EMBO/soda-nel huggingface dataset:
Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin (2021)
Figure 1

Figure 1. Characterization of binding of 27 ACE2s with GD/1/2019 RBD, GX/P2V/2017, the SARS-CoV-2 RBD and the SARS-CoV-2 NTD by FACS.

His-tagged GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins were incubated with BHK 21 cells expressing eGFP-fused ACE2s. Anti-His/APC antibody was used to detect cells expressing His-tagged protein. Cells stained with the GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins are shown in green, orange, marine and red, respectively. The SARS-CoV-2 NTD was used as negative control.
Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin (2021)
Figure 2

Figure 2. Binding affinity assay between ACE2s and the RBDs of the SARS-CoV-2, GD/1/2019 and GX/P2V/2017 by SPR.

A The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively.

B The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) and the 13 ACE2s are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.
Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin (2021)
Figure 3

Figure 3. Infectivity of the pseudoviruses of SARS-CoV-2, GX/P2V/2017 and GD/1/2019 to HeLa cells expressing hACE2.

A Hela-hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS-CoV-2, GX/P2V/2017 and GD/1/2019, respectively. Green fluorescence indicates Hela-hACE2 cells infected with pseudovirus. Untransfected HeLa cell is used as negative control. The scale bar indicates 400 μm.

B Statistic for the infectivity of the three pseudoviruses. Data represent the results of five replicates. All data are presented as mean ± SD. ***p < 0.001 (Student's t-test). ns, no significant differences.
Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin (2021)
Figure 6

Figure 6. Binding affinity assay between different SARS-CoV-2 RBD mutants and ACE2s by FACS and SPR.

A Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control.

B The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H).

C The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.

Table 1. Comparison of hACE2 binding to SARS-CoV-2 RBD, GD/1/2019 RBD and GX/P2V/2017 RBD.

A475 (4), G476 (5),

N487 (15, 1)

A475 (4), G476 (7),

N487 (11,1)

A475 (6), G476 (8),

N487 (15,1), Y489 (1)

F456 (13). Y473(2), A475(1),

Y489 (6)

Q498 (8), T500 (7, 1),

N501 (8, 1)

H498 (16), T500 (6),

N501 (13,1)

G446 (1), Y449 (10,1),

H498 (1)

Numbers in the parentheses beside hACE2 residues represent the number of vdw contacts between the indicated residue with the SARS-CoV-2 RBD, GD/1/2019 RBD or GX/P2V/2017 RBD. Numbers in parentheses beside either ligand residues represent the number of vdw contacts the indicated residues conferred. The numbers with underline suggest numbers of potential H-bonds between the pairs of residues. vdw contact was analyzed at a cutoff of 4.5 Å and H-bonds at a cutoff of 3.5 Å.

Expanded View
Comparative chromatin accessibility upon BDNF stimulation delineates neuronal regulatory elements (2022)
Figure 1

Figure 1. Transcriptional dynamics in mouse cortical neurons upon neuronal stimulation with BDNF and KCl defines early and late gene programs.

(a) (top) Experimental design. Cultured cortical neurons are stimulated with BDNF, KCl or no treatment (Control) and activated neurons are prepared in three specific time points for RNA-seq and ATAC-seq at early (1h) and late time points (6 and 10h); (bottom) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF (Con = no stimulation). 44 and 42 KDa bands indicated with lines. Nucleolin shown as internal control.

(b) Hierarchical clustering for all differentially-expressed genes (DE-genes, adjusted P-value < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared to unstimulated control samples at each time point.

(c) Number of DE-genes at each time-point and treatment combination (above and below zero indicates up-regulated and down-regulated genes, respectively). Darker shades indicate genes newly up/down regulated at a given time point (new).

(d) Partitioning around medoids clustering of expression dynamics using genewise-scaled Z-scores of top 5,000 DE-genes from (c) with lowest adjusted p-value (k = 10 clusters). Numbers indicate clusters. Left line plots indicate expression levels for signature genes selected for each cluster. 4,907 DE-genes are also selected with FDR=1% and more than 100 counts across samples.

(e) Gene Ontology enrichment analysis for clusters shown in (d). Each ribbon shows clusters with their respective significant gene groups using topGO(Alexa et al, 2006). Up to five significant terms per cluster are shown (P-values adjusted with BH). Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters. Names on the right y-axis indicate ontology common names.
Comparative chromatin accessibility upon BDNF stimulation delineates neuronal regulatory elements (2022)
Figure 3

Figure 3. TFs linked to differentially accessible chromatin regions identify common and stimulus-specific regulatory TFs.

(a) (i) DA-peaks obtained at each time point are analyzed with GENRE (Genomically Equivalent Negative REgions) software to generate regions used as negative controls in enrichment tests(Mariani et al, 2017). (ii) A library of 8-mers and position weight matrices (both motifs) are used to classify positive sequences (DA-peaks) versus negative control regions as a receiver operating characteristic area under the curve value (ROC-AUC).

(b) Enrichment of regulatory motifs in gained (left) and closing (right) DA peaks. Circle size indicates ROC-AUC for recovery of positive versus negative regions. Circle color indicates P-value significance (one-sided Wilcoxon rank sums test). Red lines indicate adjusted P < 0.1 after Benjamini Hochberg's correction. ROC-AUC values lower than 0.5 are shown as gray circles. Barplots indicate maximum value observed for each TF group across all time points for BDNF (orange bar) and KCl (green bar). In relevant cases for main text fold changes are labeled.

(c) RNA expression changes for TFs related to 8-mer groups bZIP, CTCF, HIC1, and EGR. Significant changes relative to control samples are shown with asterisks (Wilcoxon rank sums test, two-sided).

(d) Proteomics of the chromatin-bound fraction from mESC-derived neurons 1h after stimulation show significant enrichment of TFs upon stimulation with BDNF compared to KCl (boxplot, lower panel, wilcoxon two-tailed rank sum test, *P < 0.0001; central band indicates the median, boxes depict third and first quartiles and whiskers show the 1.5× IQR above and below the box). Out of 5,508 detected proteins, Fos shows a significant abundance increase after BDNF stimulation compared to KCl (|log2 fold change| ≥ 1, adjusted P-value < 0.05). High levels of BDNF (blue dot) are associated with experimental stimulation.
Comparative chromatin accessibility upon BDNF stimulation delineates neuronal regulatory elements (2022)
Figure 4

Figure 4. BDNF transcriptional up-regulation is linked to bZIP and EGR interactions in promoters and enhancers.

(a) Scheme indicating annotation of ATAC-seq peaks based on the presence of a bZIP motif (bZIP) and another TF (TF2). Peaks are linked to genes based on closest genomic distance ("closest gene") or only if present in gene TSS ("TSS"). A log2-fold change median indicates the difference in accessibility or expression log2-fold changes between all peaks with bZIP+TF2 motifs versus only bZIP peaks, and equivalently for the expression values of genes connecting to those peaks.

(b) Genome-wide changes at bZIP+TF2 and bZIP measured as log2-fold change distributions for accessibility ("consensus peaks"), genome-wide closest genes ("closest gene"), and proximal target gene RNA levels ("genes with TSS peaks") when bZIP motifs are co-present with other TFs (TF2) in ATAC-seq peaks versus bZIP alone. Significance is assessed using the one-sided Wilcoxon rank sums test between peaks with k-mers co-present versus peaks with only bZIP. Absolute changes lower than 0.1 and not significant are shown as white boxplot bars.

(c) Chromosome 15 genome tracks neighboring Arc, displaying ATAC-seq read counts per million (CPM); RNA-seq CPM; H3K27ac normalized signal upon KCl stimulation, H3K4me1(Malik et al, 2014); CTCF(Sams et al, 2016); and Cortical neurons Hi-C data(Bonev et al, 2017). Red bars in ATAC-seq tracks indicate gained DA-peaks in BDNF, and red bars in RNA-seq tracks indicate Arc differential expression in BDNF and KCl 1h. Green blocks in Hi-C tracks indicate anchor points for the calculation of contact scores, using shaman(Cohen et al, 2017). Line and Spearman's rho value indicate counts rank-based correlation between highlighted peaks across all samples (Appendix Figure S4c). TF module names indicate the presence of 8-mers in those peaks.
Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition (2020)
Figure 1

Figure 1: EMT-related genes are dysregulated in crizotinib-resistant cells

A) Table reporting all drug-resistant cell lines used in this study.

B) Proliferation assay of H3122 parental and isogenic drug-resistant cells, treated with the indicated concentrations of crizotinib for 72h. P < 0.0001 was calculated for IC50 shift as indicated in the methods.

