Journal: Neuron

Article Title: Polyserine-mediated targeting of FAF2/UBXD8 ameliorates tau aggregation

doi: 10.1016/j.neuron.2025.08.002

Figure Lengend Snippet: ( A ) Immunostaining of DAPI (blue), phosphorylated tau (AH36) (green) and NeuN (magenta) in hippocampi of WT or PS19 animals injected with AAV9-GFP, AAV9-FAF2 or AAV9-FAF2 PS . ( B ) Quantification of AH36+ hippocampal area as in (A). One-way ANOVA and Tukey’s MCT. n=3 sections per animal averaged for n=10 animals per group. ( C ) Volcano plot of protein expression by Tandem mass tag pro (TMTpro) mass spectrometry in brain extracts from AAV9-FAF2 animals compared to AAV9-GFP at 6-months-old. Shaded areas represent fold change >2 and p-value >0.01. ( D ) Volcano plot of protein expression by TMTpro mass spectrometry in brain extracts from AAV9-FAF2 PS animals compared to AAV9-GFP at 6-months-old. Shaded areas represent fold change >2 and p-value above 0.01. ( E ) Percent of time spent on closed arms during elevated plus maze assay for 10-month-old animals as in (A). Boxplot whiskers by Tukey’s method. One-way ANOVA. WT+GFP(n=16), PS19+GFP(n=18), PS19+FAF2(n=11), PS19+FAF2 PS (n=12). ( F ) Percent freezing during training period of fear conditioning assay for animals as in (E). Grey boxes denote cued stimulus (tone) and red line denotes aversive stimulus (foot shock). WT+GFP(n=18), PS19+GFP(n=19), PS19+FAF2(n=11), PS19+FAF2 PS (n=11). ( G ) Average percent of time spent freezing during periods of cued stimulus for animals as in (F). Boxplot whiskers by Tukey’s method. One-way ANOVA and Tukey’s MCT. WT+GFP(n=18), PS19+GFP(n=19), PS19+FAF2(n=11), PS19+FAF2 PS (n=11). ( I ) Average percent of time spent freezing during contextual testing for animals in (F). Boxplot whiskers by Tukey’s method. One-way ANOVA and Tukey’s MCT. WT+GFP(n=18), PS19+GFP(n=19), PS19+FAF2(n=11), PS19+FAF2 PS (n=11).

Article Snippet: Rabbit anti-p-tau (AH36) , StressMarq Biosciences , Cat#SMC-601; RRID:AB_2820300.

Techniques: Immunostaining, Injection, Expressing, Mass Spectrometry, EPM Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) The nucleosome core particle (NCP) (PDB: 3LZ0): histone N- and C-terminal tails (as defined by trypsin digest) are depicted as dotted lines and to relative scale. ( b ) Secondary domain architecture of BPTF [Uniprot Q12830; 3,046 aa; 338 kDa]. Region covered by the C-terminal tandem PHD-BD (aa 2865–3036; as used through this study) in blue. ( c, d ) dCypher assay Alpha counts plotted as a function of GST-PHD-BD Query concentration to histone peptide ( c ) or NCP ( d ) Targets. ( e ) Relative EC 50 (EC 50 rel ) and 95% confidence interval (CI95) values from dCypher curves (in c , d ) and ; for calculation see ’Materials and methods’. Targets are color coded as per legends. ND, Not Detected, Not Testable.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Concentration Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) dCypher is a no-wash bead-based proximity assay on the Alpha platform to interrogate chromatin readers (see ’Materials and methods’). PTM-defined biotinylated peptides or NCPs (the potential Targets) are immobilized to streptavidin donor beads and tagged reader domain(s) (the Queries) to anti-tag Acceptor beads. Donor beads are excited at 680 nm causing the release of singlet oxygen and quantitative fluorescence emission (520–620 nm) from proximal acceptor beads (i.e., bridged by a [Query:Target] interaction). ( b,c ) GST-PHD-BD selectivity (Query concentration as noted; chosen from dCypher curves in [ EC 80 rel for H3K4me3 peptide; EC 50 rel for H3K4me3tri ac NCP]) for a panel of histone peptides (~100 nM final; b ) and NCP (10 nM final; c ). ( d,e ) GST-PHD-BD binding to histone H3 lysine 4 (H3K4) methyl states (me0 /me1/me2/me3) on peptides ( d ) or NCPs ( e ). Alpha Counts are plotted as a function of Query concentration. ( f ) dCypher assay alpha counts plotted as a function of GST-PHD-BD concentration to NCPs. ( g ) EC 50 rel values with 95% confidence intervals (CI95) (see ’Materials and methods’ for calculation of same) from dCypher curves in ( f ) with Targets color coded as per legend (see also ). ND, Not Detected.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Proximity Assay, Fluorescence, Concentration Assay, Binding Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a–b ) EC 50 rel (with CI95) values calculated from dCypher curves of GST-PHD, 6His-PHD, or 6His-BD Query binding to peptide ( a ) or nucleosome core particle (NCP) ( b ) Targets from curves in ( c–g ), with same color code as per each legend. ND, not detected. -, not tested. ( c–e ) Alpha counts plotted as a function of 6His-PHD Query concentration for peptide ( c ) or NCP ( d ) Targets, or for GST-PHD Query concentration for NCP ( e ). ( f, g ) Alpha counts plotted as a function of 6His-BD Query concentration for peptide ( f ) or NCP ( g ) Targets. See previously and ’Materials and methods’ regarding comparison of EC 50 rel across different affinity tags.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Binding Assay, Concentration Assay, Comparison

