Journal: bioRxiv

Article Title: Treacle and MDC1 coordinate rDNA break repair by homologous recombination

doi: 10.1101/2025.04.21.649841

Figure Lengend Snippet: (A) Localisation of BRCA1 and RAD51 after 1h or 2h of I-Ppo1 WT treatment or 2h of I-Ppo1 H98A treatment, respectively, in U2OS cells. (B) Localisation of BRCA1 and RAD51 in siLuc, siTreacle, siTopBP1, and siNBS1 transfected U2OS cells after 2h of I-Ppo1 WT treatment. All scale bars = 10 µ m. (C) Quantification of the experiment in (B) showing the number of BRCA1 and RAD51, respectively, nucleolar caps (n= 100 cells). Graphs show a single representative replicate (out of three), and bars represent mean. (D) Co-immunoprecipitation from 293T cells transfected or non-transfected with HA-tagged Treacle and treated with WT I-Ppo1 for 2h. (E) SIM of Treacle and BRCA1 after 2h of I-Ppo1 WT transfection of Treacle-GFP expressing U2OS cells. Scale bar= 5 µ m.

Article Snippet: MDC1 PST-mNeon U2OS cells were cultured in the presence of 1 µg/ml Puromycin (Invivogen).

Techniques: Transfection, Immunoprecipitation, Expressing

Journal: bioRxiv

Article Title: Treacle and MDC1 coordinate rDNA break repair by homologous recombination

doi: 10.1101/2025.04.21.649841

Figure Lengend Snippet: (A) SIM of H2AX and BRCA1 in U2OS cells after 2h of I-Ppo1 WT transfection. Scale bar = 5 μm. (B) SIM of U2OS cells stably expressing Treacle-GFP and treated with I-Ppo1 WT for 2h, stained with BRCA1 and MDC1 antibodies. A reconstruction of nucleolar caps using 3D surface is shown. Scale bar = 1μm. (C) Localisation of BRCA1 and RAD51 in WT, ΔMDC1, and H2AX S139A RPE1 cells 2h after I-Ppo1 WT transfection. All scale bars = 10 µ m. (D) Quantification of the experiment in (C) showing the number of BRCA1 (upper panel) and RAD51 (lower panel) caps per cell (n= 90 cells). Graph shows a single replicate (experiment was performed in triplicates) and bars represent mean.

Article Snippet: MDC1 PST-mNeon U2OS cells were cultured in the presence of 1 µg/ml Puromycin (Invivogen).

Techniques: Transfection, Stable Transfection, Expressing, Staining

Journal: bioRxiv

Article Title: Treacle and MDC1 coordinate rDNA break repair by homologous recombination

doi: 10.1101/2025.04.21.649841

Figure Lengend Snippet: (A) A scheme of various MDC1 mutations introduced to ΔMDC1 U2OS cell line. (B) Localisation of BRCA1, RAD51, and mNeon/GFP-MDC1 after 2h of I-Ppo1 WT transfection. U2OS ΔMDC1 cells expressing wild type MDC1 (WT-mNeon), S168A/S196A MDC1 (DM-GFP), SDT → ADA MDC1 (12A-GFP), MDC1 lacking the entire PST repeat region (ΔPST-mNeon), and TQXF → AQXF (AQXF-GFP). All scale bars = 10 μm. (C) Quantification of the experiment in (B) showing the number of BRCA1 (left panel) and RAD51 (right panel) nucleolar caps per cell (n= 100 cells). Graph represents a single replicate (experiment was performed in duplicates for RAD51 and in quadruplicate for BRCA1) and bars represent mean.

Article Snippet: MDC1 PST-mNeon U2OS cells were cultured in the presence of 1 µg/ml Puromycin (Invivogen).

