EMBO/soda-nel huggingface dataset:
Coenzyme A corrects pathological defects in human neurons of PANK2 associated neurodegeneration (2016)
Figure 4

Figure 4. PKAN human neurons show impaired iron dependent mitochondrial biosynthesis.

A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry. B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry. C Heme quantification by absorbance at 400 nm of the soluble cell lysates. D Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).
Coenzyme A corrects pathological defects in human neurons of PANK2 associated neurodegeneration (2016)
Figure 5

Figure 5. CoA treatment recovers PKAN human neurons abnormal phenotype and functionality.

A Representative images of co-cultures of control and PKAN NPCs infected with GFP-LV and tdT-LV expressing vectors, respectively, at the beginning (Day 1), after 120 (Day 120) and 150 days of differentiation (Day 150). Scale bar 20 μm. Plots show the number of green and red human neurons counted at different time points. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA). B Example of electrophysiological properties of cultured PKAN human neurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test). C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN human neurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA). D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA). E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 1

Figure 1. The CENP-A epigenetic self-assembly mechanism is not required for de novo CENP-A deposition at native human centromere.

(A) Schematic illustration of the two-step CENP-AOFF/ON assay using the Auxin (IAA) inducible degradation system.

(B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells.

(C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm.

(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.

(E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005.

(F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images. Scale bar, 10 µm.

(G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation. Unpaired t test, ns.
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 2

Figure 2. Previously deposited CENP-A is dispensable for precise de novo CENP-A incorporation.

(A) Schematic of DNA sequence organization at centromeres with more than one higher order repeat array (HOR) and experimental set-up of the CENP-AOFF/ON cycle performed in experiments B-D.

(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.

(C) Venn diagram showing the number of CUT&RUN-seq peaks that are NT specific (left, blue), WO specific (right, red) or overlapping (center).

(D): Venn diagram showing the total length in Mb of CUT&RUN-seq peaks that are NT specific (left, blue), WO specific (right, red) or shared between NT and WO (center).

(E) Illustration of genomic make-up of DLD-1 with an exogenous CENP-AmRFP-DHFR overexpression system.

(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.

(G) Schematic illustration for the experiments shown in H, I.

(H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm.

(I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 3

Figure 3. De novo CENP-A deposition is impaired in the absence of CENP-B.

(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C.

(B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm.

(C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments. Unpaired t test, ***p=0.0003.

(D) Schematic representation of experiments shown in E, F.

(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm.

(F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 4

Figure 4. Neocentromere formation arises at the CENP-B negative Y-chromosome.

(A) Concept of experiments shown in B-D.

(B) Timeline of experiment performed in C, D.

(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.

(D) Quantification of CENP-C (as read-out of CENP-A) presence at the X (green) or Y (magenta) centromere in the indicated cell lines. Each dot represents one experiment. Error bars represent SD from 3 independent experiments. Unpaired t test, **p=0.0042.

(E) Schematic illustration of CRISPR/Cas9-mediated genome editing of the non-essential RPS4Y gene on the Y chromosome with the insertion of a neomycin resistance cassette.

(F) Experimental set-up for G, H.

(G) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm.

(H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039.
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 5

Figure 5. Ectopic CENP-B is capable to recruit CENP-C independently of CENP-A at LacO arrays.

(A) Experimental schemes for B-G. LacI-CENP-B or LacI control (baits) were expressed into CENP-AEA/- lacO-tetO U-2OS cells. Immunostaining against CENP-C or CENP-A (preys) was performed following LacI-CENP-B transfection in untreated, IAA treated and IAA wash-out (WO) conditions.

(B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets. Scale bar, 5 µm.

(C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t test, ****p<0.0001.

(D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments. Mann-Whitney test was performed on pooled single cell data of three independent experiments, ****p<0.0001.

(E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset. Scale bar, 5 µm.

(F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments. Unpaired t test, ***p=0.0001.

(G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 6

Figure 6. CENP-B drives de novo CENP-A and CENP-C assembly at endogenous centromeres.

(A) Schematic representation of experiments in B-E.

(B) Time line of experiment in C-E.

(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells. Scale bar, 5 µm.

(D) Bar plot showing the percentage of cells with CENP-A and CENP-C co-localization after a CENP-A/COFF/ON cycle in the indicated conditions. Error bars show SD from 5 independent experiments. Unpaired t test, ***p=0.0002. Each dot represents one experiment (>30 cells analyzed for experiment).

(E) Box plot of CUT&RUN qPCR for CENP-A (green) or CENP-C (orange). Box plot shows median, 25th and 75th percentiles, whiskers show minimum and maximum values. Results from 3 independent experiments including combined data from qPCR reactions using alpha-satellite primers and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Mann-Whitney test, CENP-A: **p= 0.0087, *pp= 0.0411, CENP-C: **p= 0.0022.
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 7

Figure 7. CENP-B is capable to initiate the CENP-A-mediated epigenetic loop via de novo recruitment of CENP-C at the native centromere.