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) The PHD-H3K4me3 (top) and BD-Kac (bottom) binding pockets on previously solved structures of the individual domains in complex with histone peptides (PDB: 2FUU and 3QZT). Binding pockets are circled/labeled: on PHD for A1, R2, and K4me3; on BD for Kac. Relative location of PTM-binding residues W2891 (PHD) and N3007 (BD) also indicated and mutated to alanine in ( b, c ). ( b ) dCypher assay Alpha counts plotted as a function of GST-PHD-BD N3007A (GST-PHD-BD mut ; top) or GST-PHD W2891A -BD (GST-PHD mut -BD; bottom) Query concentration to NCP Targets. ( c ) EC 50 rel (CI95) values from dCypher curves in ( b ). Targets color coded as per legends. ND, Not Detected.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Binding Assay, Labeling, Concentration Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) 1 H, 15 N-HSQC overlays of 15 N-PHD-BD apo (black) or in the presence of H3tri ac (green), H3tetra ac (blue), or H3K4me3tri ac (red) peptides. Arrows denote trajectory of chemical shift perturbation (CSP) and are colored by peptide. Shown are representative resonances for the bromodomain (BD) (left) and plant homeodomain (PHD) (right) binding pockets. ( b ) Histone H3-A1 is essential for 6His-PHD-BD binding to nucleosome core particles (NCPs) (compare H3K4me3tri ac to NΔ1H3K4me3tri ac [integrity of each target confirmed with anti-H3K4me3 ]). dCypher assay Alpha counts are plotted as a function of Query concentration to indicated NCP Targets.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Binding Assay, Concentration Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a–c ) Full spectral overlays of 15 N-PHD-BD on titration with histone H3 tail peptides (aa 1–34): H3K4me3tri ac ( a ); H3tri ac ( b ); or H3tetra ac ( c ). Molar ratios of each peptide are denoted by color in panel legends. ( d ) Validation of N-terminally truncated H3 NCP (NΔ1H3K4me3tri ac ). Alpha counts plotted as a function of anti-H3K4me3 ( RevMab Cat# 31-1039-00, lot # P-09–00676) concentration: binding of this antibody is dependent on H4K4me3 (compare H3K4me3 and NCP-unmodified) but independent of residue A1 (NΔ1H3K4me3tri ac ) or acetylation at K9, K14 or K18 (H3K4me3tri ac ). Table shows EC 50 rel (with CI95) values calculated from dCypher curves. Targets color coded as per legend. ND, not detected.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Titration, Concentration Assay, Binding Assay, Residue

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a–d ) Histones (including 15 N-H4) for NMR were individually purified and validated by MALDI mass spectrometry prior to nucleosome core particle (NCP) reconstitution (see ’Materials and methods’). ( e ) The 15 N-H4 containing NCP was resolved by native PAGE and stained with ethidium bromide to demonstrate reconstitution/absence of free DNA. ( f ) 15 N-H4 containing NCPs were resolved by reducing SDS-PAGE and Coomassie stained to interrogate histone stoichiometry. Figure 4—figure supplement 1—source data 1. Full native gel image for 15 N-H4-NCP reconstitution, stained with ethidium bromide. Figure 4—figure supplement 1—source data 2. Full SDS-PAGE image for 15 N-H4-NCP reconstitution, stained with coomassie blue. Figure 4—figure supplement 1—source data 3. All gel images for labeled and with regions cropped for figure denoted with dashed boxes.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Purification, Mass Spectrometry, Clear Native PAGE, Staining, SDS Page, Labeling

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: Nucleosome crystal structure (PDB ID: 3LZ0) showing interaction of the H4 tail basic patch (residues: K16-R17-H18-R19-K20) with DNA.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques:

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a–c ) Installation of the acetyl-lysine analogue was validated by MALDI mass spectrometry and purity of protein by SDS-PAGE (see ’Materials and methods’). *denotes contaminants ( d ) 15 N-H4K S 16ac containing nucleosome core particle (NCPs) were resolved by reducing SDS-PAGE and Coomassie stained to interrogate histone stoichiometry. ( e ) The 15 N-H4K S 16ac containing NCP was resolved by native PAGE and stained with ethidium bromide to demonstrate reconstitution/absence of free DNA. ( f ) Bromodomain PHD finger transcription factor (BPTF) Bromodomain (BD) purity was assessed by SDS-PAGE. Figure 4—figure supplement 3—source data 1. Full SDS-PAGE image of histone H4K16C protein purification for H4K s 16ac installation. Figure 4—figure supplement 3—source data 2. Full SDS-PAGE image for 15 N-H4K s 16ac-NCP reconstitution, stained with coomassie blue. Figure 4—figure supplement 3—source data 3. Full native gel image for 15 N-H4K s 16ac-NCP reconstitution, stained with ethidium bromide. Figure 4—figure supplement 3—source data 4. Full SDS-PAGE image for BPTF BD, stained with coomassie blue.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Mass Spectrometry, SDS Page, Staining, Clear Native PAGE, Protein Purification

