EMBO/soda-nel huggingface dataset:
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 2

Figure 2. A muscle-specific decrease in α-dystroglycan glycosylation and laminin binding upon POGLUT1 mutation.

A Muscle sections show variable labeling using an antibody against glycosylated α-dystroglycan (αDG-IIH6), whereas labeling using antibodies against α-dystroglycan core protein (αDG-Core), β-dystroglycan (βDG) and laminin-2 is similar to control (Scale bar, 100 μm).

B Western blots and ligand-overlay (O/L) of wheat-germ agglutinin–enriched muscle and fibroblasts lysates from PII.2, PII.5, the healthy sibling (HII.3) and healthy controls (Ctr, Ctr1, and Ctr2) (Muscle: 250μg protein/lane, Fibroblasts: 800μg/lane). In muscle, expression of αDG-IIH6 is reduced but the αDG-Core shows similar expression as controls, with a slight reduction in molecular weight; laminin overlay assay detected some binding activity but was diminished compared to controls, whereas agrin-binding activity showed no difference with controls. In fibroblasts, both αDG-IIH6 and αDG-Core expression and laminin-binding activity were normal, suggesting that unlike other muscular dystrophies, α-dystroglycan glycosylation defect is muscle-specific in POGLUT1 patients.

C No ultrastructural alterations are observed in muscle by electron microscopy (6-7 fields were captured from PII.1, PII.3, and PII.5). Note normal basement membrane compactation (arrowheads). Original magnification: 26,700.
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 4

Figure 4. POGLUT1D233E has lower enzymatic activity than wild-type.

A Protein O-glucosyltransferase activity of wild-type (WT) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat (hFIX-EGF). Wild-type shows higher O-glucosyltransferase activity than D233E, and this activity is dependent on the concentration of the acceptor substrate hFIX-EGF repeat (left) and on the concentration of donor substrate UDP-glucose (UDP-Glc) (right). Values indicate mean ± SEM from three independent assays.

B O-glucosyltransferase activity towards five different single EGF repeats from mouse Notch1. Wild-type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E. The Y-axis shows the normalized activity relative to wild-type POGLUT1. EGF repeats were at 10 μM. Values indicate mean ± SEM from three independent assays.

C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC. hFIX-EGF repeat was incubated with wild-type or D233E mutant POGLUT1 and donor substrate, UDP-Glc or UDP-xylose (UDP-Xyl), at 37ºC overnight. Values on top of the peaks indicate the measured masses. Addition of Glc (162 Da) or xylose (132 Da) to hFIX-EGF (5696.2 Da) by wild-type POGLUT1 caused a shift to an earlier retention time (left). Similarly, the products exhibited a similar shift after incubation with POGLUT1D233E (right). These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX-EGF repeats and thus has residual enzymatic activity.
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 5

Figure 5. Muscles from D233E patients show decreased Notch signaling and pool of satellite cells.

A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain (NICD) in patients.

B-C qRT-PCR on RNA extracted from skeletal-muscle shows that expression of HES1 (B) and PAX7 (C) was significantly lower in D233E patients compared to healthy controls. Mean ± SEM, Student´s t-test (B) and Mann-Whitney U test (C).

D PAX7+ cells in skeletal muscle sections, demonstrating that satellite cells are less abundant in D233E patients than in controls (n = 3 muscles with 10-15 fields analyzed per muscle). Mean ± SEM; Mann-Whitney U test; Scale bar, 50 μm.
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 6

Figure 6. Loss of Rumi results in temperature-sensitive Drosophila muscle defects and is only weakly rescued by POGLUT1D233E expression.

A-A” Staining of Drosophila indirect flight muscles at 25% pupal development with 22C10 antibody (myotube marker) and anti-Twist antibody (myoblast marker) indicates that in wild-type animals, indirect flight muscles are formed from 6 myotubes, and a large number of Twist+ myoblasts are present.

B-B” rumi79/79 mutants raised at 18C show no obvious defects in myoblast number or myotube morphology.

C-D” rumi79/79 mutants exhibit decreased number of myoblasts at 25°-30°C and either an incomplete set of short myotubes when raised at 25C or aberrant morphology of myotubes when raised at 30C.

E-E” rumi79/79 animals overexpressing FLAG-tagged POGLUT1WT in the muscle compartment using Mef2-GAL4 and raised at 25C during the pupal stage show a rescue of the rumi79/79 muscle phenotype.

F-G” Overexpression of FLAG-tagged POGLUT1D233E only partially rescues the rumi79/79 muscle phenotype, with some variation in the myotube length and the number of restored Twist+ cells (compare G and F). Note that the number of Twist+ cells restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT. Scale bar in A’ is 50 µm and applies to A-G”.

