRCT structure to highlight the basic patch on the H‑BRCT surface. Sir4 H‑BRCT is shown in two orientations rotated by 180° and selected residues are labeled.

B) Superpositions of Sir4 H‑BRCT-Esc1pS1450 (grey cartoon, peptide as sticks in atom colors), Sir4 H‑BRCT-Ty5pS1095 (blue cartoon, peptide as sticks in atom colors), and Sir4 H‑BRCT-Ubp10pT123 (green cartoon, peptide as sticks in atom colors) peptide complex structures reveal the highly similar conformation of peptide binders. Esc1pS1450, Ty5pS1095, and Ubp10pT123 peptides used for co-crystallization are indicated with residues modeled in the structures in bold and residues that contribute most binding to Sir4 H‑BRCT boxed.

C) Detailed view of the Sir4 H‑BRCT-Esc1pS1450 peptide interaction. Sir4 H‑BRCT is displayed as orange model with residues involved in peptide binding shown as sticks (orange, atom colors). The Esc1pS1450 peptide is presented as sticks in grey (atom colors). Electrostatic interactions are highlighted with green dotted lines while main hydrophobic contacts are shown as dotted lines in black.

D) Detailed view of the Sir4 H‑BRCT-Ty5pS1095 peptide interaction. Sir4 H‑BRCT is displayed as in (C) while the Ty5pS1095 peptide is presented as sticks in blue (atom colors). Protein-peptide interactions are labeled as in (C).

E) Detailed view of the Sir4 H‑BRCT-Ubp10pT123 peptide interaction. Sir4 H‑BRCT is displayed as in (C) while the Ubp10pT123 peptide is presented as sticks in green (atom colors). Protein-peptide interactions are labeled as in (C).

F) MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1, Ty5 and Ubp10. KD = equilibrium dissociation constant. ΔFnorm values were divided by the amplitude of the saturation level, resulting in the fraction bound (from 0 to 1) for each data point. Data are represented as the mean ± SEM from ≥3 independent measurements.
The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin (2019)
Figure 4

Figure 4: Sir4 RKR residues are important for heterochromatin silencing and proper localization of Sir4 at the nuclear periphery

A) Example images of Nup49-RFP Sir4-GFP in wild-type (wt) cells (GA-10372; row 1); sir4 RKR cells (GA-10373; row 2); wt cells transformed with either empty plasmid (row 3), plasmid overexpressing wt Sir4 H‑BRCT (row 4), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 5). The bar indicates 2 μm.

B) Quantification of mean fluorescence intensity of Sir4-GFP using >100 cells. Error bars show the SD from 3 independent experiments.

C) Quantification of cells containing Sir4 clusters at the nuclear envelope (NE). Data are represented as mean ± SD from 3 independent experiments where >100 cells were counted.

D) Top: Schematic representation of the TEL7L::URA3 reporter used to assay silencing at telomeres. Bottom: wt cells (GA-503) transformed with either an empty plasmid (row 1), plasmid overexpressing wt Sir4 H‑BRCT (row 2), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 3). Cells lacking endogenous sir4 (GA-5822) (row 4). Cells were grown in the absence of uracil and plated as 1:5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days.

E) Top: Schematic representation of the HMR-EΔB::TRP1 reporter gene locus used for assaying silencing at the HMR locus. Bottom: wt cells (GA-485) transformed with plasmids as in panel D. Cells lacking endogenous sir4 (GA-6888) (row 4). Cells were grown in the absence of tryptophan and plated as 1:5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days.

Data information: (B, C) ***p < 0.001 (t-test).
The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin (2019)
Figure 5

Figure 5: Sir4 H‑BRCT interacts with Tom1 and Cbf1 in a phospho-dependent manner

A) Scheme of experimental design for IP-MS. Cells lacking endogenous sir4 and expressing the Myc-tagged H‑BRCT domain of Sir4, either wt or carrying mutations (RKR) were subjected to anti-Myc immunoprecipitation followed by LC-MS/MS analyses.

B) Volcano plot showing proteins that interact with wt Sir4 H‑BRCT but not with the RKR mutant (upper right quadrant).

C) MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Tom1 and Cbf1. KD represents the equilibrium dissociation constant. The KD for interactions with the non-phospho peptides could not be determined (n.d.). ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are represented as the mean ± SEM from at least 3 independent measurements.

D). Extracts from asynchronous cultures of strains Sir4-Myc Tom1-HA (GA-10645), Sir4-RKR-Myc Tom1-HA (GA-10646), Tom1-HA (GA-10647) and Sir4-Myc (GA-10199) were subjected to anti-Myc IP and western blotting with indicated antibodies in the presence and absence of lambda phosphatase.
The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin (2019)
Figure 6

Figure 6: Comparison between Sir4 and Dbf4 H‑BRCT domains and phospho-peptide binding to Dbf4 H-BRCT

A) Superposition of Sir4 (orange) and Dbf4 (PDB 3QBZ, cyan) H‑BRCT domains highlighting the structural conservation of the Sir4 phospho-epitope binding region in Dbf4. The only change is the Arg1066 (Sir4 H‑BRCT) replacement to Lys200 (Dbf4 H‑BRCT), highlighted by an arrow, while all other residues are conserved.

B) MST analysis of the binding interactions between Dbf4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1 and Ubp10. KD = equilibrium dissociation constant, which could not be determined (n.d.) for non-phospho peptides. ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are the mean ± SEM from ≥3 independent measurements.

Table 1: Binding motifs of phospho-peptides

Table listing interacting protein residues around the phospho group of phospho-peptides for which KD values, from MST measurements, for Sir4 H‑BRCT and Dbf4 H‑BRCT binding were determined. A consensus motif is listed below the table.

Figure legends expanded view figures
Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells (2017)
Figure 3

Figure 3: Karyotypic characterization of the late passage ICL lines

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12, (B) Ch14, (C) Ch9 and (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.
CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo (2015)
Figure 2

Figure 2. CRISPR/Cas9-induced disruption of GFP expression in the daughter cells of single microinjected aRGCs in organotypic slices of telencephalon of Tis21::GFP mouse embryos.

Single aRGCs in neocortex in organotypic slices of telencephalon of mouse E14.5 Tis21::GFP embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ (Control, Con) or GFP (gGFP) and with Dextran 10,000-Alexa 555 (Dx-A555), followed by analysis after 24 h of culture.

(A) Scheme of the Cas9/gRNA complex microinjection.

(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555 immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.

(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.

(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).
CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo (2015)
Figure 4

Figure 4. Sequencing analysis of the CRISPR/Cas9-induced disruption of the Tbr2 locus.

(A-C) Next-generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off-target loci generated from PaprikaRFP-expressing cells isolated at E15.5 from neocortex of mouse embryos in utero electroporated at E13.5 with plasmids encoding Cas9_T2A_PaprikaRFP and either gTbr2 (black) or control gRNA (Con, white) as in Fig 3 A-D.

(A) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off-target sites (OT1-4). Data are expressed as percentage of the total number of reads and are the mean of two independent experiments (8 and 5 embryos per condition, respectively).

(B, C) Analysis of one of the experiments of (A) for deletions (B) and insertions (C) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9/gTbr2 plasmid electroporation.
Myeloid cell-based delivery of IFN-γ reprograms the leukemia microenvironment and induces anti-tumoral immune responses (2021)
Figure 1

Figure 1. B-ALL growth in the presence of gene therapy-based delivery of IFNγ.

(A, B) B-ALL progression measured as (A) absolute number or (B) percentage of OFP+ cells in peripheral blood of transplanted mice (mean ± SD, each dot represents an individual mouse, CTRL = 6 mice, IFNγ = 10 mice; **** p ≤ 0.001, Two-Way ANOVA; mice in blue and yellow (B) were also analyzed by single-cell RNA sequencing; two additional independent experiments are shown in EV2).

(C, D) Mean Fluorescence Intensity (MFI) of MHC II on macrophages (C) and their percentage (D) within the BM immune infiltrate at 12 and 17 days after B-ALL injection (mean ± SD, each dot represents an individual mouse; * p = 0.0145, Two-Way ANOVA).

(E) MFI of MHC class II on OFP+ B-ALL cells at the experimental endpoint in the BM of transplanted mice, 12- and 17-days post-leukemia injection (mean ± SD, each dot represents an individual mouse; ** p ≤ 0.01, Two-Way ANOVA).

(F, G) Percentage of CD4+ and CD8+ cells within OFP- BM cells (F) and maturation state (G) of CD8+ lymphocytes (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) in the BM immune infiltrate at 12- and 17-days after B-ALL (mean ± SD, each dot represents an individual mouse; ** p = 0.005, **** p ≤ 0.001, Two-Way ANOVA).