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) dCypher assay Alpha counts plotted as a function of GST-PHD-BD Query concentration to homotypic (e.g. [H3 • H3]) or heterotypic (e.g. [H3 • H3K4me3]) NCP Targets (created as in ’Materials and methods’). ( b ) Relative EC 50 (EC 50 rel ) and 95% confidence interval (CI95) values from dCypher curves (in a ; for calculation see ’Materials and methods’). • indicates a heterotypic Target. Limited testing data from a 6His-PHD-BD query added for comparison. Targets are color coded as per legends. ND, Not detected; -, Not tested.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Concentration Assay, Comparison

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) Heatmap of CUT&RUN signal (see ’Materials and methods’) aligned to the transcription start site (TSS, +/−2 kb) of 18,793 genes in K562 cells. High and low signal (red and blue, respectively) are ranked by / linked to H3K4me3 (top to bottom). ( b–c ) CUT&RUN RPKM normalized tracks at representative loci using Integrative Genomics Viewer (IGV, Broad Institute). Note the general co-localization of BTPF (endogenous) with H3K4me3 and H3K18ac. ( d ) Venn overlap for H3K4me3, H3K18ac and BPTF peaks. Peaks were called using SEACR, designed for the S parse E nrichment A nalysis of C UT& R UN data , and intersected using bedtools . To compare data sets, the top peaks (number noted for each) were considered. ( e ) BPTF signal distribution for SEACR peaks represented as a Violin plot, and derived from Venn diagram groups. Column 1 depicts the signal distribution for all 8,405 BPTF peaks. Other columns depict BPTF peaks that overlap with peaks for H3K4me3, H3K18ac, or both PTMs (as noted). The Fisher exact test was used to determine if BPTF co-occupancy with specific PTMs or combinations were statistically significant (**** denotes p-value <10 –4 ). See for sequencing statistics; all project files available at Gene Expression Omnibus (GEO) accession GSE150617 .

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Derivative Assay, Sequencing, Expressing

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) CUTANA nucleosome spike-ins contain a 5’biotin for immobilization to magnetic beads and a DNA barcode to define post-translational modification (PTM) status/monitor release into the CUT&RUN eluate. A four-member panel was assembled to explore GST-PHD-BD binding (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; on 80- N -25 DNA containing a central 147 bp 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode). ( b ) GST-PHD-BD shows strong preference for spike-in nucleosome containing H3K4me3tri ac . Table shows relative release of spike-ins (percent barcoded nucleosome/total barcode reads) in Reader CUT&RUN (’Materials and methods’). Antibodies are noted by column; GST-BPTF (PHD-BD) is detected by α-GST to facilitate pAG-MNase recruitment. ‘Nucleosome bandwidth’ is the percentage of total sequence reads taken up by spike-in standards. ( c ) Heatmap of CUT&RUN signal aligned to the transcription start site (TSS, +/−2 kb) of 18,793 genes in K562 cells. Rows were k-means clustered into four groups (boxed) using ChAsE chromatin analysis tool . High and low signal (red and blue, respectively) are ranked by / linked to H3K4me3 (top to bottom). ( d ) CUT&RUN RPKM normalized tracks at representative loci using Integrative Genomics Viewer (IGV, Broad Institute). Note the co-localization of BTPF (endogenous) or GST-PHD-BD (exogenous) with H3K4me3 and H3K18ac; that H3K18ac alone is insufficient to recruit BTPF or GST-PHD-BD; and that GST-PHD-BD shows robust recruitment at some locations where BPTF is absent (e.g. B4GALT2 promoter; see ‘Discussion’).

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Magnetic Beads, Modification, Binding Assay, Sequencing

