bethyl recognizing phospho kap1  (Bethyl)

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Domain architecture of HP1α and HP1γ: nte, amino-terminal extension; CD, chromodomain; CSD, chromoshadow domain; cte, carboxyl-terminal extension. (B) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled HP1γ CSD recorded in the presence of increasing amounts of KAP1 Hbox1 peptide. Spectra are color coded according to the protein:peptide molar ratio. (C) Binding affinities of wild type or mutated HP1γ CSD for the indicated KAP1 peptides. (a) from Gaurav et al. and (b) determined by NMR. (D) Representative binding curve used to determine the binding affinity of HP1γ CSD for KAP1 Hbox by tryptophan fluorescence. K d is represented as the average ± SD of three independent experiments. n = 3. (E and F) The crystal structure of the HP1γ CSD dimer in complex with KAP1 Hbox peptide. Two protomers of the HP1γ CSD dimer are shown in a ribbon diagram and labeled CSD1 and CSD2. KAP1 Hbox is depicted as a ribbon in (E) or sticks in (F). (G) A close view of the KAP1 Hbox -binding site of the HP1γ CSD dimer. Residues involved in the interaction between HP1γ CSD and KAP1 Hbox are labeled. CSD2 residues are labeled with the apostrophe. Dashed lines represent hydrogen bonds. (H and J) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled wild-type or mutated HP1γ CSD recorded in the presence of increasing amounts of wild-type or mutated KAP1 Hbox peptide. Spectra are color coded according to the protein:peptide molar ratio. See also .

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Labeling, Binding Assay, Fluorescence

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled HP1γ CSD (purple) and FL HP1γ. The FL HP1γ spectra were recorded in the presence of increasing amounts of KAP1 Hbox peptide and are color coded according to the protein:peptide molar ratio. HP1γ CSD resonances in the spectrum of FL HP1γ broadened beyond detection, indicating that the size of this part of HP1γ becomes too large, i.e., HP1γ CSD multimerization (higher order than detectable dimerization) reduces the tumbling rate. (B) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled S95D/S97D and Δhinge mutants of FL HP1γ recorded in the presence of increasing amounts of KAP1 Hbox peptide. (C) Superimposition of the crystal structure of one protomer of HP1γ CSD (wheat) in complex with KAP1 Hbox (green) and the AlphaFold model of FL HP1γ (gray, with CD and cte colored pink and light cyan, respectively, from UniProt, # Q13185 ). (D) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled I165E HP1γ CSD recorded in the presence of increasing amounts of unlabeled wild-type HP1γ CSD . Spectra are color coded according to the protein:ligand molar ratio. (E) Molecular mass distribution histograms of FL HP1γ at indicated concentrations in mass photometry assay. Maxima of the fits are labeled (kDa). (F) Molecular mass distribution histogram of the 2:1 mixture of FL HP1γ and KAP1 Hbox1 peptide in mass photometry assay. Maxima of the fits are labeled (kDa). (G) Two KAP1 Hbox -bound dimers of HP1γ CSD (one dimer is colored wheat, and the other is colored light blue) interact via their β-sheet regions. KAP1 Hbox is shown as green ribbon and, due to symmetry, can be bound in either direction. The α-helix interface and β-sheet interface in the tetrameric organization of the complex are labeled.

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Labeling

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Superimposition of the crystal structure of the KAP1 Hbox -bound dimer of HP1γ CSD (wheat) with two molecules of the AlphaFold model of FL HP1γ (one FL HP1γ is colored gray, and the other FL HP1γ is colored magenta). Regions of FL HP1γ are labeled. (B) Two peripheral protomers from the KAP1 Hbox -bound dimer of dimers of HP1γ CSD (colored as in ) are superimposed with two molecules of the AlphaFold model of FL HP1γ (one FL HP1γ is colored gray, and the other FL HP1γ is colored magenta). (C–E) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled HP1γ CSD recorded in the presence of increasing amounts of HP1γ CD (C), HP1γ hinge (D), or HP1γ cte (E). Spectra are color coded according to the protein:peptide molar ratio.

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Labeling

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) A close view of the β-sheet interface between two KAP1 Hbox -bound dimers of HP1γ CSD (colored as in ). Only one protomer per dimer is shown for clarity. KAP1 Hbox is depicted as green ribbon. The residues involved in the formation of the β-sheet interface are shown as sticks and labeled. (B) Molecular mass distribution histograms of K141E HP1γ (top), R125E/K141E HP1γ (middle), and the 1:3 mixture of R125E/K141E HP1γ and KAP1 Hbox (bottom) in mass photometry assay. Maxima of the fits are labeled (kDa). (C) Molecular mass distribution histograms of HP1α at indicated concentrations in mass photometry assay. Maxima of the fits are labeled (kDa). (D) A close view of the β-sheet interface between two KAP1 Hbox ′-bound dimers of HP1α CSD (Gaurav et al. ). KAP1 Hbox ′: aa 468–496 of KAP1. Only one protomer per dimer is shown for clarity. KAP1 Hbox is depicted as a yellow ribbon. The residues involved in the formation of the β-sheet interface are shown as sticks and labeled. (E) The crystal structures of dimers of the KAP1 Hbox -bound dimers of HP1γ and HP1α CSD are superimposed. Two protomers of one HP1γ CSD dimer (CSD1 and CSD2) are colored wheat, and two protomers of the second HP1γ CSD dimer (CSD3 and CSD4) are colored light blue. Both dimers of HP1α CSD are colored green. The dimers interact through their β-sheets. (F) A model of limited multimerization of HP1γ CSD and robust unlimited multimerization of HP1α CSD . (G) Bright-field images of concentration series of WT HP1γ, WT HP1α, and L139E/L150E HP1α with 30 nM of 2.7 kbp linearized PUC19 plasmid DNA. Scale bar represents 20 μm.

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Labeling, Concentration Assay, Plasmid Preparation

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) AI662270 expression was measured in HP1α −/− , HP1β −/− , HP1γ −/− , and KAP1 −/− mouse ESCs using quantitative reverse transcription PCR (qRT-PCR). Data are presented as average between two independent biological replicates. (B) Generation of the entry mouse ESC line. The attP sequence (MIN tag) is inserted directly after the transcription start site of HP1γ. Schematic illustrating the CRISPR-Cas9-mediated genome editing strategy, with the gRNA and PAM sequences highlighted. The donor single-strand DNA contains the MIN tag sequence with a HincII restriction cut site for screening and homology arms flanking the translational start site. The positions of screening PCR primers are indicated, which produce 509 and 557 bp products in WT and HP1γ attP/attP cells, respectively. (C) PCR-based validation of two individual HP1γ attP/attP ESCs using primers indicated in (B), followed by HincII digestion. PCR products showing a reduced size after HincII digestion are considered positive. (D) Schematic outlining the Bxb1-mediated recombination strategy used to generate the HP1γ −/− ESC line. A cassette containing the attB site, RFP, a stop codon, and a polyA signal was inserted directly after the transcription start site by attP-attB recombination to produce HP1γ −/− ESCs. (E and F) Characterization of two individual HP1γ −/− ESC clones, A3 and C3, by quantitative RT-PCR analysis (E). Data are presented as mean ± standard deviation (SD) from three technical replicates. Western blot analysis to detect HP1γ in the two individual HP1γ −/− clones (F). The tubulin blot was used as a loading control. See also 1 and .

