EMBO/soda-nel huggingface dataset:
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 6

Fig. 6 CREBH ablation rescues the phenotypes induced by hepatic HRD1 deletion.

(A) Flowchart of the study design for the knockdown CREBH in vivo.

(B) Hepatic CREBH protein levels 5 weeks after AAV-shCrebh injection. (n=5 for each group).

(C) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).

(D) Serum FGF21 protein levels in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).

(E) Body height and tibia length of the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection (n=5 for each group).

(F) Hepatic Igf1, Cyp2d9 and Cyp4a12 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).

(G, H) Percentage of the mated and estrus cycle of the WT and L-HRD KO 5 weeks after AAV-shCrebh injection.

Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 7

Fig. 7 Hepatic FGF21 ablation rescues the phenotypes induced by hepatic HRD1 deletion.

(A) Examples of the body sizes of the WT, L-HRD KO and HRD1/FGF21 DKO mice.

(B, C) Body weight and body height of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group).

(D) Tibia lengths of the WT, L-HRD KO and HRD1/FGF21 DKO mice fed with normal chow diet. (n=5 for each group).

(E) Hepatic Hsd3b5, Cyp2d9 and Cyp4a12 mRNA in the WT, L-HRD1 KO and HRD1/FGF21 DKO mice. (n=5 for each group).

(F) Estrus cycle of L-HRD KO and HRD1/FGF21 DKO mice at the age of 4 months.

(G) Percentage of the Activity, feeding and water intake of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group).

Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids (2018)
Figure 1

Figure 1. ParBF binding outside of parS centromere on plasmid and chromosome

(A) Schematic representation of the 'Nucleation & caging' model. Most ParB dimers (green dots) are highly confined in a cluster (dotted circle) centered on the parS sites (black rectangles) onto which some ParB are stably bound (red dots). The DNA entering the cluster is bound stochastically by ParB. Red and blue lines represent DNA present at small and large (or on a different molecule) genomic distance from parS, respectively.

(B) ParB clusters on F plasmid in vivo. Typical E. coli cells (DLT3594) displays foci of ParBF-mVenus protein (top) expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels. Over 99 % of cells harbor ParBF foci. White bars: 1 µm.

(C) ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq data (see text and Fig. EV2). As a benchmark for simulations, the analytics are obtained from Eq.(1) with the same parameters. Inset; The ParBF binding profile (black line) is represented as the number of nucleotide reads over 80-Kbp centered at parS. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample.

(D) and (E) Same as B and C with parSF inserted at the xylE locus on E. coli chromosome from DLT3584 and DLT2075, respectively. Cells were grown in the presence of 100 µM IPTG. The Kuhn length was adjusted to a=22-bp in the simulations and analytics. The characteristics of the A-F genetic loci are presented in Appendix Fig. S1A. Note that a highly similar ParBF DNA binding pattern is obtained when ParBF was expressed in trans from a plasmid (strain DLT3567; Appendix Fig. S1D).
A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids (2018)
Figure 2

Figure 2. ParBF DNA binding pattern is robust over a large range of intracellular ParBF concentrations.

(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling.

(B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm.

(C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of 'Nucleation & caging' with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlapping profiles.
A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids (2018)
Figure 3

Figure 3. The box II motif of ParBF is crucial for ParBF binding in the vicinity of parSF and cluster formation. (A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B&quot;2 and B&quot;3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al, 2015). (B) ParBF cluster formation requires the box II motif. Epifluorescence microscopy of ParBF-3R*-mVenus from DLT3566 is displayed as in Fig. 1D. White bars: 1 µm. (C) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif. ChIP-seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF-3R* variant. ParBF-3R* DNA binding profile displayed the number of nucleotide reads as a function of the E. coli genomic coordinates. The peak at parSF covered approximately 950-bp, which corresponds to the 402-bp between the 1st and 10th specific binding sites and ~280-bp on each sides (representing the average size of the DNA library; see Appendix Fig. S3E). No ParBF-3R* enrichment was found on parSF-flanking DNA and elsewhere on the chromosome. Inset, zoom in on the right side of parSF over 5-Kbp with the ParB density, normalized to 1 at the first bp after the last parS binding repeat, plotted as a function of the distance from parSF. Note that a highly similar DNA binding pattern is obtained with ParBF -3R*-mVenus (strain DLT3566; Appendix Fig. S3C).
A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids (2018)
Figure 4

Figure 4. ParB of V. cholerae assembled in cluster similarly to ParBF.

(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123.

(B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample.

(C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA library (304-bp; for comparison, a simulation without is provided in Appendix Fig. S4B).
A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids (2018)
Figure 5

Figure 5. Robust dips and peaks signatures in ParB DNA binding profiles.

ChIP-sequencing assays were performed on DLT2075 (xylE::parSF) expressing ParBF grown in exponential (expo) or stationary (stat) phases with addition of rifampicin when indicated (+Rif). The assays have been performed in duplicate for the +Rif and once for the stationary phase experiments.

(A) ParBF DNA binding around parSF is independent of active transcription. The color-coded ParBF profiles are represented over 50-Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site. Loci A, C, E and F are defined in Appendix Fig. S1A.

(B) The dips and peaks are highly similar in the three indicated conditions. Same as in (A) with zoom in on the right side of parSF up to 9-Kpb and normalization to 1 at genomic coordinate 230. The dotted line corresponds to the analytics description of 'Nucleation and caging' (see details in Fig. 1C-E).

(C) ParBF binding profile upstream of the locus A. Same as in (A) with zoom in from -6.5 to -16.5-Kbp by normalization to 1 at genomic coordinate -6.5-Kbp (upstream of the dip at the locus A). The ParBF DNA binding profile remains compatible to a power-law, represented by the analytics description (dotted line), upstream of the locus A in stationary phase (black) and in exponential phase (blue). Also, the dips and peaks are highly similar in both conditions. These data are not in favor of the '1D-spreading' or the 'Spreading and bridging' models that predicts a basal uniform distribution or a linear decrease after a barrier, respectively (Broedersz et al, 2014).

(D) The promoter region at locus A prevents ParBF DNA binding. Chip-seq assays were performed in isogenic xylE::parSF strains (DLT2075; black curve) in which the locus A is replaced by a kanamycin gene (DLT3651; red curve). The assay in the ∆(locus A) genomic context has been performed once. The relative ParB density as a function of the distance from parSF is drawn and normalized as in (A). The promoter region is depicted as in Appendix Fig. S1A.
The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma (2022)
Figure 1

Fig. 1. Identification of OTUD6B as a dependency in MM that promotes the G1/S cell cycle transition

A, Averaged sgRNA representation of a DUB CRISPR drop out screen in MM1.S cells clustered per gene. The ratio of normalized sgRNA read-counts on day 14 versus day 0 was determined for each sgRNA and the average fold change blotted per gene (except for non-targeting controls). Lines represent the median. Essential genes included as controls are: POLR2, PRPF8, RPL32, RPA3, RPL8, RPS19. OTUD6B is marked in red.

B, Cell proliferation analysis of different MM cell lines, expressing shRNAs targeting OTUD6B as counted by trypan blue exclusion method. Numbers are depicted as fold of shCtrl on day 8 after infection (n = 3 independent experiments).

C, Cell cycle analyses of JJN3, MM1.S, RPMI8226, L363 and U266 cells expressing the indicated shRNAs by BrdU/PI flow cytometry (n = 3 independent experiments).

D, Flow cytometric analysis of PI stained MM1.S cells transduced with the indicated shRNAs before and after release from G1/S block. Left: Exemplary FACS-plots. Right: Quantification of three independent experiments showing the percentage of cells in G1 phase.

E, Immunoblot analysis of cells described in (D), harvested at the indicated time points after G1/S release. Exemplary blot from three independent experiments.

F, DUB activity assay for OTUD6B in asynchronous, G1/S- or mitotically synchronized and released A549 cells using HA-ubiquitin vinyl-sulfone to isolate active forms of DUBs. Analysis was performed by immunoblotting using the indicated antibodies.

Data information: In (B-D), data are mean ± s.d. *. P < 0.05; **, P < 0.01; ***, P < 0.001; by paired Student's t-test (B-D) corrected for multiple testing (B, D).
The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma (2022)
Figure 2

Fig. 2. LIN28B is a direct deubiquitylation substrate of OTUD6B

A, Results of mass spectrometric (MS) based screening for OTUD6B substrates correlating the results from a FLAG- and a Bio-ID-purification. Intensities (LFQ) of co-immunoprecipitated proteins identified by MS were log2 transformed and the differences between OTUD6B and EV plotted against LFQ intensities of proteins identified in the OTUD6B sample. The red dotted line represents the cut off for two-fold enrichment in the OTUD6B sample compared to control. Proteins enriched more than two-fold in the BioID-proximity screen are highlighted in green. OTUD6B (bait) and LIN28B are depicted in red.

B, In-vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA. Cells were transfected with the indicated siRNAs and overexpression constructs, then treated with MG132 for 3 hrs. Lysis and IP was done under denaturing conditions followed by WB analysis.

C, In-vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B (OTUD6B-C158A) overexpressing cells. HEK293T cells were transfected with indicated combinations of FLAG-LIN28B, HA-Ubiquitin, OTUD6B, OTUD6B-C158A and EV control and treated with MG132 for 3 hrs 24 hrs later. Denatured WCE were subjected to FLAG-IP. WCE and IP were analyzed by immunoblotting including a K48-specific ubiquitin antibody. Exemplary blots from three independent experiments are shown.

D, Pull-down of endogenous LIN28B from HEK293T WCE using bacterially purified GST-OTUD6B. Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies.

E, Immunoblot analysis of an in-vitro deubiquitylation assay using GST-purified OTUD6B from bacteria and FLAG-purified ubiquitylated LIN28B from HEK293T. FLAG-IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG-LIN28B and HA-Ubiquitin. Purified FLAG-LIN28B was eluted from the beads and incubated with GST or GST-OTUD6B proteins followed by immunoblot analysis.

Data information: For B, D, and E: exemplary blot of two independent experiments.
The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma (2022)
Figure 3

Fig. 3. OTUD6B stabilizes LIN28B at the G1/S cell cycle transition point

A, Co-IP of FLAG-OTUD6B with endogenous LIN28B from MM1.S cells synchronized in G1/S and 12 hrs post release. MM1.S cells stably expressing FLAG-OTUD6B or FLAG-EV were synchronized at G1/S and harvested at the indicated time points after release. IPs were preformed and analyzed together with WCE by immunoblotting.

B, C, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. RPMI (B) and A549 (C) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies.

D, Immunoblot analysis of LIN28B protein half-life in G1/S-synchronized (upper panel) and asynchronous (lower panel) A549 cells upon OTUD6B depletion. Cells transfected with the respective siRNAs were synchronized in G1/S-phase or not and treated with cycloheximide (CHX) as indicated.

Data information: A-D, All blots are representations of three independent experiments.
The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma (2022)
Figure 5

Fig. 5. OTUD6B activates MYC via LIN28B

A, GSEAs showing an enrichment of MYC V1 target genes in RPMI8226 MM cells depleted of OTUD6B and LIN28B as compared to control cells. RPMI8226 cells expressing the indicated shRNAs from three independent experiments were subjected to RNA-seq analysis followed by GSEA.

B, Heat map of MYC-regulated genes which were commonly downregulated (P < 0.05) in OTUD6B and LIN28B knockdown cells compared to control cells in the RNA-Seq experiment shown in (A) (n = 3).

C, Immunoblot analysis of RPMI8226, KMS12BM and MM1.S cells expressing the indicated shRNAs. Depicted are examples of three independent experiments per cell line. Specification of MYC rearrangements: RPMI8226 (der(17)t(?8;17)(q21.2;q25)), KMS12BM (der(1;8)(q10;q10)x2), MM1.S (der3t(3;8)).

D, Real-time qPCR of RPMI8226 cells in which OTUD6B expression was silenced by the indicated shRNAs.