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) versaNucs (see ’Materials and methods’) (Lanes 1–7; post-translational modification (PTM) identity noted, 400 ng total DNA mass) were resolved by native PAGE (6%, 0.4 X TBE) and integrity confirmed by staining with ethidium bromide (vs. Lane 8; 147 × 601 biotinylated free DNA, 200 ng total DNA mass). ( b ) versaNucs (Lanes 1–7; 2 μg total protein mass) were resolved by reducing SDS-PAGE (4–20%) and histones visualized by Coomassie staining. Control lanes include unmodified nucleosome (native full length H3; EpiCypher #16 – 6006 ) and versaNuc precursor (H3.1 N∆32 before enzymatic ligation of H3 peptide; ). ( c ) Indicated purified recombinants (see ) were resolved by SDS-PAGE and visualized by Coomassie staining. Figure 1—figure supplement 2—source data 1. Full native gel image for modified versaNuc reconstitution, stained with ethidium bromide. Figure 1—figure supplement 2—source data 2. Full native gel image for wild-type versaNuc reconstitution, stained with ethidium bromide. Figure 1—figure supplement 2—source data 3. Full SDS-PAGE image for modified versaNuc reconstitution, stained with coomassie blue. Figure 1—figure supplement 2—source data 4. Full SDS-PAGE image for wild-type versaNuc reconstitution, stained with coomassie blue. Figure 1—figure supplement 2—source data 5. Full SDS-PAGE image for wild-type and mutant 6His-BD, stained with coomassie blue. Figure 1—figure supplement 2—source data 6. Full SDS-PAGE image for wild-type 6His-PHD-BD, stained with coomassie blue. Figure 1—figure supplement 2—source data 7. Full SDS-PAGE image for wild-type GST-PHD-BD, stained with coomassie blue. Figure 1—figure supplement 2—source data 8. Full SDS-PAGE image for wild-type 6His-PHD, stained with coomassie blue. Figure 1—figure supplement 2—source data 9. Full SDS-PAGE image for mutant GST-PHD and GST-PHD-BD, stained with coomassie blue. Figure 1—figure supplement 2—source data 10. All gel images for labeled and with regions cropped for figure denoted with dashed boxes.

Article Snippet: A biotinylated CUTANA nucleosome mini-panel (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; each on 80- N -25 DNA containing a central 147 bp Widom 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode: ) was synthesized, individually coupled to magnetic streptavidin beads ( NEB #S1421S ) at saturation, and spiked into each CUT&RUN reaction (final concentration 0.8 nM) with Con-A immobilized cells just prior to antibody addition.

Techniques: Modification, Clear Native PAGE, Staining, SDS Page, Control, Ligation, Purification, Mutagenesis, Labeling

Journal: Epigenetics & Chromatin

Article Title: The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

doi: 10.1186/s13072-022-00462-7

Figure Lengend Snippet: High-confidence interaction partners of ZFP57 in mouse ESCs. a Western blot analysis of ZFP57 and KAP1 in wild-type (WT), Zfp57 -AviTag-transfected and Zfp57 -/- ESCs. Note that AviTag-ZFP57 migrates as a 60 kDa band while the endogenous ZFP57 migrates as a 50 kDa-band. b Experimental workflow of the tagged protein-mass spectrometry approach used for identification of the ZFP57 interactors. The Zfp57 cDNA was cloned into the expression vector pEF6-Avitag-GGGx2-Avitag. Zfp57 -AviTag was transfected in stably BirA-expressing mouse ESCs and biotin-labelled pulled proteins were pulled down using streptavidin. Precipitated protein complexes were digested with trypsin for LC–MS/MS analysis. Proteins in common between Zfp57 -AviTag-transfected and Mock-transfected BirA-expressing E14 ESCs were excluded from further analyses. Images of petri dishes and eppendorf tubes are taken from https://doi.org/10.7875/togopic.2020.104 and https://doi.org/10.7875/togopic.2022.115 , respectively. BirA is depicted in light blue, biotin in red, ZFP57 in dark blue and ZFP57-interactors in grey, green, and olive. Zfp57 -AviTag-transfected and mock-transfected BirA-expressing E14 ESCs are depicted in red and green, respectively. c Chord diagram showing enriched GO clusters of ZFP57 interactors. The proteins identified by LC–MS/MS analysis ordered according to their relative enrichment are shown on the left, and the enriched GO clusters are indicated with different colours on the right. The number of peptides by which proteins were recognized by LC–MS/MS analysis is displayed in gradient red (2–20 peptides). d Western blot of proteins pulled down with streptavidin in WT and Zfp57 -AviTag-transfected ESCs and revealed with anti-MSH2 antibody. e Western blot of proteins pulled down with streptavidin in Msh2 -AviTag-transfected ESCs and revealed with anti-KAP1 antibody. f Western blot of proteins immunoprecipitated with anti-KAP1 antibody in WT and Zfp57-/- ESCs and revealed with anti-MSH2 antibody. Input corresponds to 1% of the cell lysate used for immunoprecipitation

Article Snippet: Streptavidin beads (Dynabeads MyOne streptavidin T1, ref 65,601, Invitrogen) and anti-MSH6 (Santa Cruz sc-137015), anti-H3K9me3 (Abcam 8898), anti-H3K4me3 (Abcam ab8580) antibodies or rabbit/mouse IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel.

