mutant open reading frame  (OriGene)

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , Schematics introducing the workflow. b , SDS–PAGE of the purified PRC1 C8 complex that includes RING1b-, BMI1- and MBP-tagged CBX8. c , Size exclusion chromatography of the purified PRC1 C8 . d , An in vitro ubiquitylation assay comparing PRC1 C8 with a RING1b–BMI1 heterodimer. Samples in lanes 2 and 3 included E1, E2, ubiquitin and ATP. Ubiquitylation is detected by western blot using an anti-H2A antibody. Representative of two independent repeats. e , Chromatin condensates induced by the PRC1 C8 complex and the individual proteins, visualized by confocal (left and center) and phase contrast (right, from independent experiments) microscopy. CBX8 is GFP labeled, and chromatin is Cy5 labeled. Representative results of three repeats. f , Cryo-confocal microscopy of vitrified PRC1 C8 –chromatin condensates. Representative of two independent repeats. g , Cryo-ET of a PRC1 C8 –chromatin condensate. Shown is a central slice through the reconstruction (left image) and subtomogram averages (center, bottom) placed in a volume the size of the tomographic slice, at the determined position and orientation (right image). See Supplementary Table for a list of cross-correlation peaks. h , A surface representation of the volume of a subset of the PRC1 C8 –chromatin condensate structure that is inaccessible to probes of given radii. i , Inaccessible volumes for given probe radii are plotted, with exemplary molecules indicated (in gray) and selected probes colored as in h . For the indicated complexes, the hydrodynamic radius was estimated using resolved domains from published structures (see for PDB accessions) as a minimum size estimate. j , As in g but without PRC1 C8 . See Supplementary Table for a list of cross-correlation peaks. k , Pairwise distances of each individual nucleosome to its nearest three neighboring nucleosomes in three-dimensional space in tomograms with and without PRC1 C8 . Only unique pairs are plotted from two tomograms with (+PRC1) and three tomograms without (−PRC1). Box boundaries extend from the 25th to the 75th percentile, the center line indicates the median, and whiskers extend from the 5th to the 95th percentiles. All points are plotted. Significance was tested using a one-way Brown–Forsythe analysis of variance with a Games–Howell post hoc test. **** P < 0.0001 ( P values: first <0.0000000000001, second 0.0000000000017 and third 0.0000000000099).

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: SDS Page, Purification, Size-exclusion Chromatography, In Vitro, Ubiquitin Assay, Ubiquitin Proteomics, Western Blot, Microscopy, Labeling, Confocal Microscopy, Tomography

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: Sequencing, Agarose Gel Electrophoresis, SDS Page, Staining, Activity Assay, Western Blot, Ubiquitin Proteomics, Concentration Assay

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , Titration of chromatin against PRC1 C8 . Condensates were assessed with a fluorescence widefield microscope imaging a Cy5 label on the chromatin. Presented are representative micrographs of three replicates, including two with MBP-tagged PRC1 C8 (presented) and one with GFP-tagged PRC1 C8 . b , Representative micrographs of FRAP recorded in PRC1 C8 –chromatin condensates. CBX8 is GFP labeled, and chromatin is Cy5 labeled. The mean fluorescence intensity of the bleached area, normalized to prebleach mean signal, is plotted for every time point. The error bars show the standard deviation from n = 7 (GFP) and n = 8 (Cy5) FRAP measurements recorded from two independent experiments. The GFP signal recovery was fit with an exponential association model, and best-fit values for plateau and fluorescence recovery half time ( T 1/2 ) are shown with standard error. n.d., non-determined. c , A schematic representation of the FRAP experiment. Scale bars, 10 μm ( a ) and 2 μm ( b ).

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: Titration, Fluorescence, Microscopy, Imaging, Labeling, Standard Deviation

