EMBO/soda-nel huggingface dataset:
RIF1 and KAP1 differentially regulate the choice of inactive vs. active X chromosomes (2021)
Figure 3

Figure 3. RIF1 associates with Xist promoter on the future Xi.

(A, B). RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) and at 2 days of EB differentiation (B). P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant.

(C). Rif1 deletion decreases the efficiency of upregulation of a Luciferase reporter under the control of Xist promoter (Xist-2p-luc), at 2 days of EB differentiation. As a control (Luc), empty luciferase reporter vector, was transfected in parallel. The average of three independent experiments is shown. Error bars indicate the standard deviation. p calculated by Student's two-tailed, unpaired t test, for comparison of fold activation of Xist-2p-Luc normalised to empty vector (Luc) in Rif1+/+ versus Rif1-/- cells. **p ≤ 0.01. See Appendix Material and Methods for details about the normalisation.

(D). Association of RIF1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line (grey), also harbouring one castaneus and one 129 X chromosome. Allele-specific ChIP-qPCR primers were used, cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2, ***p ≤ 0.001. Below, schematic of the Xist/Tsix alleles in the Fa2L undifferentiated cells.

(E). Association of RIF1 with Xist P2 in the Fa2L cells (black) upon differentiation. The analysis was performed as in (D). Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2. *p ≤ 0.05. Below, schematic of the Xist/Tsix alleles in the Fa2L differentiated cells.

(F). Quantification by ChIP-qPCR of RIF1 association with the indicated regions in the Fa2L cell line, following treatment with DMSO only (black) or flavopiridol (grey). Primers as in (E).

(G). Same as in (F) but for a wild type female mESC line. All enrichments are presented relative to input DNA. Mean ±standard deviation from three (F) and two (G) independent experiments are presented. Statistical significance was determined using Student's two-tailed, paired t test (*p ≤ 0.05, **p ≤ 0.01 and ns=not significant).
RIF1 and KAP1 differentially regulate the choice of inactive vs. active X chromosomes (2021)
Figure 4

Figure 4. KAP1 regulates the Xa/Xi choice through Tsix

(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05).

(B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant.

(C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.

(D). RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase (control) or against Kap1 (Kap1 KD). Left: Cells with no (0, red), one (1, grey) and two or more (2, black) Tsix foci were counted in two independent experiments, shown averaged. Statistical significance was determined by χ2. A minimum of 110 cells were counted per each time point for each line (**p ≤ 0.01, ****p ≤ 0.0001). Right: examples of cells with one (top) or two (central and bottom) Tsix FISH signals. Scale bars: 5μm.
RIF1 and KAP1 differentially regulate the choice of inactive vs. active X chromosomes (2021)
Figure 5

Figure 5. RIF1 negatively regulates KAP1 association with Xist P2.

(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations.

(B). Using allele-specific primers, ChIP-qPCR was used to measure the association of KAP1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line also harbouring one castaneus and one 129 X chromosome (grey). cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments. Statistical significance was determined by Student's two-tailed, paired t test. Below, schematic of the Xist/Tsix alleles in the Fa2L cells.

(C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01).

(D). Allele-specific KAP1 association with Xist P2 in Fa2L cells following knock down of Luciferase (Control, black) and Rif1 (Rif1 KD, grey). cast indicates association with the castaneus Xist P2 promoter and 129 indicates association with the 129 Xist P2 promoter. Enrichments are presented relative to input DNA. Average ±standard deviation of two independent experiments.
RIF1 and KAP1 differentially regulate the choice of inactive vs. active X chromosomes (2021)
Figure 6

Figure 6. KAP1 recruits the 7SK complex to the Xist P2 promoter/Tsix terminator of the future Xa.

(A, B). H3K9me3 association with the Xist promoter (P1 and P2) in two independent Rif1+/+ and Rif1-/- cell lines, analysed by ChIP-pPCR in mESCs (A) and 2d EBs (B). Myf5 serves as a positive control region, β actin negative. Average ±standard deviation of two independent experiments.

(C). HEXIM1 association with the Xist promoter (P1 and P2) in two independent Rif1+/+ and two Rif1-/- mESC lines, analysed by ChIP-qPCR. Tkbp1 and Myf5 are two control regions. As in the case of KAP1 association, deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2. Average ±standard deviation of two independent experiments. p values were calculated by two-tailed, unpaired, equal variance t-test. (**p ≤ 0.01, ***p ≤ 0.001).

(D). Western blot analysis of KAP1 levels in protein extracts from Fa2L cells after Kap1 knock down. SMC1: loading control. Quantification of KAP1 protein levels normalised to SMC1 and relative to Luciferase control cells are shown below.

(E). Upon infection of Fa2L cells with shRNA against Kap1 or control, against Luciferase, HEXIM1 association with P2 was analysed, on both alleles, by ChIP-qPCR. As in the case of KAP1, HEXIM1 shows preferential association with Xist P2 on the Cast allele (future Xa, Control). The association is lost upon knock down of Kap1 (Kap1 KD). Myf5 serves as a negative and Neat1 as a positive control regions.
Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli (2020)
Figure 1

Figure 1 - In vivo and in vitro proteomics-based assays link NlpI to several classes of PG hydrolases.

A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity. Full results can be found in Tables EV 1 and 2.

C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins. Full results can be found in Tables EV 3 and 4. GO enrichments can be found in Tables EV 9 and 10.
Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli (2020)
Figure 2

Figure 2 - NlpI interacts with several EPases and is able to form trimeric complexes with them.

A. Dissociation constants for interactions between NlpI with MepM, MepS, PBP4, PBP7 as determined by Microscale thermophoresis (MST). The values are mean ± standard deviation of three independent experiments. The corresponding MST binding curves are shown in Appendix Fig. S2B.

B. MepS-NlpI interaction by MST as an example plot for Fig 2A. The same plot is also shown in Appendix Fig. S2B. MST curve plotted is the mean data +/- SD of three independent experiments. Fl, fluorescently labelled; FNorm, normalized fluorescence.

C-D. NlpI has different binding sites for MepS and PBP4, and MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI-PBP4 (C) and NlpI-PBP7 (D) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001), all p-values can be found in the Table EV 7.
Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli (2020)
Figure 4

Figure 4 - NlpI localizes along the entire cell envelope and associates with PG machineries

A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm. See Appendix Fig S5 for further details.

B. Genetic interactions of NlpI with PG machineries. Strains were arrayed and assessed as in Fig. 3b. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean. Full results can be found at Table EV 5.

C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The same images for control and NlpI mutant strains have been reused in Fig. 3C. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB. Cell length of mutants are displayed in Appendix Fig. S12d.

D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4. Binding curves are shown in Appendix Fig. S6.
Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli (2020)
Figure 5

Figure 5 - PBP4 forms a PG multienzyme complex with NlpI and PBP1A/LpoA.

A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.
Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli (2020)
Figure 6

Figure 6 - Proposed model for a role of NlpI in coordinating formation of PG multi-enzyme complexes containing EPases.

A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.

B. Hypothetical model of NlpI scaffolding endopeptidases during cell elongation. Black arrows indicate interacting proteins with numbers indicating apparent EC50 / KD values. OM, outer membrane; CM, cytoplasmic membrane. MepS-PBP1A interaction is not represented due to illustrative restrictions.
Distinct roles of a- and b-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration (2021)
Figure 1

Figure 1. TTLL1 and TTLL7 have distinct enzymatic specificities in brain and in neurons

A) Alignment of sequences of all human α- and β‑tubulin isotypes. Only the C-terminal tail region of the tubulins are shown. Isotypes with identical amino acid sequence in these regions are pooled. A large number of glutamate residues in the C-terminal tails would, if modified by glutamylation, be detectable with GT335 (yellow), which requires one additional acidic amino acid residue after the modification site (Wolff et al, 1992). By contrast, the GEF motif (red/purple), which is the main glutamylation site in β2‑tubulin (TUBB2) in the brain (Rüdiger et al, 1992), is present only in a few β‑tubulin isotypes, but not in α-tubulin.