(H) Experimental overview of a therapeutic B-ALL model. Replicate experiments are shown in Figure EV3 A,B.

(I) Absolute numbers of B-ALL cells in the peripheral blood (PB) of CTRL and IFNγ animals at the indicated timepoint (mean ± SD, each dot represents an individual mouse, CTRL = 9 mice, IFNγ = 10 mice; **** p ≤ 0.001, Two-Way ANOVA).

Statistical analyses of panels A and I are shown in Appendix Table S4 and S5, respectively.
Myeloid cell-based delivery of IFN-γ reprograms the leukemia microenvironment and induces anti-tumoral immune responses (2021)
Figure 2

Figure 2. IFNγR1 knock-out HSPCs display loss of IFNγ anti-tumoral activity.

(A) Engraftment of CD45.2 donor cells and lineage composition following transplantation of wild-type (WT) or IFNγ receptor 1 knock out (KO) HSPCs transduced with either the control Tie2.NGFR (CTRL) or Tie2.IFNγ (IFNγ) LV (mean ± SD, each dot represents an individual mouse, WT CTRL = 7 mice, WT IFNγ = 7 mice, KO CTRL = 7 mice, KO IFNγ = 7 mice).

(B) Vector copy number (VCN) in peripheral blood eight weeks post-transplantation (mean ± SD, each dot represents an individual mouse).

(C) B-ALL progression measured as absolute number of OFP+ cells in the peripheral blood (mean ± SD, each dot represents an individual mouse; * p = 0.0286 at day 10; p = 0.0174 at day 12; *** p = 0.0002; **** p ≤ 0.0001; Ordinary Two-Way ANOVA).

(D) MFI of MHC class II positive macrophages identified by F4/80 expression in the BM (mean ± SD, each dot represents an individual mouse; WT IFNγ vs WT CTRL: * p = 0.0221, Mann-Whitney test).

(E) Percentage of CD8+ T lymphocytes within OFP- CD45+ BM cells (mean ± SD, each dot represents an individual mouse).

(F) Maturation state (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) of CD8+ lymphocytes (mean ± SD, each dot represents an individual mouse; WT IFNγ vs WT CTRL: **** p ≤ 0.0001, ordinary One-Way ANOVA).

Statistical analysis of panel C is shown in Appendix Table S6.
Myeloid cell-based delivery of IFN-γ reprograms the leukemia microenvironment and induces anti-tumoral immune responses (2021)
Figure 5

Figure 5. IFNγ-driven changes in B-ALL and possible mechanisms of efficacy loss.

(A) UMAP visualisation of the B cell/B-ALL compartment. From left to right panel, cells are coloured according to: unsupervised clustering (Louvain - resolution 0.6), experimental condition and marker-based custom cell-type classification, unequivocally identifying leukemic cells by the presence of the tTA transcript.

(B, C) Violin plots show the distribution of IFNγ (B) and MHC-II gene (C) signatures (module score value for each cell - see gene list details in methods) for each experimental condition, within each custom cell-type. Wilcox Rank Sum Test for IFNγ signature, condition IFNγ d12 vs. CTR d12: B-ALL p= 2.68x10-19, B cells p=0.5, plasma cells p= 0.78; Wilcox Rank Sum Test for MHC-II signature, condition IFNγ d12 vs. CTRL d12: B-ALL p= 4.26x10-46, B cells p=1x10-9, plasma cells p= 0.32.

(D) Reduction of OFP expression in B-ALL cells in the IFNγ treated versus CTRL (mean).

(E) Viability of OFP+ B-ALL cells at sorting before undergoing single-cell RNA sequencing processing (mean, CTRL = 4 mice, IFNγ = 6 mice; * p = 0.0333, Mann-Whitney test).

(F) Expression of Mki67 transcripts in B-ALL cells from CTRL d12 and IFNγ d12 conditions, extracted from the scRNAseq dataset (**** p ≤ 0.0001, Welch's test).

(G) IFNγ d12 B-ALL cells were split according to MHC II gene signature values and compared for gene expression profile in order to identify significantly enriched terms (within the hallmarks gene set MsigDB collection) by using the GSEA approach (H). On the left, hallmarks terms downregulated (negative NES - blue labels) in the MHCII high leukemia cell population are shown, whereas the right shows upregulated hallmarks terms (positive NES - red labels) in the MHC II high leukemia cell population, as compared to the MHC II low counterpart.

(I) Loss of IFNγ response over time in B-ALL cells from the IFNγ group, potentially facilitated by reduced expression of Ifngr1, Ifngr2, Jak1, Stat1 and Irf1 (data extracted from the scRNAseq dataset statistical analysis by Two-Way ANOVA).
Myeloid cell-based delivery of IFN-γ reprograms the leukemia microenvironment and induces anti-tumoral immune responses (2021)
Figure 6

Figure 6. Combination of IFNγ gene therapy with other immunotherapies enhances anti-tumoral activity.

(A-D) Combination gene therapy with IFNα, TNFα and IFNγ, expressed from the Tie2 vector platform. CTRL (Tie2.NGFR), 2 independent experiments, n = 9 mice; IFNα, 1 experiment, n = 2 mice; IFNα/IFNγ, 1 experiment, n= 4 mice; IFNα/TNFα, 1 experiment, n = 3 mice; IFNγ/TNFα, 2 independent experiments, n = 19 mice.

(A) Vector copy number (VCN) in peripheral blood at eight weeks post-transplantation (mean ± SD).

(B) B-ALL progression in peripheral blood measured as absolute number of OFP+ cells (mean ± SD, each dot represents an individual mouse; * p = 0.0308, ***p = 0.0004 ****p ≤ 0.0001, Two-Way ANOVA with Geisser-Greenhouse correction).

(C) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; *p = 0.0484, **p = 0.0049, ordinary One-Way ANOVA).

(D) Percentage of CD8+ T lymphocytes within CD45 positive and OFP negative splenic cells (mean ± SD, each dot represents an individual mouse; *p = 0.0356, ordinary One-Way ANOVA).

(E-G) Combination of IFNγ gene therapy with immuno-oncology drugs. CTRL (Tie2.NGFR) + antibody isotype, n = 6 mice; CTRL + αCTLA4 antibody, n= 5 mice; CTRL + αLAG3 antibody, n = 5 mice; CTRL + 1-Methyltryptophan (1-MT), n = 5 mice; IFNγ (Tie2.IFNγ) + antibody isotype, n = 7 mice; IFNγ + αCTLA4 antibody, n = 7 mice; IFNγ + αLAG3 antibody, n = 7 mice; IFNγ +1-MT, n = 5 mice.

(E) B-ALL progression in peripheral blood measured as absolute number of OFP+ cells (mean ± SD, each dot represents an individual mouse; ns = not significant, **p = 0.001 (CTRL isotype vs IFNγ + αCTLA4); **p = 0.0022 (CTRL isotype vs CTRL + αLAG3), ***p ≤ 0.001, ****p ≤ 0.0001; comparisons are against the corresponding isotype, or as indicated by the lines; Two-Way ANOVA).

(F) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001 as compared to CTRL isotype, ordinary One-Way ANOVA).

(G) Percentage of CD8+ T lymphocytes within OFP negative BM cells (mean ± SD, each dot represents an individual mouse).

Statistical analyses of panels C and E are shown in Appendix Table S7 and S8, respectively.

Expanded View Figure Legends
Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms (2021)
Figure 1

Fig 1. Cytoplasmic compartmentalization of the silkworm piRNA pathway

(A, B) Colocalization of AcGFP-Siwi, AcGFP-BmAgo3 and mCherry-BmVasa in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant dissociates from BmVasa foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.

(C, D) Colocalization of AcGFP-Siwi, AcGFP-BmAgo3 and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.

(E) Heatmap of colocalization ratio between fluorescence protein-tagged piRNA factors. Each colocalization ratio is quantified by using ImageJ plugin Comdet (see Materials and Methods) and averaged from n=3 independent z-stacks. Darker color represents more frequent colocalization. Known nuage proteins (BmVasa, BmAgo3, Siwi) form a cluster at the top-left corner. Siwi-D670A mutant, but not BmAgo3-D697A, clusters with BmSpnE, BmQin and BmDcp2 at the bottom-right corner, representing piP-bodies. See also Figures EV1 and EV2.

(F) Schematic diagram showing the proposed spatial compartmentalization of the BmN4 piRNA pathway. Nuage components: BmVasa, BmAgo3, Siwi; piP-body components: BmSpnE, BmQin, BmDcp2. Siwi-D670A mutation leads to the accumulation of the mutant itself in piP-bodies and colocalization with BmSpnE and BmQin. See also Figure EV1.