Techniques: Western Blot, Transfection, Mass Spectrometry, Clone Assay, Expressing, Plasmid Preparation, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation

Journal: Epigenetics & Chromatin

Article Title: The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

doi: 10.1186/s13072-022-00462-7

Figure Lengend Snippet: Parental origin-specific binding of MSH2 and MSH6 to the ICRs. a Box plot showing the methylation level of non-repetitive MSH2-bound regions (left) and MSH2-bound ICRs (right). b Electropherograms showing the DNA methylation level of the Inpp5f and Gnas ICRs in genomic DNA, ChIP DNA obtained with anti-H3K9me3, anti-H3K4me3 antibodies and Bio-ChIP DNA pulled down with streptavidin in Msh2 -AviTag-transfected ESCs, and revealed by bisulfite sequencing. Methylation levels were determined from the ratio between methylated (C) and unmethylated (T) cytosines. c Electropherograms showing the allele-specific binding of MSH6 in hybrid WT and Zfp57 -/- ESCs at the Plagl1 and Inpp5f ICRs. The SNVs associated with the maternal JF1 or paternal Black/6 (B6) alleles are shown at the top. The relative enrichment of the maternal over the paternal allele is reported below the electropherogram. Black arrows indicate the SNVs. d Bar plot showing relative enrichment of Biotin-tagged MSH2 protein to ICRs in WT and Zfp57 -/- ESCs. ChIP values were expressed as % input and normalized to the Smg7 region that is used as a positive control

Article Snippet: Streptavidin beads (Dynabeads MyOne streptavidin T1, ref 65,601, Invitrogen) and anti-MSH6 (Santa Cruz sc-137015), anti-H3K9me3 (Abcam 8898), anti-H3K4me3 (Abcam ab8580) antibodies or rabbit/mouse IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel.

Techniques: Binding Assay, Methylation, DNA Methylation Assay, Transfection, Methylation Sequencing, Positive Control

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: CircEZH2 interacts with m 6 A reader IGF2BP2 and blocks its ubiquitination-dependent degradation. A RNA pulldown was performed using biotin-labeled circEZH2 probe, followed by mass spectrometry analysis. B RNA pulldown assay was performed to verify the interaction between circEZH2 and IGF2BP2 in HCT116 and SW620 cells. C RBP immunoprecipitation (RIP) assay was performed using IGF2BP2 or IgG antibodies, followed by qRT-PCR assay for circEZH2 expression in HCT116 and SW620 cells. D Western blot was performed to detect the expression of IGF2BP2 in negative control (NC) and circEZH2-depleted (shRNA-1 and shRNA-2) HCT116 and SW620 cells. Hsp 70 served as a loading control. E qRT-PCR assay was performed to detect the mRNA levels of IGF2BP2 in negative control and circEZH2-depleted HCT116 and SW620 cells. F CircEZH2 knockdown accelerated the ubiquitination modification of IGF2BP2 in HCT116 cells transfected with ubiquitin (Ub) and treated with MG-132, which was detected by Western blot. G IHC staining of IGF2BP2 in tumors from control and circEZH2-OE AOM/DSS mice. Scale bar = 25 μm. H The mRNA level of IGF2BP2 in CRC ( n = 275) and adjacent normal ( n = 349) tissues was analyzed using TCGA database. I IHC staining of IGF2BP2 in CRC and adjacent normal tissues. Scale bar = 50 μm. J H score of IGF2BP2 in CRC and adjacent normal tissues ( n = 124). K Spearman’s correlation analysis revealed a positive association between IGF2BP2 protein expression and circEZH2 expression in CRC tissues (r = 0.4886; P < 0.01). L Kaplan-Meier analysis (log-rank test) of overall survival according to IGF2BP2 expression level ( P < 0.01). L Data were showed as mean ± SD. # P > 0.05, ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Ubiquitin Proteomics, Labeling, Mass Spectrometry, Immunoprecipitation, Quantitative RT-PCR, Expressing, Western Blot, Negative Control, shRNA, Control, Knockdown, Modification, Transfection, Immunohistochemistry

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: IGF2BP2 is a direct target of miR-133b in CRC. A Schematic illustration of IGF2BP2 3′-UTR wild-type (WT) and miR-133b binding site mutated (Mut) IGF2BP2 3′-UTR luciferases reporter vectors. B (Left panel) Relative luciferase activities were determined in HCT116 and SW620 cells transfected with IGF2BP2 3′-UTR WT or Mut luciferase reporter vectors and miR-133b or NC mimics. ( Right panel ) Relative luciferase activities were determined in HCT116 and SW620 cells transfected with IGF2BP2 3′-UTR WT or Mut luciferase reporter vectors and miR-133b inhibitor or inhibitor NC mimics. C IGF2BP2 mRNA expression was determined by qRT-PCR in HCT116 and SW620 cells transfected with miR-133b or NC mimics. D IGF2BP2 protein expression was determined by Western blot in HCT116 and SW620 cells transfected with miR-133b (50 nM and 100 nM) or NC mimics. Hsp 70 served as a loading control. E IGF2BP2 protein expression was determined by Western blot in control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE-treated HCT116 and SW620 cells. Hsp 70 served as a loading control. F-J CCK-8, plate colony-formation, EdU incorporation, wound healing and transwell assays were performed to determine the proliferation and migration abilities of control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE-treated HCT116 and SW620 cells. K Image of subcutaneous tumor xenografts in control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE groups. L The tumor growth curves of xenografts were plotted in control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE-treated HCT116 groups. M The tumor weights of xenografts were evaluated. Data were showed as mean ± SD. ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Binding Assay, Luciferase, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, CCK-8 Assay, Migration

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: CircEZH2/IGF2BP2 enhances the stability of CREB1 mRNA in CRC. A MeRIP-seq (accession number: GSE179042) analysis was performed to discover the potential m 6 A modification profile in CRC tissues. m 6 A sites were displayed in 5′-UTR, CDS and 3′-UTR. B Schematic diagram based on MeRIP-seq showed the remarkable m 6 A modification site in the mRNA of CREB1. C qRT-PCR assay was performed to detect the expression of RGMA, FCGR1A, B4GALT3, NEK9, CLDN2, RTN4RL1, ZNF74, CENPL, RAB44 and CREB1 mRNAs in negative control (NC) and circEZH2-depleted HCT116 and SW620 cells. D Symbol showed the m 6 A motif in CRC tissues. E Stability of CREB1 mRNA was assessed by RNase R treatment followed by qRT-PCR in HCT116 and SW620 cells. F RBP immunoprecipitation (RIP) assay demonstrated the CREB1 mRNA enrichment precipitated by anti-IgG or anti-IGF2BP2 antibodies in negative control and circEZH2-depleted HCT116 and SW620 cells. G RIP assays demonstrated the CREB1 mRNA levels precipitated by anti-IgG or anti-m 6 A antibodies in NC and circEZH2 shRNA, control and miR-133-OE and NC and IGF2BP2 siRNA HCT116 cells. H Relative expression of CREB1 mRNA in CRC tissues and matched adjacent normal tissues was analyzed by qRT-PCR assays ( n = 124). I Spearman’s correlation analysis revealed a positive association between CREB1 mRNA expression and circEZH2 expression in CRC tissues (r = 0.5903; P < 0.01). J IHC staining of CREB1 in CRC and adjacent normal tissues. Scale bar = 50 μm. K H score of CREB1 in CRC and adjacent normal tissues ( n = 124). L Spearman’s correlation analysis revealed a positive association between CREB1 protein expression and circEZH2 expression in CRC tissues (r = 0.2831; P < 0.01). M The expression levels of miR-133b and CREB1 in tumors from control and circEZH2-OE AOM/DSS models were determined by qRT-PCR assay. N IHC staining of CREB1 in tumors from control and circEZH2-OE AOM/DSS mice. Scale bar = 25 μm. Data were showed as mean ± SD. # P > 0.05, ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Modification, Quantitative RT-PCR, Expressing, Negative Control, Immunoprecipitation, shRNA, Control, Immunohistochemistry

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: CircEZH2 aggravates CRC progression through modulating CREB1 expression. A CREB1 mRNA expression was determined by qRT-PCR in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 and SW620 cells. B CREB1 protein expression was determined by Western blot in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 and SW620 cells. C-G CCK-8, plate colony-formation, EdU incorporation, wound healing and transwell assays were performed to determine the proliferation and migration abilities of control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 and SW620 cells. H Image of subcutaneous tumor xenografts in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 groups. I The tumor growth curves of xenografts were plotted in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 groups. J Schematic illustration indicates the mechanism by which circEZH2/miR-133b/IGF2BP2/CREB1 axis aggravates CRC progression. Data were showed as mean ± SD. ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Expressing, Quantitative RT-PCR, Control, shRNA, Western Blot, CCK-8 Assay, Migration

Journal: eLife

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

doi: 10.7554/eLife.14530

Figure Lengend Snippet: ( A ) Glutamate release by synaptosomes, monitored by enzymatic conversion of glutamate by glutamate dehydrogenase (GluDH) (see Materials and methods). The functionality of isolated synaptosomes was also assessed by immunoblot detection of phosphorylated Ser603 of synapsin1 and Ser774 of dynamin1 ( B and C ). ( A ) Synaptosomes were preincubated in buffer containing either Ca 2+ (final concentration 1.3 mM) or EGTA (final concentration 0.5 mM), followed by sequential addition of GluDH (200 U) and KCl (K + ) for depolarization (final concentration 50 mM) or not stimulated (no K + was added). Data represented as mean of three replicates, with the bars indicating the range of values. ( B and C ) Immunoblot analyses of phosphorylation changes in synapsin1 (Ser603) and dynamin1 (Ser774). Synaptosomes monitored and treated 2 min after addition of KCl as described in ( A ) were analyzed by immunoblotting using phosphospecific antibodies. ( B ) Representative immunoblots. To ensure equal loading, all samples were also blotted using antibodies insensitive to phosphorylation change, and tubulin as a loading control. ( C ) Quantification of the blot signals obtained from three independent experiments shown as the mean of the three replicates, with the error bars indicating the range of values. DOI: http://dx.doi.org/10.7554/eLife.14530.003

Article Snippet: The following primary antibodies were used: synapsin1, dynamin1-3 and α-tubulin (Synaptic Systems, Göttingen, Germany), phospho S774-dynamin1, phospho S603-synapsin1 (Rockland, Pottstow, PA).

