Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: TRIP12 overexpression modifies the partitioning of chromatin independently of its catalytic activity (A) Graphical representation of GFP fusion proteins. The yellow star locates the ubiquitin ligase catalytic site. IDR, intrinsically disordered region; ARM, armadillo repeats; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; GFP, green fluorescent protein; RFTD, replication foci targeting domain; BAH, bromo-adjacent homology; and MTase, methyltransferase. Black rectangles represent NLSs. Numbers indicate the length in amino acids of the proteins. (B) Representative images of TRIP12-GFP (left) and GFP-TRIP12 (right) overexpressing HeLa S3 cells visualized by immunofluorescence after transient transfection. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Blue arrows indicate the thickening of perinuclear and perinucleolar regions. Red arrows indicate chromatin condensates. Scale bars represent 2 μm. (C) Determination of DNA granularity relative to GFP expression level in TRIP12-GFP and GFP-TRIP12 expressing HeLa S3 cells. For each cell, the DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (D) Representative images of ΔHECT-GFP, TRIP12(C1959A)-GFP, H2B-GFP, and GFP-DNMT1 expressing HeLa S3 cells by immunofluorescence after transient transfection. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Scale bars represent 2 μm. (E) Determination of DNA granularity relative to GFP expression level in ΔHECT-GFP, TRIP12(C1959A)-GFP, H2B-GFP, and GFP-DNMT1 expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated.

Article Snippet: HeLa S3 , ATCC , CCL-2.

Techniques: Over Expression, Activity Assay, Ubiquitin Proteomics, Immunofluorescence, Transfection, Expressing, Two Tailed Test

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The IDR of TRIP12 is responsible for the formation of chromatin condensates (A) Prediction of TRIP12 3D-structure by AlphaFold model. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange, and yellow represent very high, confident, low, and very low model confidence, respectively. (B) Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial NLSs. IDR, intrinsically disordered region; ARM, armadillo domain; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; and GFP, green fluorescent protein. (C) Representative images of TRIP12-domains fused to GFP expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP, and ΔIDR-GFP constructs allowed for nuclear localization with a faint presence in the cytoplasm. Scale bars represent 2 μm. (D) Determination of DNA granularity relative to GFP expression level in TRIP12-domains fused to GFP expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (E) Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS, and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (F) Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in D, 2F, and C. A Spearman r coefficient test and a two tailed-p value are indicated. (G) Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HeLa S3 cells (left). The cytoplasmic expression of 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity relative to GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in “ ” on more than 40 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated. (H) Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope), and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was assessed from the slope values obtained in G. A Spearman r coefficient test and a two tailed-p value are indicated. (I) Representative image of cellular fractionation of HeLa S3 cells expressing 1-107-GFP, 108-207-GFP, 208-324-GFP, and 325-445-GFP constructs. GFP expression in soluble and chromatin-bound fractions was determined by western blot analysis. The level of HSP90 and PanH3 protein expression were used as loading and enrichment controls. The graph represents the percentage of GFP expression in the different fraction. Results are expressed as mean ± SEM of four different experiments. (J) Electric net charge of [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR at pH 7.4 determined by Prot pi|Protein Tool (left graph). Percentage of basic, acidic, and uncharged residues in [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR determined by Prot pi|Protein Tool (right graph). (K) Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle indicates artificial NLSs. Representative images of MED1-IDR-GFP expressing HeLa S3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is represented in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated.

Article Snippet: HeLa S3 , ATCC , CCL-2.

Techniques: Expressing, Immunofluorescence, Construct, Transfection, Two Tailed Test, Over Expression, Comparison, Cell Fractionation, Western Blot

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The chromatin condensates induced by TRIP12-IDR are enriched in heterochromatin marks (A) Representative images of localization of TRIP12-IDR mediated condensates and HP1α, H3K9me3, EZH2, H3K27me3, and H2AK119Ub heterochromatin marks in HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (B) Representative fluorescence intensity profiles and averaged horizontal fluorescent intensity profiles corresponding to (A). Number of analyzed condensates is indicated. Error bars represent the standard deviation of the mean. (C) Representative images of localization of TRIP12-IDR-mediated condensates and phosphorylated RNA polymerase II on serine 2 and serine 5. White arrows show the localization of RNA polymerase II staining at the periphery of the chromatin condensates. Nuclei were counterstained with DAPI. Scale bars represent 2 μm (top) and 0.5 μm (bottom). (D) Representative fluorescence intensity profiles and averaged horizontal fluorescent intensity profiles corresponding to (C). Number of analyzed condensates is indicated. Error bars represent the standard deviation of the mean.