H Quantification of myotube lengths in rumi79/79 mutants with or without POGLUT1 expression indicates a weak rescue of the phenotypes by POGLUT1D233E compared to POGLUT1WT. Mean ± standard deviation is shown. The differences in myotube lengths are statistically significant (n = 22-54). Mean ± standard deviation is shown; One-way Anova with Bonferroni´s multiple comparisons test.
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 7

Figure 7. D233E primary myoblasts exhibit decreased proliferation and increased differentiation.

A-F Proliferation assays examined proliferating (PAX7+ MyoD+, arrows), self-renewing (PAX7+ MyoD−, open arrows), and differentiating (PAX7− MyoD+, arrowheads) cells from D233E patients (n = 2), healthy controls (n = 3) and disease controls (n = 3) at 1, 4, and 8 days growing in proliferation medium. (A) shows representative images from day 8. The number of patient myoblasts is lower than that of controls at all three time points, indicating a slower proliferation rate in patient myoblasts (B), and accordingly, the percentage of proliferating cells (C) is smaller in the patient. (D) The percentage of self-renewing cells is small in patient´s culture, reflecting a poor capacity for maintaining the pool of quiescent SCs. In addition, the percentage of PAX7+ cells (E) is smaller in patient´s culture, similarly to what was found in adult D233E muscle (shown in Fig 5D). (F) The percentage of differentiating cells is higher in the patient. We examine proliferation capturing 7-12 randomly fields per day and condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).

G-I When myoblasts started to be confluent, the proliferation medium was replaced with differentiation medium, and the myoblasts started to fuse yielding myotubes, which were analyzed after four days of differentiation. (H) The fusion index, which measures the percentage of nuclei in myotubes with respect to the total number of nuclei in myogenic cells, was higher in D233E cultures than in controls. (I) Besides, myogenin showed increased expression in D233E myoblast cultures compared to controls. The data support that differentiation process is facilitated in patient´s muscle. Data was collected from 3-10 randomly chosen areas per condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test (H) and One-way Anova with Bonferroni´s multiple comparisons test (I); Scale bar, 50 µm).
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 8

Figure 8. Rescue experiments on immortalized primary myoblasts by lentiviral infection.

A-C We overexpressed GFP-tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts. (B) We analyzed the proliferation at 1, 3 and 5 days, and proliferation rate in patient-LV-NICD-GFP reached the level observed in control-LV-GFP, which were higher than in patient-LV-GFP. (C) However, the percentage of PAX7+ cells in patient-LV-NICD-GFP showed the same reduced value as in patient-LV-GFP, which were significantly lower than in control-LV-GFP. The majority of control myoblasts were proliferating cells (PAX7+ MyoD+, arrows), with no quiescent cells and few differentiating cells detected. Data were collected from n = 2 independent assays (5-8 images per condition). Mean ± SEM is shown; One-way Anova with Tukey multiple comparisions test (B) and Kruskal-Wallis with Dunn´s multiple comparisons test (C); Scale bar, 50 µm).

D-F As previously described in patient’s primary myoblasts, patient-LV-GFP myoblasts showed a striking facilitated differentiation compared to control-LV-GFP-myoblasts. This phenotype fully disappeared in patient-LV-GFP-NICD myoblasts, as quantification of the fusion index (E), and myogenin expression (F) demonstrated. The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients. Data were collected from n = 3 independent assays (13-17 images per condition). Mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).
A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss (2016)
Figure 9

Figure 9. Effects of decreased Notch signaling on α-dystroglycan glycosylation during differentiation.

A Immortalized primary myoblasts from a healthy control showed a progressive pattern of α-dystroglycan glycosylation during five days of differentiation (D0-D5), as has been previously described by Goddeeris et al. Immortalized primary myoblasts from patients displayed an irregular pattern of glycosylation, with a final level of glycosylated α-dystroglycan that was lower than control.

B α-dystroglycan glycosylation of infected immortalized myoblasts from patient-LV-NICD-GFP at differentiation day 5 (D5) showed a striking reduction, in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression. α-dystroglycan in myoblasts from patient-LV-GFP showed a reduction of glycosylation compared to control-LV-GFP at D5, similar to the results obtained in the adult muscle from patients (Fig. 2).

C-D C2C12 myogenic cells showed a reduced level of glycosylated α-dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium. This indicates that reducing Notch signaling can affect α-dystroglycan glycosylation in wild-type C2C12 myoblasts cells, which do not harbor any known mutations in α-dystroglycan glycosyltransferases or Poglut1.

E-F qRT-PCR experiments show that upon DAPT or LY3039478 treatment, a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation.
The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency (2021)
Figure 1

Figure 1. Identification of factors regulating divergent non-coding (DNC) transcription by a synthetic genetic array (SGA) and flow cytometry analysis.