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Expressing, Reverse Transcription, Quantitative RT-PCR, Sequencing, CRISPR, Biomarker Discovery, Clone Assay, Standard Deviation, Western Blot, Control

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Expression levels of AI662270 were measured by quantitative reverse transcription PCR (RT-PCR) in KAP1 −/− ESCs stably expressing GFP-tagged KAP1 fragment (aa 114–834 of KAP1, GFP-KAP1 FR ) (WT or the V488E mutant). The expression data in rescue cell lines were normalized to the protein levels of GFP-KAP1 FR and are presented as mean ± SEM from three independent biological replicates. n = 3. Statistical analyses were performed using an unpaired, two-tailed Student’s t test; *, **, and **** stand for p values < 0.05, 0.01, and 0.0001, respectively. (B) Representative immunofluorescence images show the cellular distribution of GFP-KAP1 FR WT and the V488E mutant stably expressed in KAP1 −/− ESCs. Endogenous HP1γ was visualized by immunostaining with an anti-HP1γ antibody. (C and D) Boxplots show the variation of coefficient for GFP-KAP1 FR intensity (C) and the Pearson correlation coefficient between GFP-KAP1 FR and endogenous HP1γ (D). Statistical analyses were performed using an unpaired two-tailed Student’s t test; **** stands for p value < 0.0001. (E) Expression levels of AI662270 were measured by quantitative RT-PCR in HP1γ −/− ESCs stably expressing GFP-tagged HP1γ (WT or the W174A mutant). The expression data in rescue cell lines were normalized to the protein levels of GFP-HP1γ and are presented as mean ± SEM from three independent biological replicates. n = 3. Statistical analyses were performed using an unpaired two-tailed Student’s t test; *, **, and **** stand for p values < 0.05, 0.01, and 0.0001, respectively. (F) Representative immunofluorescence images show the cellular distribution of GFP-HP1γ WT and the W174A mutant (together with mCherry-KAP1 FR ) stably expressed in HP1γ −/− ESCs. (G and H) Boxplots show the variation of coefficient for GFP-HP1γ intensity (G) and the Pearson correlation coefficient between GFP-HP1γ and mCherry-KAP1 FR (H). Statistical analyses were performed using an unpaired, two-tailed Student’s t test; *** and **** stand for p values < 0.001 and 0.0001, respectively. (I) Genome browser representation of enrichments of HP1γ and KAP1 ChIP-seq in WT ESCs and H3K9me3 CUT&Tag in WT and KAP1 KO ESCs at AI662270 . The y axis represents read density in counts per million mapped reads (CPM). Putative AI662270 enhancers were identified using the ABC model. (J) The expression of AI662270 in NPCs derived from WT, KAP1 −/− and HP1 −/− NPCs by quantitative RT-PCR analyses. Data are presented as mean ± SD, based on two biological replicates for HP1 knockout cells and four technical replicates for WT and KAP1 −/− cells. Statistical analyses were performed using an unpaired, two-tailed Student’s t test. *, **, and *** indicate p values < 0.05, 0.01, and 0.001, respectively, while n.s. denotes no significant difference. See also .

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Mutagenesis, Two Tailed Test, Immunofluorescence, Immunostaining, Quantitative RT-PCR, ChIP-sequencing, Derivative Assay, Knock-Out

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) AI662270 expression was measured in mouse tissues using quantitative RT-PCR. Data are presented as mean ± SD from four technical replicates. n = 4. Statistical analyses were performed using an unpaired, two-tailed Student’s t test to compare HP1γ with HP1α and HP1β. *, **, and *** indicate p < 0.05, 0.01, and 0.001, respectively, while n.s. denotes no significant difference. (B) A heatmap illustrating the expression profiles of shared dysregulated genes in HP1 knockout cells, with expression levels indicated by color intensity. (C) Gene Ontology (GO) analysis of shared dysregulated genes in HP1α −/− and HP1γ −/− mouse ESCs. (D and E) Gene Ontology (GO) analysis of up- and downregulated genes in KAP1 −/− mouse ESCs.

Article Snippet: KAP1 Hbox (aa 483–493) , SynPeptide , N/A.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Knock-Out