Techniques: Transfection, Expressing

Journal: bioRxiv

Article Title: Treacle and MDC1 coordinate rDNA break repair by homologous recombination

doi: 10.1101/2025.04.21.649841

Figure Lengend Snippet: (A) Localisation of BRCA1 and RAD51 in doxycycline inducible RNF8 shRNA U2OS cell line treated with or without doxycycline after 2h of I-Ppo1 WT transfection. All scale bars= 10 μm. (B) Quantification of the experiment in (A) showing the number of BRCA1 (left panel) and RAD51 (right panel) nucleolar caps per cell (n= 100 cells). Graph represents a single replicate (experiment was performed in triplicates) and bars represent mean. (C) Localisation of BRCA1 and RAD51 in doxycycline inducible RNF168 shRNA U2OS cell line treated with or without doxycycline after 2h of I-Ppo1 WT transfection. All scale bars= 10 μm. (D) Quantification of the experiment in (C) showing the number of BRCA1 (left panel) and RAD51 (red panel) nucleolar caps per cell (n= 150 cells). Graph represents a single replicate (experiment was performed in triplicates) and bars represent mean.

Article Snippet: MDC1 PST-mNeon U2OS cells were cultured in the presence of 1 µg/ml Puromycin (Invivogen).

Techniques: shRNA, Transfection

Journal: bioRxiv

Article Title: Treacle and MDC1 coordinate rDNA break repair by homologous recombination

doi: 10.1101/2025.04.21.649841

Figure Lengend Snippet: (A) Localisation of MDC1 and γH2AX in siLuc, siTreacle, siTopBP1, and siNBS1 transfected U2OS cells after 2h of I-Ppo1 WT treatment. All scale bars = 10 µ m. (B) Quantification of the experiment in (A) showing the number of MDC1 (left panel) and H2AX (right panel) nucleolar caps per cell (n = 120 cells). Graphs represent a single replicate (experiment was performed in duplicates) and bars represent mean.

Article Snippet: MDC1 PST-mNeon U2OS cells were cultured in the presence of 1 µg/ml Puromycin (Invivogen).

Techniques: Transfection

Journal: bioRxiv

Article Title: Treacle and MDC1 coordinate rDNA break repair by homologous recombination

doi: 10.1101/2025.04.21.649841

Figure Lengend Snippet: (A) Clonogenic survival assay of RPE1 WT, ΔMDC1 and H2AX S139A cells as well PRE1 ΔTP53 (Parental) and RPE1 ΔTP53 ΔBRCA1 cells after 2h of I-Ppo1 treatment. (B) Clonogenic survival analysis of I-Ppo1 WT treated RPE1 WT, ΔMDC1 and H2AX S139A or PRE1 ΔTP53 (Parental) and RPE1 ΔTP53 ΔBRCA1 cells. Each dot represents an independent replicate, black bars show means. (C) PLA assay showing the localization between EdU and γH2AX after H98A or WT I-Ppo1 transfection in U2OS or U2OS ΔMDC1 cells. All scale bars = 10 μm. (D) Quantification of the experiment in (C) showing the mean intensity of PLA signal within the nucleolar periphery U2OS or U2OS ΔMDC1 cells. Graph represents a pool of three independent experiments (n = 400 cells) and bars represent mean.

Article Snippet: MDC1 PST-mNeon U2OS cells were cultured in the presence of 1 µg/ml Puromycin (Invivogen).

Techniques: Clonogenic Cell Survival Assay, Transfection

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Model of MDC1 recruitment to chromatin and HILO microscopy for single-molecule imaging. (B-C) Fluorescence gels of cell lysates from (B) U2OS and (C) RPE1 expressing HaloTagged MDC1 from its endogenous locus labeled with JFX650 HaloTag-ligand. (D-E) Static fraction of Halo-MDC1 molecules in interphase (I) and mitosis (M) derived from single-particle tracking of MDC1 in (D) U2OS and (E) RPE1 cells (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± SD, two-tailed T-test). (F-G) Diffusion coefficient of (F) mobile and (G) static Halo-MDC1 molecules in interphase (I) and mitosis (M) derived from single-particle tracking of MDC1 in U2OS cells (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D., two-tailed T-test). (H-I) Diffusion coefficient of (H) mobile and (I) static Halo-MDC1 molecules in interphase (I) and mitosis (M) derived from single-particle tracking of MDC1 in RPE1 cells (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D., two-tailed T-test). (J) Domain organization of MDC1, sequence alignment of the thirteen 41 amino acid PST repeats, and consensus sequence surrounding the two conserved TP sites within the PST repeats. (K) Phosphorescence imaging and protein staining of and SDS PAGE gel loaded with samples of the wild type (WT) and non-phosphorylatable (T>A) GST-tagged PST repeat domain treated with CDK1-Cyclin B and ATP γ- 32 P. (L) Images of mitotic U2OS cells with MDC1 knock-out (arrested with nocodazole) transiently expressing HaloTagged wildtype MDC1 (WT) and MDC1 variants with phospo-mimicking (T>D) and non-phosphorylatable (T>A) mutations in the TP sites within the PST repeat region.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Microscopy, Imaging, Fluorescence, Expressing, Labeling, Derivative Assay, Single-particle Tracking, Two Tailed Test, Diffusion-based Assay, Sequencing, Staining, SDS Page, Knock-Out

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A-C) Displacement histograms and two state fit of single particle tracking analysis of Halo-MDC1 expressed from its endogenous locus in (A,B) U2OS cells and (C) RPE1 cells. (D) Fluorescence gel of cell lysates from U2OS expressing Halo-MDC1 from its endogenous locus and U2OS ΔMDC1 cells transiently expressing the indicated HaloTagged MDC1 variants labeled with JF646 HaloTag-ligand.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Single-particle Tracking, Fluorescence, Expressing, Labeling

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Fluorescence gel of cell lysates of U2OS ΔMDC1 cells transiently expressing HaloTagged (JFX650) wildtype MDC1 and truncations of the PST repeats. (B-D) Single particle tracking analysis of HaloTagged wildtype MDC1 and truncations of the PST repeats in interphase U2OS ΔMDC1 cells including the (B) fraction of static particles and the diffusion coefficients of (C) mobile and (D) static MDC1 molecules (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D.). (E-G) Single particle tracking analysis of HaloTagged wildtype MDC1 (WT), MDC1 with a deletion of the PST repeat region (ΔPST) and MDC1 variants with phospo-mimicking (T>D) and non-phosphorylatable (T>A) mutations in the TP sites within the PST repeat region in interphase U2OS ΔMDC1 cells including the (E) fraction of static particles and the diffusion coefficients of (F) mobile and (G) static MDC1 molecules (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D.).

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Fluorescence, Expressing, Single-particle Tracking, Diffusion-based Assay

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A-B) Images of fixed interphase and mitotic U2OS cells expressing Halo-MDC1 (JFX650) immuno-stained with (A) p-PST antibody #1 or (B) p-PST antibody #2 and Hoechst to label DNA. (C) Images of fixed, mitotic U2OS cells expressing Halo-MDC1 and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 (JFX650) variants untreated or treated with zeocin. Cells were immuno-stained with p-PST antibody #1 and Hoechst to label DNA. (D-E) Images of fixed U2OS cells expressing Halo-MDC1 (JFX650) (D) untreated or (E) treated with zeocin to induce DNA breaks in various phases of mitosis and early G1-Phase. Cells were immuno-stained with p-PST antibody #1, a γH2AX antibody, and Hoechst to label DNA.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Expressing, Staining, Stable Transfection

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Fluorescence gel of cell lysates from U2OS expressing Halo-MDC1 from its endogenous locus and U2OS ΔMDC1 cells expressing doxycycline (Dox.) inducible HaloTagged MDC1 variants from an expression cassette integrated using Sleeping Beauty transposase. The HaloTag was labeled with JFX650 HaloTag-ligand. (B-C) Images of fixed U2OS cells expressing Halo-MDC1 (JFX650) (B) untreated or (C) treated with zeocin to induce DNA breaks in various phases of mitosis and early G1-Phase. Cells were immuno-stained with p-PST antibody #2 and a γH2AX antibody and Hoechst to label DNA. (D) Images of fixed U2OS cells expressing Halo-MDC1 (JFX650) treated with zeocin to induce DNA breaks in various phases of mitosis and early G1-Phase. Cells were immuno-stained with antibodies against 53BP1 and γH2AX and Hoechst to label DNA.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Fluorescence, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Fluorescence gels of cell lysates from U2OS expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus labeled with JFX650 HaloTag-ligand. (B) Images of fixed, mitotic U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus untreated or treated with zeocin to introduce DNA breaks. Cells were labeled with JFX650 and Hoechst and immuno-stained with antibodies targeting TOPBP1 and γH2AX. (C) Images of fixed, mitotic U2OS cells expressing Halo-MDC1 and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 (JFX650) variants untreated or treated with zeocin. Cells were immuno-stained an antibody targeting CIP2A and stained with Hoechst to label DNA. (D) Quantification of the accumulation of knock-in HaloTagged MDC1 and MDC ΔPST at laser micro-irraditation induced DNA damage in living mitotic U2OS cells.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Fluorescence, Expressing, Labeling, Introduce, Staining, Stable Transfection, Knock-In