(A) NET-seq data of wild type (WT) yeast at the GCG1/SUT098 and ORC2/SUT014 loci. NET-seq reads in black and grey represent the Watson (+) and Crick (-) strands, respectively. The NET-seq track combines remapped data from previous publications (see Material and Methods for more details).

(B) Schematic representation of a shared promoter region initiating a coding (blue) and a DNC (purple) transcript in the opposite orientation. Fluorescent protein reporter (FPR) construction replaces the endogenous coding and DNC genomic regions with sequences encoding mCherry and YFP, respectively.

(C) SGA and flow cytometry analysis in budding yeast. The array involves the crossing of MATα query strain harboring the FPR construct with the MATa non-essential gene deletion mutant library. The systematic selection as described in Baryshnikova et al, 2010 results in haploids containing the gene deletion and FPR. Quantification of the resultant haploids by high-throughput flow cytometry records fluorescent signals in up to 50,000 cells for each gene deletion mutant.

(D, E) Scatter plot visualization of YFP vs mCherry fluorescence signals in the GCG1 and ORC2 promoter FPR screens. Each data point represents the median signal of a deletion mutant normalized to individual plate median. Highlighted data points represent the mutants of Hda1C (purple), SAGA acetylation module (blue), SAGA deubiquitination module (orange), and cac2∆ (yellow). The regression line is marked in black. The mutants favoring DNC transcription are found above the regression line. The mutants favoring coding transcription are below the regression line. Technical repeat = 1, biological observations = 50,000 individual cells before filtering.
The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency (2021)
Figure 3

Figure 3. NET-seq identifies the genome-wide effect of Hda1C on nascent divergent non-coding (DNC) transcription.

(A) Metagene analysis of NET-seq signal at all DNC loci (n=1517). The genomic intervals were centered at the transcription start site (TSS) of either protein-coding gene (left panel) or DNC transcript (right panel).

(B) NET-seq data at the YPL172C locus. The signal represents NET-seq reads showing nascent transcription at the genomic position of the divergent non-coding strand (black) and the coding strand (grey). The bar graph depicts NET-seq reads as FPKM values for the coding and DNC transcript in the strains.

(C) NET-seq data at the YDR261C locus with a novel DNC transcript. Annotations as in (B).

Data information: (B, C) Error bars represent SEM. Statistical significance of mutant transcript expression as compared to WT was assessed by unpaired two-tailed student's t-test for two biological NET-seq replicates. The * and n.s indicate p-value <0.05 and non-significance, respectively.
The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency (2021)
Figure 4

Figure 4. Single-molecule live-cell imaging reveals hda3∆ altering SUT098 transcription frequency.

(A) Schematic representation of MS2/PP7 stem-loop repeats at the 5'UTR of GCG1/SUT098 sequence, respectively. Upon transcription, respective coat proteins (dark blue and dark purple) with fluorescent proteins (red and green) bind to the MS2/PP7 repeats at the 5' UTR of GCG1 and SUT098 transcript. The ON and OFF state of transcription versus the time determines the duration and frequency (time between transcription events) of transcription.

(B) An example trace of SUT098 transcript fluorescence quantified at the transcription site in a single cell (wild type). The track shows an overlay of the raw trace (green) with the binarized trace (black). The binarized peaks represent transcription initiation.

(C) A representative image of transcription initiation observed as fluorescence spots in the recorded live-cell movie of wild type strain. The movie output using Python were analyzed and the snapshot was obtained using Image J software. The red and green channel denotes GCG1 and SUT098 transcript initiation, respectively. Images represent maximum intensity projections, and the right and bottom sidebars indicate sideviews in the yz and xz directions, respectively. Scale: 5 µm.

(D, E) Quantification of transcription dynamics by the duration and frequency (time between initiation) of transcription. Bar graphs show the transcription parameters of SUT098 in candidate mutants compared to the wild type strain. The error bars represent SEM calculated by live-cell image analysis of 179, 135, 60 and 129 single-cell biological repeats for WT, hda1∆, hda3∆ and cac2∆ strains, respectively. Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python (the asterisk * denotes p-value <0.05).
The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency (2021)
Figure 5

Figure 5. Hda1 affects the H3 acetylation levels at divergent non-coding (DNC) region independently from H3K56ac.

(A) Flow cytometry data analysis. Bars represent the mCherry/YFP fluorescence ratio of the respective yeast mutants normalized to the signal values of the H3 wild type FPR strain. The error bars show SEM calculated from biological triplicates with three technical repeats. Unpaired two-tailed student's t-test analysis indicates the statistical significance of the mutant compared to the respective wild type. The asterisks *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively.