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a Chromatin condensation in response to variations in salt and PRC1 concentration measured by confocal microscopy using Cy5-labelled chromatin at a constant concentration of 50 ng/μl DNA (approximately 400 nM nucleosomes). Micrographs from one experiment. b , Representative micrographs of FRAP recorded in PRC1 C8 -chromatin condensates, where within this complex CBX8 is untagged. PRC1 C8 is labelled with ATTO488 and chromatin is Cy5 labelled. The mean fluorescence intensity of the bleached area, normalised to the pre-bleach mean signal, is plotted for every time point. Error bars show standard deviation from n = 6 (ATTO488) and n = 7 (Cy5) FRAP measurements that were recorded from four independent experiments that were carried out on different days. The ATTO488 signal recovery was fit with an exponential association model, best fit values for Plateau and fluorescence recovery half time (T 1/2 ) are shown. The lower limits of the 95% confidence interval are presented in parentheses (the upper boundaries could not be determined confidently). c , Representative micrographs of FRAP recorded in PRC1 C8 -chromatin condensates, where within this complex CBX8 includes an N-terminal MBP-tag. PRC1 C8 is labelled with ATTO488 and chromatin is Cy5 labelled. The mean fluorescence intensity of the bleached area, normalised to the pre-bleach mean signal, is plotted for every time point. Error bars show standard deviation from n = 7 (ATTO488) and n = 6 (Cy5) FRAP measurements recorded from four independent experiments that were carried out on different days. The ATTO488 signal recovery was fit with an exponential association model, best fit values for Plateau and fluorescence recovery half time (T 1/2 ) are shown with SEM. d , Images of PRC1 C8 -chromatin condensates, where CBX8 is MBP-tagged. Transmitted light (greyscale), Cy5 fluorescence (magenta, chromatin) and ATTO488 fluorescence (green, PRC1 C8 are shown. The PRC1 C8 -chromatin condensates were imaged in radical scavenger buffer (top) and in chromatin condensation buffer (bottom). e , FRAP as in ( c ) using a radical scavenger buffer. Means represent the average signal over 3 and 5 replicates of chromatin and PRC1 C8 FRAP, respectively, and error bars represent SEM. The recovery curve was fitted with an exponential association model, best fit values for Plateau and fluorescence recovery half time (T 1/2 ) are shown. f , As in (e) but using PRC1 C8 with no MBP tag on CBX8. Scale bars in all panels are 5 μm.

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: Concentration Assay, Confocal Microscopy, Fluorescence, Standard Deviation

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , Chromatin condensation in response to the whole PRC1 C8 complex (RING1b, BMI1 and CBX8) or the individual components CBX8 and the RING1b–BMI1 heterodimer. Representative images from two replicates. b , Intramolecular (purple lines) and intermolecular (green lines) protein–protein interactions mapped within PRC1–chromatin condensates using XL-MS. The data are from three independent replicates. c , PRC1 C8 or PRC1 core binding to a fluorescein-labeled 24 bp DNA probe measured by fluorescence polarization. The data points show the mean (baseline subtracted) of three independent replicates, and the error bars indicate the standard error. The continuous line represents the fit to a Hill binding model, when applicable. K d , dissociation constant. d , Titration of PRC1 C8 to unmodified chromatin (top) and H3K27me3-MLA chromatin (bottom) at an identical chromatin concentration (50 ng μl −1 DNA) and 150 mM KCl. The micrographs are representative of two independent replicates. Scale bars, 5 μm.

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: Protein-Protein interactions, Structural Proteomics, Binding Assay, Labeling, Fluorescence, Titration, Concentration Assay

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , Schematics depicting the different CBX8 mutants used, drawn to scale. b , PRC1 C8 –chromatin condensates in the context of different CBX8 mutants. Varying concentrations of PRC1 C8 were titrated to a constant concentration of C5-labeled reconstituted chromatin (50 ng μl −1 ). Widefield fluorescence and DIC micrographs are representative of three replicates. c , Quantification of the total area covered by condensates per micrograph for different PRC1 C8 mutants and concentrations. The bars represent the means from three independent replicates, and the error bars represent the standard deviation. Scale bar, 5 μm. d , A fluorescence polarization assay measuring the affinity of different PRC1 C8 mutants for a fluorescein-labeled 24 bp DNA probe. The data points are the mean (baseline subtracted) of three independent replicates, and the error bars indicate the standard error. The continuous line represents the fit to a Hill binding model, when applicable. e , Model for chromatin condensation by PRC1 C8 : electrostatic interaction between the CBX8 IDR with DNA provides charge screening and promotes phase separation.

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: Concentration Assay, Labeling, Fluorescence, Standard Deviation, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , Schematics of the experimental setup (left) and anti-CBX8 western blot (right) of wild-type and Cbx8 -KO mES cells after 48 h of RA treatment. Representative of two independent replicates. b , Overlap of ATAC-seq peaks and CBX8 and H3K27me3 ChIP-seq peaks. ATAC-seq peaks are defined from two biological replicates. c , ChIP-seq traces for H3K27me3 and CBX8 in wild-type mES cells and representative ATAC-seq traces at four genes in wild-type and CBX8-KO mES cells after 48 h of RA treatment. d , Accessibility changes at all ATAC-seq peaks in wild-type (WT) mES cells, in response to RA treatment. e , Accessibility changes at all CBX8-target sites in wild-type mES cells, in response to RA treatment. f , A comparison of accessibility at CBX8-target sites between wild-type and Cbx8 -KO cells after RA treatment. g , Top: schematic representation of the chromosome-integrated reporter. Left: ChIP–qPCR using FLAG antibody (CBX8 is FLAG tagged) at indicated distances from the TetO array, in the presence and absence of Dox treatment for 6 h. The bars represent the mean bound over input (Bd/In) normalized to intracisternal A particle retrotransposon (IAP), and the points represent two replicates. See Extended Data Fig. for ChIP–qPCR using additional antibodies. Right: brightfield and GFP-fluorescence images of the mECS cells before and after Dox treatment. h , ATAC-seq signal reporting accessibility of the integrated locus before and after Dox treatment for 6 days. From left to right: annotated are the TetO array and its proximal PGK promoter that controls the puromycin–GFP reporter gene, and the distal PGK promoter. i , A model in which PRC1 forms multivalent interactions with chromatin, thereby stabilizing chromatin condensates potentially through charge screening of negatively charged DNA by positive charges in the CBX8 IDR. These interactions dynamically change as PRC1 diffuses through condensates.

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: Western Blot, ChIP-sequencing, Comparison, ChIP-qPCR, Fluorescence

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , A GFP reporter system, where in the absence of doxycycline (Dox), the TetR-CBX8 fusion is recruited to the TetO array, but not in the presence of Dox. ChIP-qPCR in the presence and absence of Dox treatment using RING1B antibody at indicated distances from a chromosome-integrated TetO array (left), at control genes (right) and using FLAG (CBX8) antibodies at control genes (middle). Bars represented the bound over input (Bd/In) normalised to the IAP gene and the dots represent individual data points. b , ATAC-seq signal at the HoxA locus of the reporter-integrated mECS cell line before and after dox treatment. Two independent replicates are shown.

Article Snippet: The CBX8 KR21A mutant open reading frame was codon optimized for expression in Trichoplusia ni insect cells and synthesized (GenScript).

Techniques: ChIP-qPCR, Control

Journal: Nucleic Acids Research

Article Title: RNase W, a conserved ribonuclease family with a novel active site

doi: 10.1093/nar/gkae907

Figure Lengend Snippet: X-ray diffraction and refinement statistics

Article Snippet: The open reading frames of RNase W (FAU-1) from P. furiosus (full-length protein, constructs and mutants; PF0022) and S. acidocadarius (Saci_1213) were synthesized commercially by GenScript Corp. (Piscatawy, NJ) and inserted in pET24(a+) (Novagen), pET24- Pf RNase W and pET24- Sa RNase W, respectively.

Techniques: X-ray Diffraction

Journal: Nucleic Acids Research

Article Title: RNase W, a conserved ribonuclease family with a novel active site

doi: 10.1093/nar/gkae907

Figure Lengend Snippet: Structures of RNase W from P. furiosus and S. acidocaldarius . ( A and B ) Structures of RNase W proteins colored by domains: N-terminal domain in orange, S1 domain in dark green, 5′-sensor domain in light green, α helical bundle domain in red and DUF402 domain in blue. ( A ) Structure of RNase W from P. furiosus ( Pf RNase W). A dinucleotide UU with 5′ and 3′-phosphate extremities is bound to the 5′-sensor domain. In addition, a dinucleotide UA with 5′-hydroxyl and 3′-phosphate extremities is bound to the DUF402 domain. ( B ) Structure of RNase W from S. acidocaldarius ( Sa RNase W). ( C ) Pf RNase W surface representation with residues conservation score in archaeal RNase W proteins mapped on the surface. The encircled central domain includes the N-terminal domain and one helix of the α helical bundle domain annotated in panel A. ( D ) Electrostatic surface potential mapped on the surface of Pf RNase W protein.

Article Snippet: The open reading frames of RNase W (FAU-1) from P. furiosus (full-length protein, constructs and mutants; PF0022) and S. acidocadarius (Saci_1213) were synthesized commercially by GenScript Corp. (Piscatawy, NJ) and inserted in pET24(a+) (Novagen), pET24- Pf RNase W and pET24- Sa RNase W, respectively.

Techniques: