gfp hp1β  (Addgene inc)

Journal: bioRxiv

Article Title: HP1α-driven Phase Separation and Repair Pathway Choice in Response to Heterochromatin Damage

doi: 10.1101/2024.09.16.613371

Figure Lengend Snippet: (A) Representative images showing dynamics of GFP-tagged HP1α and HP1β at different time points of a FRAP experiment with Hoechst sensitization. The magenta circle represents the heterochromatin node that was bleached/damaged with a 405 nm laser. The scale bar is 5μm. (B) FRAP curves showing the dynamics of GFP-tagged HP1α and HP1β in undamaged control (No Hoechst, green) and damaged cells (Hoechst, blue). The curve depicts the mean and standard error of mean (SEM) from three experiments (N=3, n>15 cells, each condition). (C) Representative images from immunofluorescence experiment showing endogenous HP1α (green) and HP1β (blue) staining at the site of damage marked by PARP1(red). The scale bar is 5μm. The yellow line represents the line across which intensity profile in (D) is plotted. (D) The intensity of PARP1, HP1α, and HP1β along the yellow line shown in 1C is plotted. The site of damage has higher intensities of PARP1 and HP1α while the intensity of HP1β is low. (E) The enrichment of HP1α and HP1β at the site of damage quantified from the image in 1C. is shown. The distribution of cells combined from three immunofluorescence experiments is shown (N=3, n>50 cell, each condition). The line depicts the mean. The p-value is calculated using the Kolmogorov-Smirnov test. Enrichment is defined as the ratio of intensity at the site of damage to the intensity in the whole nucleus. (F) Bar graph showing enrichment of HP1α (orange) and PAR (green) in control cells (undashed) and AZD2461 treated cells (dashed) show no significant difference. Means and standard deviations (SD) from two repeats are shown (N=2, n>60 cells, each condition). The p-value is calculated using the student’s t-test. Enrichment is defined as the mean of the ratios of intensity at the site of damage to the intensity in the whole nucleus.

Article Snippet: GFP-tagged HP1α and HP1β were a gift from Tom Misteli (Addgene #17652 and #17651).

Techniques: Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: HP1α-driven Phase Separation and Repair Pathway Choice in Response to Heterochromatin Damage

doi: 10.1101/2024.09.16.613371

Figure Lengend Snippet: (A) Representative images (left) from the immunofluorescence experiment showing 53BP1(red) localization at the site of damage in cells transfected with GFP-tagged HP1α (top) and HP1β (bottom). Localized spot-bleaching was performed within the region marked by the magenta box. The site of damage is marked by γH2AX (blue). The scale bar is 5μm. The merged image shows a zoomed in view of the magenta box. Scale bar for the merge (inset) is 1μm. Quantification of 53BP1 enrichment at the site of damage is shown on the right for HP1α-GFP or HP1β-GFP overexpression. The distribution of cells combined from three immunofluorescence experiments is shown (N=3, n>70 cells, each condition). The line depicts the mean. The p-value is calculated using the Kolmogorov-Smirnov test. Enrichment here is defined as the ratio of intensity at the site of damage to intensity in the ring/annulus around the site of damage (see Supp. Fig 2 for details). Enrichment values < 1 indicate depletion, while enrichment values >1 indicate enrichment. (B-F) Scatter plot showing DDR factor enrichment at the site of damage in control cells and cells transfected with GFP-tagged HP1α or HP1β. The distribution of cells combined from three immunofluorescence experiments is shown (N=3, n>50 cells, each condition). The line depicts the mean. The p-value is calculated using the Kolmogorov-Smirnov test. Enrichment is defined as the ratio of intensity at the site of damage to the intensity in the whole nucleus. Enrichment is shown for (B) NBS1, (C) pRPA2, (D) BRCA1, (E) RAD51, (F) XRCC4 (G) Representative images from immunofluorescence experiment showing BrdU localization (in blue indicated by white arrow) at the site of damage in cells transfected with HP1α (top) and HP1β (bottom). The site of damage is marked by γH2AX (red). The scale bar is 5μm. (H) Scatter plot showing BrdU enrichment at the site of damage in control cells and cells transfected with GFP-tagged HP1α or HP1β. The distribution of cells combined from three immunofluorescence experiments is shown (N=3, n>65 cells, each condition). The line depicts the mean. The p-value is calculated using the Kolmogorov-Smirnov test. Enrichment is defined as the ratio of intensity at the site of damage to the intensity in the whole nucleus.

Article Snippet: GFP-tagged HP1α and HP1β were a gift from Tom Misteli (Addgene #17652 and #17651).

Techniques: Immunofluorescence, Transfection, Over Expression, Control

Journal: bioRxiv

Article Title: HP1α-driven Phase Separation and Repair Pathway Choice in Response to Heterochromatin Damage

doi: 10.1101/2024.09.16.613371

Figure Lengend Snippet: (A) Schematic showing the effect of TBB treatment (75μM for 6 hours) on HP1α and HP1β. (B) Representative images from immunofluorescence experiment showing γH2AX (red) and RPA2 (blue) localization at the site of damage in control cells (top) and TBB-treated cells (bottom). Scale bar is 5μm. (C) Representative images from immunofluorescence experiment showing γH2AX (red) and XRCC4 (blue) localization at the site of damage in control cells (top) and TBB-treated cells (bottom). Scale bar is 5μm. (D) Scatter plot showing pRPA2 enrichment at the site of damage in control cells and TBB-treated cells. The distribution of cells combined from three immunofluorescence experiments is shown (N=3, n>100 cells, each condition). The line depicts the mean. The p-value is calculated using the Kolmogorov-Smirnov test. Enrichment is defined as the ratio of intensity at the site of damage to the intensity in the whole nucleus. (E) Scatter plot showing XRCC4 enrichment at the site of damage in control cells and TBB-treated cells. The distribution of cells combined from three immunofluorescence experiments is shown (N=3, n>100 cells, each condition). The line depicts the mean. The p-value is calculated using the Kolmogorov-Smirnov test. Enrichment is defined as the ratio of intensity at the site of damage to the intensity in the whole nucleus.

Article Snippet: GFP-tagged HP1α and HP1β were a gift from Tom Misteli (Addgene #17652 and #17651).

Techniques: Immunofluorescence, Control

Journal: bioRxiv

Article Title: HP1α-driven Phase Separation and Repair Pathway Choice in Response to Heterochromatin Damage

doi: 10.1101/2024.09.16.613371

Figure Lengend Snippet: (A) A model depicting the spatial segregation of HP1α and HP1β in response to clustered DSBs in heterochromatin. HP1α mediates LLPS and faithful repair via HR without the risk of ectopic recombination. After induction of DSBs, HP1 localisation changes with HP1α (yellow) enrichment at the site of damage and HP1β (green) enrichment around the site of damage (red). HP1α enrichment could potentially seed the formation and/or enhancement of phase separation (blue circle) at the site of damage. This results in differential propensity for HR and NHEJ at and around the site of damage, restricting HR to the core of damaged heterochromatic node where HP1α-mediated LLPS is most prominent.

Article Snippet: GFP-tagged HP1α and HP1β were a gift from Tom Misteli (Addgene #17652 and #17651).