(B) Metagene profile of ChIP data (Data ref: Ha et al, 2019). The genomic intervals cover the first 500 bp of the coding gene (scaled to 100 bins), the first 1 kb of the DNC transcript (scaled to 200 bins), and the variable length gap between the coding TSS and the DNC TSS (scaled to 50 bins). The vertical lines represent transcription start site (TSS) of protein-coding gene (left) and the DNC transcript (0 bin). The H3ac ChIP-seq signal was normalized by the H3 signal in the same samples.

(C) Working model of Hda1C repressing DNC. The coding (blue) and divergent (purple) transcript arise in opposing directions of a shared nucleosome-free region (black). Nucleosomes (grey) comprise histones capable of modification by histone deacetylase 1 complex (Hda1C). In the normal state, Hda1C limits the frequency of DNC by deacetylating histones. With the loss of Hda1C function, increased acetylation of H3 favors DNC.

EV FIGURE LEGENDS
Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria (2021)
Figure 1

Figure 1. Membrane fatty acid composition-dependent growth of B. subtilis and E. coli.

A Growth of B. subtilis WT and fatty acid precursor-auxotrophic Δbkd cells in medium supplemented with precursor 2-methyl butyric acid (MB), isobutyric acid (IB) or grown precursor-free (PF).

B Fatty acid composition of B. subtilis WT cells grown in PF medium, and Δbkd grown with MB, IB or depleted for precursor for 90 min (IBPF). For detailed analyses, see Appendix Fig S1B.

C Temperature-dependent growth of B. subtilis Δbkd on solid medium in serial 10-fold dilutions. For comparison between WT, Δdes and Δbkd Δdes cells, see Appendix Fig S1C.

D Temperature-dependent growth behaviour of E. coli WT and fabA(Ts), including a shift from 30°C to 37°C as non-permissive temperature of fabA(Ts).

E Fatty acid composition of E. coli WT and fabA(Ts) cells grown at 30°C and shifted to different temperatures for 120 min. For detailed analyses, see Appendix Fig S2B and C.

Data information: (A) The diagram depicts mean and standard deviation (SD) of technical triplicates for each strain. (B, E) The histograms depict mean and SD of biological triplicates. (C, D) The experiments are representative of three independent repeats. CFA, cyclopropane fatty acid. Strains used: (A-C) B. subtilis 168, HS527; (D, E) E. coli MG1, MG4 (strains Y-Mel and UC1098, respectively, additionally encoding fluorescent ATP synthase (FOF1 a-mNG)).
Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria (2021)
Figure 2

Figure 2. Reduced membrane fluidity in cells depleted for UFAs or BCFAs.

A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity.

B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated.

Data information: (A, B) The experiments are representative of three independent repeats. The histograms depict means and SD of technical triplicates, together with P values of an unpaired, two-sided t-test. Significance was assumed with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, n.s., not significant. Strains used: (A) B. subtilis 168, HS527; (B) E. coli Y-Mel, UC1098.
Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria (2021)
Figure 3

Figure 3. Consequences of low membrane fluidity on membrane diffusion barrier function.

A Images of B. subtilis WT and Δbkd cells co-labelled with the membrane potential-sensitive dye DISC3(5) and the membrane permeability indicator Sytox Green. Membrane properties were assessed for Δbkd cells grown in the presence of MB, IB or washed precursor-free (IBPF) for the times indicated. As controls, WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC (gABC) or pore-forming lantibiotic Nisin. For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3A-C.

B Quantification of DISC3(5) fluorescence for cells (n=100-142) depicted in panel A. Median represented by red line.

C Images of E. coli WT and fabA(Ts) cells co-labelled with the same indicator dyes as in panel A. Membrane properties were assessed for fabA(Ts) at 30°C and upon transfer to non-permissive 37°C for the times indicated. As controls, WT cells were incubated with the pore-forming antibiotic Polymyxin B (PolyB). For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3D and E. The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background (see Fig EV1).

D Quantification of DISC3(5) fluorescence for cells (n=76-141) depicted in panel C. Median represented by red line.

Data information: (A-D) The experiments are representative of three independent repeats. (B, D) Red lines indicate the median. P values represent the results of unpaired, two-sided t-tests. Significance was assumed with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, n.s., not significant. (A, C) Scale bar, 3 µm. Strains used: (A, B) B. subtilis 168, HS527; (C, D) E. coli Y-Mel, UC1098.
Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria (2021)
Figure 4

Figure 4. Consequences of low membrane fluidity on and cell morphogenesis.

A Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A.

B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B.

Data information: (A, B) The experiments are representative of biological triplicates. Scale bar, 3 µm. Strains used: (A) B. subtilis HS541, HS548, HS549; (B) E. coli UC1098, BHH500, BHH501.