EMBO/soda-nel huggingface dataset:
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 6

Figure 6. Replication Contributes to sNASP and UBR7-Bound Post-Nucleosomal Histones.

A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide).

B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 7

Figure 7. UBR7 Opposes Stabilization of NASP-Bound Post-Nucleosomal Histone H3

Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.

Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.

Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.

Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 8

Figure 8. Inhibition of Degradation Does Not Cause Accumulation of UBR7 or sNASP Bound Post-Nucleosomal Histones

Changes in LAMP2A expression. RT-qPCR was performed to detect actin and LAMP2A mRNA following treatement with siCTRL or siLAMP2A in sNASP-FL and UBR7-FL 293T cells. Grey dotted line represents expression level in siCTRL treatment. Error bar represents mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiment was performed twice.

Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.

C,D. Immunoblot for total and modified histones following anti-FLAG IP from the

soluble fraction of parental 293T cells, or sNASP-FL (C) or UBR7-FL (D) 293T cells treated with siCTRL, siLAMP2A, or MG-132.

Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 9

Figure 9. Chromatin Accessibility Is Altered and H3K4me3 Is Decreased at TSS Bound By GFP-UBR7 in UBR7KO 293T Cells

Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that­­­­­ show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels.

Representative genome track examples of clusters 1-4 as defined in (A). Antibody 1 H3K4me3 data are shown.

C, D. Metaplots (left) of mean values per bin centered on all TSS, TSS not bound by GFP-UBR7, and GFP-UBR7 bound TSS. Dot plots (right) represent H3K4me3 (Antibody 1) from parental 293T cells (C) or loss of H3K4me3 in UBR7KO versus parental cells (D). Box limits represent 25th and 75th percentiles. Whiskers extend to 1.5x the interquartile range. Outliers are represented as black dots. A Kruskal-Wallis multiple comparisons test with Dunn post-hoc test was used to calculate Benjamini-Hochberg adjusted p-values (italics). Values p<0.05 are pictured. Experiment was performed with two independent anti-H3K4me3 antibodies (see also data for Antibody 2 in Fig EV6E).
GemC1 controls multiciliogenesis in the airway epithelium (2016)
Figure 1

Figure 1 - GemC1 is specifically expressed in developing multiciliated cells.

A. In situ hybridization for GemC1 and McIdas mRNA was performed on sagittal sections of E10.5 dpc mouse brain. GemC1 and McIdas mRNA is specifically detected in the developing choroid plexuses of the ventricles. Lower panels show higher magnifications. Scale bars, 100 µm.

B. In situ hybridization on sagittal (upper) and coronal (middle and lower) sections of the airways of E15.5 dpc mouse embryos, depicting specific GemC1 and McIdas mRNA expression in the respiratory epithelium of the nasal and oral cavity and in the upper bronchial tree in the mouse lungs. Inserts show higher magnification images. Scale bars, 200 µm.

C. MTECs were cultured ex-vivo and transitioned to ALI culture condition. Quantitative real time PCR was used to measure the expression levels of GemC1, McIdas, FoxJ1 and Geminin mRNA levels at different time points during multiciliogenesis. Values indicate expression levels (+/- standard error, s.e.) relative to ALI DAY 1 (marked by *), following normalization against the house-keeping HPRT mRNA. qPCR data were analyzed by the REST-MCS beta software from at least three independent experiments.

Abbreviations: lv: lateral ventricles, 3v: third ventricle, 4v: forth ventricle, cpe: choroid plexus epithelium, ch: cortical hem, re: respiratory epithelium, of: olfactory epithelium, uj: upper jaw, lj: lower jaw, tg: tongue, br: bronchiole.
GemC1 controls multiciliogenesis in the airway epithelium (2016)
Figure 2

Figure 2 - GemC1 induces multiciliate cell differentiation of airway epithelial cells ex vivo.

A. and B. MTEC cultures were infected with a lentivirus expressing either GFP-GemC1 (GemC1) or GFP as a control and two days later transitioned to ALI culture conditions. Cells harvested at different time points during differentiation were co-stained with antibodies against GFP (green) to mark infected cells and endogenous McIdas (A) or Foxj1 (B) (red). Arrows indicate infected cells that are McIdas or Foxj1 positive. Scale bars, 10 µm.

C. and D. The percentage of infected cells expressing McIdas (C) or Foxj1 (D) at each time point was quantified. Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition. Error bars indicate ± SEM. *p<0.05, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test.
GemC1 controls multiciliogenesis in the airway epithelium (2016)
Figure 3

Figure 3 - GemC1 directly activates the upstream regulatory sequences of MCIDAS and FOXJ1.

MCIDAS (A) and FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. In D, McIdas siRNAs or a control siRNA were also co-transfected. McIdas mRNA levels after McIdas RNAi were assessed by q PCR (C). All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.
GemC1 controls multiciliogenesis in the airway epithelium (2016)
Figure 4

Figure 4 - GemC1 cooperates with E2F5 and associates with MCIDAS and FOXJ1 promoter regions

A. and B. MCIDAS (A) and FOXJ1 (B) regulatory elements cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas, Geminin and E2F5, as indicated, or an empty vector (-) as a control. Luciferase values were normalized against co-transfected Renilla luciferase and depicted as fold induction over the control empty vector. Data shown are mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.

C. and D. Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1. HEK293T cells were co-transfected with vectors expressing GemC1-GFP (GemC1) or GFP as a control, E2F5 and DP1, together with the regulatory elements of either MCIDAS (C) or FOXJ1 (D). Following chromatin immunoprecipitation, DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR. Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti-GFP immunoprecipitates over control IgG immunoprecipitates. At least two independent experiments were performed.

E. Schematic representation of the promoter fragments from the MCIDAS (E) and FOXJ1 (F) regulatory elements assayed by chromatin immunoprecipitation. For FOXJ1, two putative transcription start sites (TSS) have been proposed and are indicated (see materials and methods).
GemC1 controls multiciliogenesis in the airway epithelium (2016)
Figure 5

Figure 5 - GemC1-deficient mice die within the first postnatal week.

A. Schematic representation of the wild type (WT) and knock-out (KO) GemC1 alleles. A lacZ-neo cassette has been inserted between exons 2 and 3 of the GemC1 gene. A splicing acceptor site (SA) upstream of lacZ ablates gene function (knock-out first allele, [38])

B. GemC1KO/KO mice compared to their wild type littermates (GemC1KO/WT) at P5.

C. Survival plot of mice homozygous for the GemC1 deletion (GemC1KO/KO, n=8) and control wild type or heterozygous littermates GemC1 (GemC1WT, n=14). GemC1 knock-out mice exhibit markedly reduced growth after birth and die within the first postnatal week.
GemC1 controls multiciliogenesis in the airway epithelium (2016)
Figure 6

Figure 6 - Multiciliate cell differentiation is blocked in the absence of GemC1.

A., B. Transverse sections from trachea isolated from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice, immunostained with antibodies against acetylated α-tubulin (ACT, cilia marker, red), pericentrin (PCNT, marker for nascent centrioles, green), γ tubulin (centriole marker, green) and E-cadherin (marker for cell boundaries, blue). Serial images have been merged to show the complete trachea section (left). Inserts show higher magnification images for acetylated α-tubulin and pericentrin staining in GemC1WT/WT and GemC1KO/KO trachea, respectively. Scale bars, 10 µm.

C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.

D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated α-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.

E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.
Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells (2016)
Figure 1

Figure 1. Cellular distribution of Rab22a and its recruitment to BMDC endosomes and phagosomes

A. Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562) and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 µm.

B. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 µm.

C. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3 µm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 µm.

In A, B, and C, the nuclear marker DAPI (blue) and DIC images are shown in the left panels. Overlays are shown in the right panels. Data are representative of three independent experiments.

D. BMDCs were incubated with 3 μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments.
Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells (2016)
Figure 2

Figure 2. Rab22a controls the intracellular trafficking of MHC-I molecules in DCs

A. Immunoblotting and densitometry quantification of Rab22a in JAWS-II DCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments.

B. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A. Data show mean ± SEM of triplicate values and are representative of two independent experiments. ** P = 0.0047.

C. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 µm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments.

D. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003.

E. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types.

F. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min.

G. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20 and 40 min.

In B and D, the two-tailed Student’s unpaired t test was performed. In E, F and G, a two-way ANOVA and the Bonferroni post-test were performed.
Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells (2016)
Figure 3

Figure 3. Rab22a controls cross-presentation by DCs without affecting antigen internalization, phagosomal degradation or endogenous MHC-I presentation

A-C. The cross-presentation ability after incubation with A) soluble OVA, B) OVA/BSA-coated beads and C) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS-II DCs was evaluated with the B3Z hybridoma. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. A) ***P = 0.0001 and B) P = 0.1432 (ns); **P = 0.0044. The two-tailed Student’s unpaired t test was performed.

D-E. Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS-II DCs. D) The endocytosis of fluorescent OVA after 1 h of internalization and E) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis. The antigen internalization was conducted at 37°C for effective uptake and at 4°C as negative control. Data represent mean ± SEM of triplicate values and are representative of three independent experiments.

F. The kinetics of OVA degradation, as percentage of proteases inhibitors, in isolated phagosomes at the indicated time periods post-internalization from Scramble and Rab22a KD JAWS-II DCs was assessed by FACS analysis. Data represent mean ± SEM of three independent experiments.

G. Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence (Scramble) and two shRNA specific for silencing Rab22a (Rab22a KD #1 and #2).

H-I. The cross-presentation capacity after the incubation with H) soluble and I) the SIINFEKL control peptide at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student’s unpaired t test was performed.

J. Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS-II DCs and T cell activation was determined 2 h later with the B3Z hibridoma. To control endogenous MHC-I antigen presentation specificity, DCs were also treated with befreldin A (BFA). The use of this drug markedly reduced CD8+ T cell response to similar levels obtained by DCs without any antigen (∅). Data represent mean ± SEM of triplicate values and are representative of three independent experiments.

K. Immunoblotting showing the amount of OVA incorporated by Scramble (a) and Rab22a KD (b) JAWS-II DCs after electroporation and BFA treatment.
Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells (2016)
Figure 4

Figure 4. Rab22a is recruited to the parasitophorous vacuole of Toxoplasma gondii and is necessary for cross-presentation of a parasite-derived soluble antigen

A. Scramble and Rab22a KD JAWS-II DCs were infected with OVA-YFP-expressing T. gondii (TgRH YFP SAG1-OVA) for 8 h and confocal images detecting the parasite (green), endogenous Rab22a (red) and GRA6 (magenta) were taken. Top panels: Scramble cells; bottom panels: Rab22a KD cells. White boxes are shown at higher magnification in the insets. The nuclear marker DAPI (blue) and DIC images are shown in the left panels. Overlays are shown in the right panels. Scale bars: 5 µm. Images are representative of at least 30 analyzed from three independent experiments.

B. The cross-presentation of OVA secreted by T. gondii (TgRH YFP SAG1-OVA) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. ***P < 0.001. A two-way ANOVA and the Bonferroni post-test were performed.

C. The efficiency of infection (8 h) of TgRH YFP SAG1-OVA in Scramble and Rab22a KD JAWS-II DCs was measured by FACS analysis at the indicated MOI. Data represent mean ± SEM of duplicate values and are representative of three independent experiments.

D. Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS-II DCs after 90 min and 7 h of infection with TgRH YFP SAG1-OVA parasite at MOI 2 and 6. Fifty µg of total cell lysates were loaded onto each lane. Data are representative of two independent experiments.
Aging is associated with increased chromatin accessibility and reduced polymerase pausing in liver (2022)
Figure 5

Figure 5: Aging affects the stability of the Pol II pausing complex rather than transcription initiation.

A) Heatmap and intensity profiles of promoter accessibility over active enhancers in liver (n = 8,855). Read densities of merged biological replicates (n=3-4) were normalized to 1x coverage.

B) Raincloud plots of changes in accessibility in aged versus young liver assessed by ATAC-seq. Raincloud plots are hybrid plots. Here, the violin and boxplots visualize the log2-fold change for all accessible genomic sites ("all") or accessible sites overlapping promoters of tNET genes ("promoters") or active enhancers in murine liver ("enhancers"). The dot plot highlights regions significantly changed with age in each category (FDR < 5 %). The numbers in the plot denote significantly up- or down-regulated regions in each category.

C) Violin and boxplots of Pol II density at enhancer regions (n = 8,144) as a means of quantifying eRNA production. p-values were calculated using a two-sided Wilcoxon rank-sum test. Box plots consist of the median (central line), the 25th and 75th percentiles (box) and the highest/lowest value within 1.5 * interquartile range of the box (whiskers).

D) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies.

E) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals.

F) Heatmaps and average intensity profiles of SPT4 ChIP-seq signal at TSSs of tNET genes in young and aged animals.

Data information: n=3-4 biological replicates per age group for tNET-seq, ATAC-seq and CUT&RUN; n=2 for ChIP-seq