C) Gene-set enrichment analysis after RNA-seq of H3122 sensitive vs CrizR1 crizotinib resistant cells.

D) Epithelial to mesenchymal transition enrichment plot from the RNA-seq of C), using the HALLMARK gene collection.

E) H3122 parental and drug-resistant isogenic cell lines were RNA-extracted and gene expression was analysed by RT-qPCR. On the right hand side is a western blot analysis of the AXL and EML4-ALK protein levels from the same cell lines.AXL: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P=0.03, CeritR vs H3122 P=0.2; LOX: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P=0.008; SNAI2: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P =0.003, CeritR vs H3122 P =0.003; VIM: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P <0.001.

F) Gene expression analysis by RT-qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h. LOX: P=0.03; SNAI2: P =0.02; VIM: P =0.03.

G) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h. *** = P < 0.001 for IC50 shift, as indicated in the methods (n=4).

H) Annexin V+ apoptotic assay in CrizR1 cells treated with 1uM crizotinib, 2.5uM bemcentinib, or combination. STE-1 parental cells were used as crizotinib drug control. crizotinib: P =0.2; bemcentinib: P =0.4; combination: P=0.3.

Data information: Statistical comparisons were performed using a paired, two-tailed student's t-test. Plotted graphs show mean ± SD (n=3, unless otherwise specified). *P < 0.05, ***P < 0.001, N.S. = Not Significant P >0.05.
Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition (2020)
Figure 3

Figure 3: Cells harbouring the EML4/ALK translocation are remarkably sensitive to alvocidib, dinaciclib and THZ1.

A) Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR, and AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of THZ1 for 72h. P <0.05 was calculated for IC50 shift, as indicated in the methods.

B) Crystal violet staining of EML4/ALK parental and drug-resistant cells. Cells were treated with the indicated drugs until the vehicle control reached confluence, then fixed and stained.

C) CrizR1 cells were treated with the indicated drugs for 72h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells. Alvocidib: early apoptosis P=0.002, late apoptosis P=0.006, alive P=0.0008; Dinaciclib: early apoptosis P=0.005, late apoptosis P=0.02, alive P=0.002.

Data information: Statistical comparisons were performed using a paired, two-tailed student's t-test. Plotted graphs show mean ± SD (n=3). *P < 0.05, ** P < 0.01, ***P < 0.001, N.S. = Not Significant P >0.05.
Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition (2020)
Figure 5

Figure 5: Alvocidib or THZ1 treatment is concordant with CDK9 or CDK7 inhibition based on RNA polymerase II ChIp-seq.

A) CrizR1 cells were treated with vehicle control, 200nM alvocidib or 100nM THZ1 for 6 hrs, then chromatin was precipitated with an anti-RNA polymerase II antibody and sequenced. Plotted is the average number of peaks per condition.

B-C) GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (B) alvocidib or (C) THZ1 treatment. Shown on the right is the plot of the "MYC targets"-enriched signature.

D) Gene tracks of RNA polymerase II occupancy at the MCL1, BIRC5 (Survivin), CCND1 and MYC genes.

E) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h. The graph represents the mean fold change ± SD. MYC: alvocidib vs DMSO P =0.03, THZ1 vs DMSO P <0.0001; CCND1: alvocidib vs DMSO P =0.001, THZ1 vs DMSO P =0.003.

F) BIM is upregulated upon MYC silencing in CrizR1 cells.

G) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA. Early apoptosis P =0.002, late apoptosis P=0.04, alive P =0.0008.

H) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib, rapamycin or both. Trametinib: early apoptosis P =0.002, late apoptosis P =0.05, alive P =0.009; rapamycin: early apoptosis P =0.04, late apoptosis P =0.8, alive P =0.003; tram+rapa: early apoptosis P =0.0001, late apoptosis P =0.04, alive P =0.01.

Data information: Statistical comparisons were performed using a paired, two-tailed student's t-test. Plotted graphs show mean ± SD (n=3, unless otherwise specified). *P < 0.05, ** P < 0.01, ***P < 0.001.
Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition (2020)
Figure 6

Figure 6: Alvocidib reduces tumour growth in vivo.

A-B) Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib or alvocidib and correspondent tumour weights (P.O. control n=5; I.P. control n=4; crizotinib n=8 and alvocidib n=6). Tumour weights, crizotinib P= 0.44, alvocidib P=0.03.