B) Schematic representation of the synthetic peptide used as antigen to raise the β-monoE antibody. The modified glutamate residue is in red.

C) Representative immunoblots of brain extracts of wild-type, Ttll1-/- and Ttll7-/- mice. Polyglutamylation of α-tubulin, detected with polyE and GT335, is specifically lost in Ttll1-/- brain. By contrast, β-tubulin glutamylation, which is specifically detected with β‑monoE, is absent from brains of Ttll7-/- mice.

D) Representative immunoblots of extracts of wild-type, Ttll1‑/- and Ttll7-/- primary hippocampal neurons at DIV4. As in brain (C), α-tubulin polyglutamylation is specifically lost in Ttll1-/- neurons, while β-tubulin glutamylation lacks in Ttll7-/- neurons.

E) Schematic representation of the epitopes detected by the anti-glutamylation antibodies used in this study. For clarity, α- and β‑tubulin are shown separately.

F,G) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7‑/- mice. Microtubules in Ttll1-/- cells have reduced levels of polyE signal, indicative of lower α-tubulin polyglutamylation (F), Ttll7‑/- neurons are specifically reduced in β‑monoE reactivity, indicating the lack of β‑tubulin glutamylation (G).
Distinct roles of a- and b-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration (2021)
Figure 2

Figure 2. TTLL1 plays a key role in Purkinje-cell degeneration and tubulin hyperglutamylation

A) Representative histology images of cerebella from 3-months-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1-/-, Ttll7-/- and Ccp1-/-Ttll1-/- mice, in contrast to the complete absence of these cells in Ccp1-/- and Ccp1-/-Ttll7-/- mice. A complete representation of all analysed cerebella is shown in Fig. EV1.

B) Histology of 25-day old mice with partial degeneration of Purkinje Cells. Note that both, Ccp1-/- and Ccp1-/-Ttll7-/- brains, show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration).

C) Representative immunoblots of cerebellum extracts of wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 lead to a loss of glutamylation from β-tubulin (β‑monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both, α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE).
Distinct roles of a- and b-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration (2021)
Figure 3

Figure 3. TTLL1-mediated polyglutamylation is responsible for the degeneration of motor axons

A-D) Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A). These sections were quantified for (B) number of myelinated axons, (C) nerve area, (D) nerve perimeter (Fig. EV2) of femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control.

E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.
Distinct roles of a- and b-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration (2021)
Figure 4

Figure 4. Glutamylation patterns generated by TTLL1 and TTLL7 have distinct effects on mitochondria motility in axons

A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice (see also Movie EV1A). Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines).

B) Kymographs of one representative experiment representing mitochondrial movements (Movie EV1B).

C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots.

G - J) Statistical analyses of mitochondrial transport parameters of five independent experiments shown in (D-G and Fig. EV3A-D). Each bar represents the mean (±SEM) of medians of single experiments for (G) run length and (H) speed distributions, and average (±SEM) (I) overall mitochondrial motility and (J) density of all five experiments. Values of fold change between control and Ttll1‑/- and Ttll7-/- conditions are indicated below the graphs. Full bars are for anterograde, and empty bars for retrograde transport events. Significance was tested using unpaired t-test. Average (G) run length as well as (H) speed showed no significant differences between control and Ttll1-/- and Ttll7-/- conditions. (I) The averages of overall motilities reveal an about 1.7-fold increase in overall mitochondrial motility in Ttll1-/- neurons, while no significant differences were found in Ttll7-/- neurons. (J) Mitochondrial density was unaltered between control and knockout conditions. Total number of mitochondria analysed from five independent experiments: control (1260), Ttll1-/- (1158) and Ttll7-/- (1191). For further statistical data on transport analyses, see Appendix Table S2.