Techniques:

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 1. Nuclear distribution pattern of heterochromatin protein 1 (HP1) isoforms in non-irradiated and γ-irradiated human cervix adenocarcinoma (HeLa) cells. Morphology of (a) HP1α (see GFP-tagged HP1α, green), (b) HP1β (see GFP-tagged HP1β, green), and (c) HP1γ (see GFP-tagged HP1γ, green) was studied by laser scanning confocal microscopy. The HP1-positive foci (green) were analyzed in the vicinity of fibrillarin-positive regions of nucleoli (red fluorescence signals). DAPI staining (blue) was used for visualization of nuclei. Scale bars show 5 µm. (d) Analysis of the number of HP1α-, HP1β-, and HP1γ-positive foci in non-irradiated and γ-irradiated (5 Gy) cells. Cells were fixed for analysis 2 h after γ-irradiation. Fifty cell nuclei per sample were studied. Asterisk in panel (d) shows significantly different result.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Irradiation, Confocal Microscopy, Staining

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 2. ChIP-polymerase chain reaction (ChIP-PCR) analysis of HP1α, HP1β, HP1γ, KAP1 abundance in ribosomal genes. (a) The analysis was performed in non-treated human cervix adenocarcinoma (HeLa) cells, and cells irradiated by 5 Gy of γ-rays. Cells were harvested 2 h after γ-irradiation. The highest density in rDNA encoding 28S rRNA and rDNA promoter region was observed for HP1γ. (b) Quantification of fragment densities, shown in panel (a), was done by ImageJ software. White asterisk shows statistically significant difference, shown by Student’s t-test at p ≤0.05.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Polymerase Chain Reaction, Irradiation, Software

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 3. Characterization of HP1β protein in human cervix adenocarcinoma (HeLa) cells using MS. Glu-C endoproteinase was used for protein digestion. After filtering the LC-MS/MS data in Proteome Discoverer 2.2 (see Methods for details), 75% sequence coverage was obtained (marked in bold type). Identified PTMs, including phosphorylation (P) and acetylation (A) are indicated. The amino acid sequence corresponding to CD and CSD domain is shown in blue or orange, respectively, with hinge region in between.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Liquid Chromatography with Mass Spectroscopy, Sequencing, Phospho-proteomics

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 4. The proportion of signature marks on HP1β protein in non-treated, γ-irradiated, and SAHA treated human cervix adenocarcinoma (HeLa) cells. Box-plots of the relative abundance of particular phosphopeptides showing extremes, interquartile ranges, means and medians (N = 3). Precursor peak of phosphopeptides was quantified in Skyline SW. Data were normalized to the sum of selected non-modified peptides. Following post-translational modifications were analyzed: (a) HP1β-S88ph; (b) HP1β-Y163/S161ph; (c) HP1β-S171ph; (d) HP1β-S174ph; (e) HP1β-S171ph-S174ph. Differences between samples in normalized peptide abundances ≥1.5-fold were considered as significant, and the significance of differences was assessed using Student’s t-tests, setting the significance threshold at P < 0.05 (shown by the asterisk). Cells were harvested 2 h after the treatment; 5Gy of γ-rays or 15 µM SAHA.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Irradiation, Targeted Proteomics

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 5. Representative MS and MS/MS spectra of HP1 G81−E96 peptide carrying phosphorylation at S88 (m/z 596.586). (a) MS extracted ion chromatograms of the 3+ charged HP1β-S88ph peptide precursor showing changes in its abundance after γ-irradiation (5 Gy) and SAHA (15 µM) treatment. The three colored lines correspond to the ion chromatogram of the monoisotopic peptide mass and the first two isotopes. (b) MS/MS spectrum produced from the precursor ion of m/z 596.586. The deposition of the mass spectrometry proteomics data see at the ProteomeXchange Consortium (for detailed information, see Methodology section).

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Irradiation, Produced, Mass Spectrometry

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 7. Fluorescence Lifetime Imaging-Forster Resonance Energy Transfer (FLIM-FRET) analysis of potential interaction between heterochromatin protein 1 (HP1) protein isoforms and the KAP1 protein. (a) Analysis of a GFP-tagged HP1α-KAP1, b] GFP-tagged HP1β-KAP1, and c] GFP-tagged HP1γ-KAP1. (b) An example of a nuclear distribution of HP1γ and the KAP1 protein. HP1γ-positive regions of nucleoli (green) were absent of KAP1. Abbreviation Nu means nucleoli; arrows show HP1γ foci absent of KAP1 and red frames show HP1γ foci colocalizing with KAP1. (c) Example of STED analysis showing the location of KAP1 inside the cell nucleoli decorated by the HP1 isoforms (see HP1β in white frame). (d) FLIM-FRET analysis of a] GFP-tagged HP1β ∆CD/Alexa 594-KAP1, b] GFP-tagged HP1β ∆CSD/ Alexa 594-KAP1, and c] GFP-tagged HP1β ∆Hinge/Alexa 594-KAP1. E [%] means FLIM-FRET efficiency. Scale bars represent 4 µm.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Fluorescence, Imaging, Förster Resonance Energy Transfer

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 8. Immunoprecipitation analysis of potential interaction between HP1α, HP1β, and HP1γ. Studies of following possible interactions: (a) HP1α-HP1α, HP1α-HP1β, HP1α-HP1γ; HP1α-KAP1; (b) HP1β-HP1α, HP1β-HP1β, HP1β-HP1γ; HP1β-KAP1; (c) HP1γ-HP1α, HP1γ-HP1β, HP1γ-HP1γ, HP1γ-KAP1. Protein-protein interaction was studied in non-treated HeLa cells, and cells treated with SAHA (15 µM) and irradiated by γ-rays (5 Gy). Cells were harvested for analysis 2 h the treatment. (d) Western blot analysis of following proteins: HP1α, HP1β, HP1γ, KAP1, normalized to the total protein levels and α-tubulin, and H3K9ac, H3S10ph, γH2AX, normalized to the level of total histone H3. (e) Data from large panel (d) were quantified by ImageJ software. Levels of HP1 isoforms were normalized to the level of α-tubulin, and γH2AX or H3S10ph levels were normalized to the level of total histone H3. Profiles of HP1α, HP1β, HP1γ, KAP1, H3S10ph, and γH2AX were studied in (no. 1) non-irradiated HeLa cells, γ-irradiated Hela cells by (no. 2) 5 Gy of γ-rays/harvested after 2h; (no. 3) 2 Gy/harvested after 10 min; (no. 4) 2 Gy/harvested after 30 min; (no. 5) combination of SAHA treatment with 2 Gy/harvested after 10 min; and (no. 6) combination of SAHA treatment with 2 Gy/harvested after 30 min. A small panel in (d) is showing western blot data for non-treated HeLa cells, and cells irradiated by 5 Gy of γ-rays or SAHA (15 µM) treated HeLa cells.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Immunoprecipitation, Irradiation, Western Blot, Software

Journal: Cells

Article Title: DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

doi: 10.3390/cells8091097

Figure Lengend Snippet: Figure 10. Fluorescence lifetime imaging-Forster resonance energy transfer (FLIM-FRET) analysis of potential interaction between HP1α, HP1β, and HP1γ. Following interactions were studied: (a) a) GFP-tagged HP1α/mCherry-HP1β, b) GFP-tagged HP1β/ mCherry-HP1β, c] mCherry-HP1β/GFP-tagged HP1γ. E (%) means FLIM-FRET efficiency. FLIM-FRET analysis of (b) a) GFP-tagged HP1β ∆CD/mCherry-HP1β, b) GFP-tagged HP1β ∆CSD/mCherry-HP1β, and c) GFP-tagged HP1β ∆Hinge/mCherry-HP1β. E (%) means FLIM-FRET efficiency. Scale bars represent 2 µm.

Article Snippet: Transiently transfected HeLa cells with plasmid DNA encoding GFP-tagged HP1β (#17651 Addgene, USA; [34]) were lysed with RIPA buffer supplemented with Phosphatase Inhibitor Cocktail 2 (#P5726, Sigma-Aldrich, Prague, Czech Republic), 1mM PMSF, and 45mM sodium butyrate.

Techniques: Fluorescence, Imaging, Förster Resonance Energy Transfer