Techniques: Isolation, Western Blot, Concentration Assay, Phospho-proteomics, Control

Journal: eLife

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

doi: 10.7554/eLife.14530

Figure Lengend Snippet: Partial spectra of mass spectrometric survey scans in MS1 (precursor spectra) containing phosphopeptides of synapsin Ser603 ( A ) and dynamin S774 ( B ) in light (K + , Ca 2+ ) and heavy (K + , EGTA) isotopic pattern that is used to calculated the relative abundances. MS/MS spectra resulting from HCD fragmentation of phosphopeptides containing synapsin Ser603 m/z 538.7478 2+ ( C ) and dynamin Ser774 m/z 703.2123 2+ ( D ), respectively, showing the phosphopeptides _QAS(ph)QAGPGPR_ and _RS(ph)PTS(ph)SPTPQR. Both of these phosphopeptides were eluted in the first fraction of SCX. MaxQuant Viewer (version 1.5.0.25) was used to illustrate the spectra. DOI: http://dx.doi.org/10.7554/eLife.14530.009

Article Snippet: The following primary antibodies were used: synapsin1, dynamin1-3 and α-tubulin (Synaptic Systems, Göttingen, Germany), phospho S774-dynamin1, phospho S603-synapsin1 (Rockland, Pottstow, PA).

Techniques: Tandem Mass Spectroscopy

Journal: eLife

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

doi: 10.7554/eLife.14530

Figure Lengend Snippet: Colors indicate phosphosites that are upregulated (red), downregulated (green), or remain unchanged (black) in depolarization with Ca 2+ versus depolarization without Ca 2+ comparison (K + , Ca 2+ versus K + , EGTA). The underlined phosphosites represent sites previously reported to be physiologically relevant (for references see Supplementary file 3, available at Dryad ). Unmarked phosphosites represent sites previously reported only by proteomic discovery-mode mass spectrometry, defined as HTP in the publicly available PhosphoSitePlus database. Phosphosites marked with a star represent phosphosites reported here for the first time. For comparison, phosphosites found in the well-studied proteins dynamin1 and synapsin1 are shown. DOI: http://dx.doi.org/10.7554/eLife.14530.013

Article Snippet: The following primary antibodies were used: synapsin1, dynamin1-3 and α-tubulin (Synaptic Systems, Göttingen, Germany), phospho S774-dynamin1, phospho S603-synapsin1 (Rockland, Pottstow, PA).

Techniques: Comparison, Mass Spectrometry

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: (A) Ovaries transgenic for mGFP-tagged MU2 were examined for mGFP fluorescence (green) and (A′) counterstained with DAPI. The oocyte nucleus and cytoplasm were fluorescent although the nucleus is brighter (arrowhead). (B) Testes from the transgenic animals were examined for mGFP fluorescence and (B′) DAPI staining. Fluorescence is not visible in the male germ cells, but is visible in somatic sheath cells. (C) Polytene chromosomes from salivary glands of transgenic animals carrying mGFP-tagged MU2 were examined for fluorescence. The localization of mGFP-MU2 was similar to the DAPI staining (C′). (D) Immmunostaining of Oregon R ovaries using anti-MU2 antiserum showed that MU2 is present primarily in the oocyte nucleus. The fluorescent signal in the oocyte nucleus may be more pronounced than in panel A. (E) mu2 a oocytes immunostained with anti-MU2 antibody and (E′) with DAPI. The overall fluorescence intensity is much less than in panel D. (F) Mutant ( mu2 a ) and wild type (OreR) larval (L) and the adult (A) extracts were prepared using RIPA buffer, and the MU2 protein in the extracts was detected using mouse anti-MU2 antiserum.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Transgenic Assay, Fluorescence, Staining, Mutagenesis

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: Immunostaining of Oregon R (A) and mu2 a (B) germaria shows γH2Av in red as a mark for DSBs, MU2 in green, and DNA as identified by DAPI in blue. The anterior tip of the germarium is pointing up. MU2 foci co-localize with the γH2Av foci, as shown in the merged image of Oregon R. The small amount of MU2 signal in the mu2 a mutant does not co-localize with γH2Av. (C) Plot showing the difference in the number of γH2Av foci in the germaria of Oregon R and mu2 a ovaries. γH2Av foci were counted in at least ten germaria showing equal intensity of staining without changing any settings of the instruments. The number of foci is represented as the mean±SD.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Immunostaining, Mutagenesis, Staining