(E, F) Real-time qPCR (E) and proliferation (F) analyses of RPMI8226 cells infected with the indicated shRNA constructs.

Data information: In (D-F), values are normalized to shCtrl (n = 3 independent experiments with three technical replicates each). Data are mean ± s.d. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; by Student's t-test corrected for multiple testing.
The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma (2022)
Figure 6

Fig. 6. OTUD6B associates with MYC activity, MGUS to MM transition and poor outcome in MM patients

A, Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis. mRNA expression in primary CD138+ MM cells was quantified by real-time qPCR (n = 89 patients). Data are fit by linear regression (black line); Pearson r, Pearson correlation coefficient; P < 0.0001; by linear regression and Pearson correlation.

B, GSEAs showing an enrichment of MYC target genes in MM patients with high OTUD6B expression in two independent data sets (Left: GSE24080; Right CoMMpass-cohort - dbGaP accession: phs000748.v4.p3).

C, D, Kaplan-Meier survival curves of MM patients with high or low OTUD6B expression. Patients from the upper and lower quartiles of the two cohorts comprising the GSE24080 dataset (n = 176 for TT2 and n=108 for TT3) (C) and the CoMMpass cohort (n=385) (D) described in B were taken for analysis. P values calculated by Log-rank test.

E, Data from (Zhan et al., 2007) reanalyzed to show expression levels of OTUD6B in normal bone marrow (BM) (n=22), monoclonal gammopathy of undetermined significance (MGUS) (n = 44) and multiple myeloma (MM) (n=12). Box-and-whisker plots show the upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the 10th to the 90th percentile, and dots correspond to minimal and maximal values.

F, Kaplan-Meier curves for EFS of bortezomib treated MM patients with high or low OTUD6B expression. Patients classified as bortezomib responders (R) in the GSE9782 dataset were grouped according to their OTUD6B expression (highest and lowest 30%; n= 53) and analyzed for event free survival. P values calculated by Log-rank test.

G, H, Proliferation analysis of (G) LP-1-Cas9 and (H) MM1.S-Cas9 cells infected with indicated sgRNAs cultured without or with a sub-lethal dose of bortezomib (LP1: 7 nM; MM1.S: 12.5 nM) or carfilzomib (for MM1.S only. Sub-lethal dose of 5 nM). The ratio of sgRNA expressing (GFP+) cells to uninfected cells was measured by flow cytometry at the indicated time points after infection. Results are normalized to GFP+ cells at day 4 (n = 3 independent experiments). Values represent mean ± s.d.

Data information: *, P < 0.05; ** P < 0.01; ****, P < 0.0001; by Student's t-test , (H) corrected for multiple testing.

Extended View - Figure Legends
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 1.

Non-endosomal ubiquitin-positive structures accumulate in ESCRT depleted cells. HeLa cells were left untransfected (A) or transfected with control (B), Hrs (C), Tsg101 (D), Vps22 (E), or Vps24 (F) siRNA for 5 d and processed for immunofluorescence microscopy. Cells were labeled with antibodies against EEA1 (blue), ubiquitin (mono and poly-Ub, red), and Lamp2 (green). Colocalization between EEA1 and Ub is indicated in purple and between Ub and Lamp2 in yellow. Bar, 10 μm. Single channel images of the insets are shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200702115/DC1). (G) Knock-down efficiencies were analyzed by Western blotting. Equal loading was verified by anti-α-tubulin immunoblotting.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 2.

p62- and Alfy-positive structures accumulate in cells depleted of Tsg101 or Vps24. HeLa cells transfected with siRNA against Tsg101 or Vps24 were fixed, permeabilized, and stained with antibodies against ubiquitin (red) and p62 (green) (A) or against ubiquitin (green) and Alfy (red) (B). Colocalization is indicated in yellow. Bar, 10 μm. (C) The number and size (area) of p62-positive particles were quantified using ImageJ software. Approximately 100 cells from three different experiments (n = 334 (control), 309 (Tsg101), and 246 (Vps24)) were used for the quantification and the data is presented as average number per 100 cells. Error bars = SEM.