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Genome editing strategy to knock-in a cDNA encoding MDC1 lacking its PST repeat region into the endogenous MDC1 locus. Colored arrows indicate primer positions. (B-C) PCR using genomic DNA from clonal U2OS cell lines subjected to genome editing using the indicated primer pairs. In (B) successful genome editing of all alleles is expected to lead no product amplification due to the large insertion of the cDNA and selection cassette. In (C) successful insertion of the donor sequence is validated using a reverse primer oriented downstream of the right homology arm and a forward primer in the donor. (D) Growth curve of U2OS cells expressing Halo-MDC1 and Halo-MDC1 ΔPST from the endogenous MDC1 locus, and MDC1 knock-out cells (ΔMDC1).

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Knock-In, Amplification, Selection, Sequencing, Expressing, Knock-Out

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Still images from live cell timelapse imaging of U2OS ΔMDC1 stably expressing HaloTagged MDC1 variants labeled with JFX650. (B) Quantification of the time from nuclear envelope breakdown to anaphase onset of U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants. (C) Quantification of the time from nuclear envelope breakdown to anaphase onset, the chromosome segregation phenotype, and Halo-MDC1 expression level of U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants labeled with JFX650. The chromosome segregation phenotype was determined by manual inspection.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Imaging, Stable Transfection, Expressing, Labeling

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Quantification of clonogenic survival assays using the PARP inhibitor olaparib of U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus. (N = 3 biological replicates plated in triplicate, Mean ± S.D., Two-way ANOVA) (B) Images of fixed interphase U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with antibodies targeting RAD51 and γH2AX. (C) Images of fixed interphase U2OS cells expressing Halo-MDC1 from its endogenous locus, MDC1 knock-out cells (ΔMDC1), and U2OS ΔMDC1 stably expressing HaloTagged MDC1 variants untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with an antibody targeting RAD51. (D) Quantification of the number of RAD51 foci of the experiment in (C). An individual replicate is plotted on the left (red line = median, >100 cells per condition) and 3 biological replicates are plotted on the right (Median ± S.D., Two-way ANOVA). (E) Images of fixed interphase U2OS cells expressing Halo-MDC1 from its endogenous locus, MDC1 knock out cells (ΔMDC1), and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with an antibody targeting 53BP1. (F) Quantification of the number of 53BP1 foci of the experiment in (E). An individual replicate is plotted on the left (red line = median, >100 cells per condition) and 3 biological replicates are plotted on the right (Median ± S.D., Two-way ANOVA). (G-H) Quantification of the accumulation of (G) Halo-MDC1 and Halo-MDC1 ΔPST and (H) ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 10-19 cells, Mean ± 95% CI). (H) Quantification of the maximal accumulation of ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 10-19 cells, Mean ± 95% CI). (G) Quantification of HR efficiency using the DR-GFP reporter stably integrated into the AAVS1 locus of U2OS ΔMDC1 cells. I-Sce and MDC1 variants were transiently transfected (N = 3-4 biological replicates, Mean ± S.D., Two-way ANOVA).

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Expressing, Staining, Labeling, Knock-Out, Stable Transfection, Irradiation, Transfection

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A-B) Quantification of the accumulation of (A) Halo-MDC1 or (B) Halo-MDC1 ΔPST labeled with JFX650 HaloTag-ligand and ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 16-19 cells, Mean ± S.E.M.). (C) Accumulation of ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants. Each trace represents an individual cell.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Labeling, Irradiation, Stable Transfection, Expressing