Article Snippet: HeLa S3 , ATCC , CCL-2.

Techniques: Immunofluorescence, Fluorescence, Standard Deviation, Staining

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The formation of TRIP12 induced-chromatin condensates is a dynamic and reversible mechanism driven by PPPS (A) Visualization of CREST (top) and LAMIN B1 (bottom) and chromatin condensates in TRIP12-IDR-expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (B) Representative images of chromatin condensate formation and GFP expression in IDR-GFP expressing HeLa S3 cells by live cell microscopy. Images were acquired every hour for 24 h after transfection. For clarity, representative image of a cell 8 h after transfection and every 2 h are represented. Nuclei were counterstained with SiR-DNA-647 nm. Scale bars represent 5 μm. (C) Quantification of GFP expression and DNA granularity in IDR-GFP-expressing HeLa S3 cells obtained by live cell microscopy. Images were acquired every hour for 24 h by live cell confocal microscopy. GFP intensity and DNA granularity were determined using Columbus and Fiji software. Results are expressed as mean ± SEM of at least five independent cells. A Spearman r coefficient test and a two tailed-p value are indicated. Error bars represent the standard deviation of the mean. (D) Expression of IDR-GFP, FLAG-tagged anti-GFP degrader in VHL-NbGFP4-FLAG expressing HeLa S3 cells determined by western blot 24 h after increasing doses of doxycycline (0.25, 0.5, and 1 μg/mL). The immunoblots are representative of three independent experiments. HSP90 protein level was used as a loading control. (E) Representative images of VHL-NbGFP4-FLAG expressing in HeLa S3 cells transfected with IDR-GFP construct in the presence or not of doxycycline (1 μg/mL) for 24 h obtained by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 10 μm. (F) Determination of DNA granularity relative to IDR-GFP expression level in VHL-NbGFP4-FLAG expressing in HeLa cells transfected with IDR-GFP construct in the presence of doxycycline (1 μg/mL) for 24 h or vehicle. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated (G) Representative images of DNA condensates and IDR-GFP expression in VHL-NbGFP4-FLAG expressing HeLa S3 cells transfected with IDR-GFP construct every hour (for 13 h) visualized by cell live microscopy after addition of doxycycline (1 μg/mL). DNA is stained with SiR-DNA-647 nm. Scale bars represent 2 μm. (H) Quantification of DNA granularity and GFP intensity in VHL-NbGFP4-FLAG expressing HeLa S3 cells transfected with IDR-GFP construct every hour (for 13 h) visualized by cell live microscopy after addition of doxycycline (1 μg/mL). Results are expressed ±SEM of at least five independent cells. A Spearman r coefficient test and a two tailed-p value are indicated. Error bars represent the standard deviation of the mean. (I) Representative images of DAPI organization in IDR-GFP expressing HeLa S3 cells in the presence of 1,6-hexanediol 0.5% or vehicle for 18 h. Cells with three different GFP intensities are represented. Nuclei were counterstained by DAPI. (J) Determination of DAPI granularity in IDR-GFP expressing HeLa S3 cells in the presence of 1,6-hexanediol 0.5% or vehicle for 18 h as described in “ .” Error bars represent the standard deviation of the mean. (K) IDR-GFP mobility in DNA condensates by FRAP analysis. H2B-dsRed expressing HeLa S3 cells were transfected with IDR-GFP construct or H2B-GFP used as control. After 24 h, GFP fluorescence was photo-bleached using an FRAP module of confocal microscope. The recovery of fluorescence was measured every second for 35 s. Representative images of IDR-GFP and H2B-GFP expressing cells with bleached and non-bleached areas at indicated times are represented (left). The graph represents the mean ± SD of GFP fluorescence intensity obtained from four independent cells. (L) IDR-GFP mobility in DNA condensates by half-bleached FRAP analysis. HeLa S3 cells were transfected with IDR-GFP construct. After 24 h, GFP fluorescence in a half of a condensate was photo-bleached using an FRAP module of confocal microscope. The recovery of GFP fluorescence was measured every 0.5 s for 2 min. Representative images of an IDR-GFP expressing cell with bleached and non-bleached areas before and just after bleaching are represented (left). Recovery curves of the bleached half (green) and non-bleached half (magenta) are shown in the right figure, n = 15. Data are mean ± SEM. Scale bars represent 2 μm.

Article Snippet: HeLa S3 , ATCC , CCL-2.

Techniques: Expressing, Immunofluorescence, Microscopy, Transfection, Confocal Microscopy, Software, Two Tailed Test, Standard Deviation, Western Blot, Control, Construct, Staining, Fluorescence

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: Consequences of TRIP12-IDR mediated-chromatin condensates on cellular growth, chromatin accessibility, and genome expression (A) Cellular fate of IDR-GFP (left) and H2B-GFP (right) transfected HeLa S3 cells. Twenty-four hours after transfection, fluorescence of IDR-GFP transfected cells was determined using Incucyte apparatus to classify cells in three categories: low, moderate, and high. About 11–12 cells per category were monitored for 30 h every 2 h. Mitotic divisions were annotated with a M and nuclear fusion with an NF. Green bars represent living cells whereas black bars represent dead cells. (B) Distribution in the cell cycle of IDR-GFP (left) and H2B-GFP (right) transfected HeLa S3 cells. Twenty-four hours after transfection, the position in the cell cycle was determined by immunofluorescence using CYCLIN A and EdU staining as described in “ .” The graph represents the percentage of cells in the different phases of the cell cycle expressed as mean (±SEM) of at least three independent experiments. (C) Representative image of EdU incorporation in IDR-GFP-mediated chromatin condensates (left). HeLa S3 cells were transfected with IDR-GFP construct. Twenty-four hours after transfection, cells were incubated with EdU (1 μM) for 20 min. Incorporation of EdU was visualized by Click-It followed by confocal microscopy. Nuclei were counterstained with DAPI. The graph represents the intensity of fluorescence along the arrow (right). Scale bars represent 2 μm. (D) Determination of accessible genomic regions in IDR-GFP expressing cells by ATAC-seq experiments. Only merged regions with a Max tags >50 were considered. Results were obtained from two independent experiments and expressed as a percentage ±SEM of accessible genomic regions compared to non-transfected cells (set as 100%). (E) Percentage of accessible regions in IDR-GFP expressing HeLa S3 cells with a lower (down, fold change < 2), higher (up, fold change > 2), and unchanged accessibility compared to non-transfected cells (set as 100%). Results were obtained from two independent experiments. (F) Percentage of down (fold change < 2) and upregulated (fold change > 2) mRNA in IDR-GFP expressing HeLa S3 cells compared to non-transfected cells as determined by RNA-seq analysis. Results were obtained from two independent experiments. (G) Gene set enrichments of downregulated and upregulated genes in IDR-GFP expressing HeLa S3 cells compared to non-transfected cells using GSEA software with a nominal p value < 0.001. NES stands for Normalized Enrichment Score (left).

Article Snippet: HeLa S3 , ATCC , CCL-2.

Techniques: Expressing, Transfection, Fluorescence, Immunofluorescence, Staining, Construct, Incubation, Confocal Microscopy, RNA Sequencing, Software

Journal: bioRxiv

Article Title: Differential nucleosome organization in human interphase and metaphase chromosomes

doi: 10.1101/2025.11.11.687715

Figure Lengend Snippet: ( A ) Chemically cleaved nucleosomal DNA fragments from interphase and mitotic HeLa S3 cells visualized on an agarose gel. ( B ) Crick–Watson cleavage peak-to-peak distance plot showing three dominant distances (–12, –5, and +2 nucleotides), consistent with primary and secondary cleavage sites at –1 and +6, respectively. ( C ) Frequency of AA/TT/AT/TA dinucleotides within nucleosomes and their flanking regions, based on unique nucleosome maps from interphase and metaphase. ( D–F ) Representative genomic loci showing nucleosome occupancy scores from both clone 2 and clone 1-2. Distinct interphase–metaphase differences are observed at a CTCF-binding site on Chromosome 1 (D) and the TSS of AAR2 on Chromosome 20 (E), while similar patterns are found near an exon of C16orf46 on Chromosome 16 (F). Nucleosome organization is consistent across both clones.

Article Snippet: Parental human HeLa S3 cells (ATCC CCL-2.2) were used to generate H4S47C-expressing cell lines for chemical mapping studies.

Techniques: Agarose Gel Electrophoresis, Binding Assay, Clone Assay