C) Tumour growth of in vivo xenografts of Ste-1 AlectR cell lines in response to alvocidib and correspondent tumor weights (I.P. control n=6; alvocidib n=4). Tumour weights alvocidib P=0.004.

D) Number of cleaved caspase-3 positive nuclei in alvocidib treated tumours compared to controls.

E) Ki67 and p21 staining of CrizR1 xenograft tumours. Scale bar = 200μm. Quantitative analysis of IHC staining is presented on the right. Ki67 P=0.0004; p21 P=0.02.

F) qPCR of Survivin (P=0.0009), MCL-1 (P=0.04), MYC (P=0.007) and BIM (P=0.03) in Ste-1 AlectR cells xenografts treated with alvocidib (Control n=6; alvocidib n=4).

G) Proliferation assay of H3122 parental and isogenic drug-resistant cell lines, treated with DMSO, 100nM lorlatinib, 200nM alvocidib or 50nM dinaciclib for 72h (n=4).

H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7).

I) Model suggesting the clinical sequencing of CDK inhibitors post-ALK inhibition failure as a potential alternative to chemotherapy.

Data information: Statistical comparisons were performed using a paired, two-tailed student's t-test. Plotted graphs show mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = Not Significant P >0.05.

Expanded View Figure Legends
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly (2019)
Figure 1

Figure 1. Histone H3 is ubiquitylated in vitro by the CLRC.

A Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses.

B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species.

C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly (2019)
Figure 2

Figure 2. Histone H3 lysine 14 is ubiquitylated in vitro by CLRC.

A Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right.

B MS/MS spectrum of the histone H3 peptide corresponding to residues 10-17. The observed y and b ions and fragment map are shown.

C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly (2019)
Figure 3

Figure 3. H3K14 ubiquitylation is associated with H3K9me-enriched chromatin.

A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.

B MS/MS spectrum of the histone H3 peptide corresponding to residues 9-17. The observed y and b ions and fragment map are shown.

C Summary of the ubiquitylated peptides identified by LC-MS/MS.

D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown (see also Fig EV3F). Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly (2019)
Figure 4

Figure 4. H3K14 is critical for heterochromatic silencing.

A Diagram of the mating type loci in S. pombe. The position of the silencing reporter gene (kint2::ura4+) is shown. The target regions in the ChIP analysis are indicated by black bars above (kint2::ura4+) and below (cenH) in the diagram.

B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA).

C ChIP analysis of H3K9me2 levels associated with the mating-type loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.

D ChIP analysis of Rik1-myc levels associated with the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.

E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated as in (B).
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly (2019)
Figure 5

Figure 5. H3K14 ubiquitylation promotes H3K9 methylation.

A Experimental scheme.

B In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right).

C,D In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed as in (B). The 3H-methyl group incorporation was measured using a liquid scintillation counter (D). Error bars indicate the standard deviations calculated from three independent experiments.
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly (2019)
Figure 6

Figure 6. Clr4 N-terminal regions are involved in recognizing ubiquitylated H3 and heterochromatic silencing.

A Schematic of recombinant Clr4 proteins used in the in vitro HMTase assay: GST-fused full-length Clr4 (FL) and truncated Clr4 proteins (∆2-∆4).

B Recombinant proteins used in (C) and (D) were resolved by 10% SDS-PAGE and visualized by CBB staining.

C In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed as in Figure 5C.

D In vitro pulldown assays of GST-Clr4 proteins with biotinylated ubiquitin. Bound ubiquitin was analyzed by Western blotting with HRP-conjugated streptavidin (left). Precipitated GST proteins were analyzed by SDS-PAGE and CBB staining (right).

E Schematic of FLAG-tagged, wild-type and mutant (∆63-127) Clr4 proteins.

F Whole cell lysates prepared from control cells, cells expressing FLAG-tagged wild-type Clr4 (F-clr4) or mutant Clr4 (F-clr4∆63-127), or clr4∆ cells were subjected to immunoblotting using the indicated antibodies.

G Heterochromatic silencing assays of wild-type and clr4 mutant strains. Silencing at the mating-type Kint2::ura4+ was evaluated.

H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating-type loci (Kint2::ura4+) and the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.

I The levels of Kint2::ura4+, mating-type cenH, and centromeric dg transcripts were quantified by RT-qPCR and calculated as fold change to the wild-type expression. Error bars indicate standard errors from three technical replicates.

Expanded Figure legends