(A) Schematic to illustrate the goal of this experiment.

(B) Schematic illustration of the experiments performed in C-H.

(C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Scale bar; 5 µm. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A.

(D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments. Each dot represents one independent experiment with at least 30 cells per condition. Unpaired t test, ****p<0.0001.

(E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Error bars represent SEM from 3 independent experiments. Each dot represents one independent experiment. Unpaired t test, **p=0.0048.

(F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. Scale bar, 5 µm.

(G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition. Error bars represent SEM from 3 independent experiments.

(H) Bar plot of CUT&RUN qPCR results using CENP-C antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Each dot represents one independent experiment. Error bars represent SEM from 3 independent experiments. Unpaired t test, ***p=0.0003.
A genetic memory initiates the epigenetic loop necessary to preserve centromere position (2020)
Figure 8

Figure 8. Centromere identity is maintained by CENP-B/C in resting CENP-A-deprived CD4+ T cells.

(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots).

(B) Representative images of sorted CD4+ T cells with high or low CENP-A level. The nucleus is contoured by a white dashed line. Scale bar, 5 µm.

(C) Schematic representation of the analysis of CENP-A expression over time in CD4+ T cells, shown in D-G. T cell were activated with anti-CD3/anti-CD28-coated beads.

(D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low).

(E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted.

(F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01.

(G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors. Unpaired T test: **p=0.0064.

(H) Model of the role of CENP-B in the maintenance of centromere identity. I-III: See text for details. In III: (top) representative image of a pseudo-dicentric chromosome in PD-NC4 cells with an active (red) and inactive (green) centromere bound by CENP-C and CENP-B, respectively; (bottom) example of a rare centromere re-activation in which the inactive (red) centromere gains CENP-C binding capacity. White dashed lines contour representative chromosomes. Schematic to the right illustrate the observed CENP-C and CENP-B localization patterns on a chromosome.

Expanded View Figure Legends
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 1

Figure 1. Cnot6l expression pattern and phenotype of Cnot6l knockout mice.

A, B: Quantitative RT-PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes (GV and MII), somatic tissues, ES cells (A) and preimplantation embryos (B). n = 3 biological replicates.

C: Cumulative numbers of pups per female showing fertility of WT and Cnot6l-/- female mice. n = 5 females for each genotype.

D: Representative images results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 100 μm. Arrows indicate polar body-1 (PB1).

E: Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 20 μm. Arrows indicate spindle poles.

F: Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l-/- female mice. The numbers of analyzed oocytes were indicated (n).

G: Quantification of preimplantation embryos derived from WT and Cnot6l-/- females that develop to the indicated stages after hCG administration and mated with adult WT males. The numbers of analyzed embryos are indicated (n).

H: Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l-/- female at indicated time points after hCG administration. Scale bar, 100 μm.

Data information: Error bars, s.e.m. **P < 0.01; ***P < 0.001 by two-tailed Student's t test. n.s.: non-significant.
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 2

Figure 2. Effect of Cnot6l and Btg4 knockout on oocyte meiotic maturation.

A: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n).

B: Representative images of WT, Cnot6l-/- and Btg4-/- oocytes showing PB1 emission at 16 h after culture. Arrows indicate PB1. Scale bar, 100 μm.

C: Representative images of chromosome spreads made from WT and Cnot6l-/- oocytes after 16 h of in vitro maturation culture. Immunofluorescent staining of topoisomerase II (TOP2) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres, respectively. Numbers of paired sister chromatids are indicated. Scale bar, 5 μm.

D: Percentage (%) of aneuploidy among in vitro cultured WT and Cnot6l-/- oocytes that have released PB1s. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student's t test. n.s.: non-significant. The numbers of analyzed oocytes were indicated (n).

E: Rates of GVBD, PB1 emission, and normal spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes cultured in vitro. Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t test. n.s.: non-significant. The numbers of analyzed oocytes were indicated (n).

F: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μm.

G: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm.
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 3

Figure 3. Meiotic cell cycle of Cnot6l-/- oocytes is arrested at metaphase I.

A: Live cell imaging results showing in vitro meiotic division of WT and Cnot6l-/- oocytes. Abbreviations: PM, prometaphase; MI, metaphase I; AI, anaphase I; TI, anaphase I-metaphase II transition; MII, metaphase II; PB1: polar body-1. Hours after released from GV arrest were indicated. Scale bar, 20 μm.

B: Data represent the mean and standard deviations of mCherry-Securin fluorescence intensity in WT and Cnot6l-/- oocytes at each time point. Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest. The numbers of analyzed oocytes were indicated (n).

C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μm.

D: BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l-/- oocytes at 9.5 h after culture. More than 8 oocytes were observed for each genotype with similar results. Scale bar, 5 μm.

E: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n).