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: (A) S2 cells were transiently transfected with pAGW-MU2 (N-terminal eGFP tag), irradiated with 25 Gy at 5 Gy/min 24 hrs after transfection, and fixed immediately at the termination of treatment. Control S2 cells on the left expressing eGFP-MU2 (green) and stained with γH2Av (red). After irradiation, shown in the middle and right panels, MU2 localized to distinct foci, which co-localized with γH2Av as observed in the merged image (yellow). (B) S2 cells were irradiated with 25 Gy, and stained with anti-MU2 (green) and rabbit anti-γH2Av (red) antibodies. Unirradiated S2 cells are shown on the left; irradiated S2 cells are shown in the middle and right panels. After irradiation, MU2 protein localized to distinct foci and co-localized with γH2Av. (C) Kinetics of the formation of MU2 foci were studied at different intervals after irradiation. MU2 foci appear immediately after irradiation and co-localize with γH2Av.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Transfection, Irradiation, Control, Expressing, Staining

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: RNAi was performed in S2 cells targeting two different regions of MU2. After four days of continuous treatment cells were irradiated with 25 Gy at 5 Gy/min, fixed immediately, and stained with rabbit anti-γH2Av (red) antibodies and DAPI (blue). dsRNA treatment with the FHA or the BTCT domain of MU2 are represented as FHA RNAi and BRCT RNAi, respectively, along with a control unrelated RNAi as described earlier. The lower set of panels show γH2Av foci after a chase of 15 min.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Irradiation, Staining, Control

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: Peptide pull-down assays were performed using chemically synthesized peptides of H2Av (phosphorylated and unphosporylated) to examine the interaction between bacterially expressed domains of MU2 and the peptides of H2Av. The H2Av peptides were biotinylated at the N terminus, which was used to conjugate them to the streptavidin agarose beads. These beads were then incubated with bacterially expressed and purified GST, GST-FHA, GST-BRCT domains of MU2. Panel (A) shows a coomassie-stained SDS polyacrylamide gel of an H2Av peptide pull-down experiment (γH2Av indicates the phosphopeptide; H2Av indicates the unphosphorylated peptide). Expressed GST-FHA and GST-BRCT domains, and bands appearing at the same molecular weight in other lanes, were identified using mass spectrometry. The GST-BRCT domain is pulled down specifically by the phosphorylated form of the peptide (position of the band indicated by an asterisk). (B) Biotinylated H2Av (phosporylated or unphosphorylated) peptides were incubated with nuclear extracts from S2 cells to isolate the MRN complex. Ten mg of total protein from the nuclear extract was loaded onto agarose beads. At the end of incubation the beads were washed, and the MRN complex was detected by Western analysis.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Synthesized, Incubation, Purification, Staining, Phospho-proteomics, Molecular Weight, Mass Spectrometry, Western Blot

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: (A) Immunostaining of an Oregon R ovary with rabbit anti-RAD50 detected with secondary goat anti-rabbit IgG AlexaFluor 594 (red), and counterstained with DAPI (blue). (B) Germarium of an Oregon R ovary stained with rabbit anti-RAD50 (red), mouse anti-MU2 (green), and DAPI (blue) showing co-localization of RAD50 and MU2 in foci (yellow). (C) Immunoblot analysis of the eluates of the GST pull-down experiments detecting the physical interaction between the N-terminal FHA domain of MU2 and components of the MRN complex. (D) Genetic interaction between nbs and mu2 was assessed by studying the levels of apoptosis in Oregon R, homozygous nbs and mu2 a mutants, and mu2 a nbs double mutants. Wing imaginal discs were dissected, and propidium iodide staining was performed to detect apoptosis.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Immunostaining, Staining, Western Blot

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: Diagram of the MU2 protein showing the N-terminal FHA domain and the C-terminal tandem BRCT domain. Arrows show the positions of threonine-glutamine (TQ) dipeptides. Numbers indicate the amino acid positions. Homology to MDC1 is based on a blast2 comparison using BLOSUM45.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Comparison

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: (A) Eye imaginal discs from Oregon R and mu2 a larvae were treated with 0.05 M HU for 2.5 h. Mitotic cells were assessed by staining the discs with the mitosis-specific marker phospho-histone 3 (PH3) Ser 10 followed by fluorescence microscopy. (B) Plot showing the number of mitotic cells in each genotype in the presence and absence of HU. Five discs were assayed for the number of mitotic cells. The mu2 a mutant was assayed in two different genetic backgrounds.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Staining, Marker, Fluorescence, Microscopy, Mutagenesis

Journal: PLoS Genetics

Article Title: Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster

doi: 10.1371/journal.pgen.1000473

Figure Lengend Snippet: In a wild type genetic background a DSB induced by irradiation in a stage 14 egg chamber leads to the activation of a DNA damage response checkpoint and DNA repair. In mu2 a mutants recognition of a DSB is impaired. Thus, the damage response checkpoint is not activated and DNA repair does not ensue. This leads to incorporation of the unrepaired break into the zygote. The lack of cell cycle control in the early embryo (until cycle 10) and the presence of relatively high levels of telomeric proteins (deposited maternally) may lead to the association of the DSB with these proteins and the establishment a new telomere.

Article Snippet: Cells were incubated with primary rabbit anti-γH2Av at 1∶1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1∶100).

Techniques: Irradiation, Activation Assay, Control