Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Overlap between ZNF143/ZFP143 peaks from re-analysed publicly available data and CTCF peaks from CISTROME for human (left panel) and mouse (right panel) datasets. Box plots for each ZNF143/ZFP143 dataset represent the median overlap with CTCF peaks. Each dot represents the overlap between the indicated ZNF143/ZFP143 peak set with an individual CTCF peak set. Colours represent the antibody used for chromatin immunoprecipitation, as indicated below. (B) Venn diagram showing the overlap between ZNF143 peaks detected by Proteintech (light pink) and FLAG (light green) antibodies in K562 cells. (C) Heatmap showing the enrichment of SBS (i.e. ZNF143) and CTCF motifs in common, Proteintech-specific, and FLAG-specific peaks in K562 cells. (D) Tornado plots of ChIP-seq signals detected by Proteintech (light pink), FLAG (light green), and custom (orange) antibodies, and CTCF signal (blue) in K562 cells. The ChIP-seq signals are centred on common (top) and Proteintech-specific (bottom) peaks. (E) Genomic tracks showing ChIP-seq signals for CTCF (blue) and signals detected by Proteintech (pink), FLAG (light green), and custom12 (orange) antibodies in K562 cells. Rectangles indicate common (left) and Proteintech-specific (middle and right) peaks in the region. (F) Scatter plot of the percentage of loop anchors overlapping the peak (x-axis) against the fold enrichment of peaks in loop anchors (y-axis) for a number of DNA binding proteins and for Proteintech-specific, FLAG-specific and common peaks in K562 cells.

Article Snippet: As loop annotation and peak sets were for the hg19 human reference genome assembly, the coordinates of the common, Proteintech-specific and FLAG-specific ZNF143 peaks were lifted over from the hg38 to the hg19 assembly using liftOver.

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing, DNA Binding Assay

Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Tornado plots of CTCF ChIP-seq signal from two biological replicates in wild-type (WT) and ZNF143-knockout (KO) haematopoietic stem and progenitor cells (HSPC) centred at ZNF143-related (top) and ZNF143-unrelated (bottom) CTCF peaks . (B) Same as in (A) but for the CTCF ChIP-seq signal in HSPC from two orthogonal studies , . (C) Rolling mean of the normalised CTCF motifs scores, annotated for the ZNF143-related (top) and ZNF143-unrelated (bottom) CTCF peaks. (D) Violin plots showing the fraction of ZNF143-related (left) and ZNF143-unrelated (right) CTCF peaks overlapping CTCF peaks from the CISTROME database . (E) GC bias scores calculated for CTCF ChIP-seq data generated from WT and ZNF143-KO HSPC samples . Note the divergence of the first WT CTCF replicate from the rest of the samples. (F) Genomic tracks showing CTCF ChIP-seq signal from two biological replicates in WT and ZNF143-KO HSPC , CTCF ChIP-seq signal from two other HSPC samples , , and GC content. Horizontal bars indicate ZNF143-related and ZNF143-unrelated CTCF peaks . Note the overlap of ZNF143-related peaks with GC-rich regions. (G) Tornado plots of ZNF143 ChIP-nexus signal from control and CTCF-depleted HEC1B cells centred at ZNF143-only (top) and shared ZNF143 and CTCF (bottom) peaks . (H) Genomic tracks showing ZNF143 ChIP-nexus signal from control and CTCF-depleted HEC1B cells . Horizontal bars indicate ZNF143-only and shared ZNF143 and CTCF peaks. Note the specific loss of signal at shared peaks upon CTCF depletion. (I) Venn diagram showing the overlap between ZNF143-CTCF motif pairs located 37 bp apart from each other and SINE/B2 repeat elements in the mouse genome from RepeatMasker. (J) Tornado plots of CTCF and ZNF143 ChIP-seq signal centred at ZNF143-CTCF motif pairs located 37 bp apart from each other .

Article Snippet: As loop annotation and peak sets were for the hg19 human reference genome assembly, the coordinates of the common, Proteintech-specific and FLAG-specific ZNF143 peaks were lifted over from the hg38 to the hg19 assembly using liftOver.

Techniques: ChIP-sequencing, Knock-Out, Generated, Control

Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Gene ontology (GO) terms, overrepresented in ZFP143-bound genes, identified based on ZFP143-HA ChIP-seq data in ZFP143-FKBP cells. Coloured areas represent gene sets involved in various cellular functions. (B) Bar plots showing the number of ZNF143/ZFP143 peaks overlapping between datasets (top panels), the fraction of ZNF143/ZFP143 peaks with SBS motifs present (middle panels), and the number of cell types sharing ZNF143/ZFP143 peaks (bottom panels) in the re-analysed publicly available human and mouse ChIP-seq datasets. (C) Venn diagram showing the overlap between conserved ZNF143-bound genes in human, conserved ZFP143-bound genes in mouse, and ZFP143-bound genes identified in ZFP143-FKBP cells. (D) Venn diagram showing the overlap between GO terms significantly overrepresented in conserved ZNF143-bound genes in human, conserved ZFP143-bound genes in mouse, and ZFP143-bound genes identified in ZFP143-FKBP cells. (E) GO terms, overrepresented in conserved ZNF143-bound genes in human (left panel) and conserved ZFP143-bound genes in mouse (right panel).

Article Snippet: As loop annotation and peak sets were for the hg19 human reference genome assembly, the coordinates of the common, Proteintech-specific and FLAG-specific ZNF143 peaks were lifted over from the hg38 to the hg19 assembly using liftOver.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation