Journal: Life Science Alliance

Article Title: Huntingtin (HTT) interactome in regulation of DNA repair/remodeling and RNA processing pathways

doi: 10.26508/lsa.202503424

Figure Lengend Snippet: (A) Representative images of ISPNs treated with bleomycin (10 μg/ml, 2 h) costained with TCERG1 and HTT (MCA2050) antibodies. Orthogonal projections of z-stacks (right panels) demonstrate colocalization. Scale bar, 2 μm. (B) Graph shows Pearson’s and Manders’ colocalization coefficients. The data are presented as the mean ± SD. t test with equal variances: ** P < 0.001, n = 5 (five images taken from independent wells with 5–10 cells per image). (C) Representative co-IP experiment from control and HD (180) ISPNs treated with bleomycin (10 μg/ml, 2 h) and untreated (NT). Total cell lysates were prepared, and HTT complexes were immunoprecipitated using antibodies to total HTT (MCA2050). TCERG1 and HTT proteins were detected in the IPs. IgG negative control IPs are shown at the bottom panel. The inputs for HTT, TCERG1, and beta-tubulin are shown (right panels; the same lysates were reused for HTT/BRG1/ARID1A co-IPs shown in ). (D) Proximity ligation assay (PLA) in normal (33CAG) and HD (180CAG) ISPNs treated with bleomycin (10 μg/ml, 2 h) and untreated using TCERG1 and HTT antibodies. Images shown for 180CAG ISPNs were taken at higher intensity for illustrative purpose, whereas quantitation was done with the same settings as for 33CAG ISPNs. Scale bar, 10 μm. (E) Graphs show quantification of PLA signals using MetaXpress software (Molecular Devices). The data are presented as the mean ± SEM of the number of nuclear PLA sites per cell, sum intensity of nuclear PLA sites per cell, and average nuclear PLA site intensity relative to technical negative control within each experiment. Y-axis labels are displayed at the top of each graph. One-way ANOVA with pairwise multiple comparison procedures (Holm–Sidak’s method): * P < 0.01, ** P = 0.001, n = 3 (three images taken from independent wells, with 15–20 cells per image). t test with equal variances: ^ P = 0.005, ^^ P < 0.001, # P = 0.016, ## P = 0.003, n = 3 (three images taken from independent wells with 15–20 cells per image).

Article Snippet: Other antibodies used were as follows: ATM (2C1) from Genetex (#GTX70103), 1:1,000 dilution; phospho-ATM (Ser1981) from R&D Systems (#AF1655), 1:1,000 dilution; phospho-histone H2A.X (S139) (3F2) from Genetex (#GTX80694), 1:1,000 dilution; histone H2A.X (D17A3) from Cell Signaling Technology (#7631), 1:1,000 dilution; phospho-DNA-PKcs (S2056) from Abcam (#18192), 1:1,000 dilution; DNA-PKcs (E6U3A) from Cell Signaling Technology (#38168), 1:1,000 dilution; TCERG1/CA150 from Bethyl Laboratories (#A300-360A), 1:1,000 dilution; BRG1 (BLR106H) from Bethyl Laboratories (#A700-106), 1:1,000 dilution; ARID1A/BAF250 (BLR279L) from Bethyl Laboratories (#A700-279), 1:1,000 dilution; and b-actin (C4) from Santa Cruz Biotechnology (#sc-47778), 1:2,000 dilution.

Techniques: Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Negative Control, Proximity Ligation Assay, Quantitation Assay, Software, Comparison

Journal: Life Science Alliance

Article Title: Huntingtin (HTT) interactome in regulation of DNA repair/remodeling and RNA processing pathways

doi: 10.26508/lsa.202503424

Figure Lengend Snippet: (A) BAF chromatin remodeling and Mediator complexes identified among HTT-associated proteins (Metascape analysis). (B) Representative co-IP experiments from control and HD (180) ISPNs treated with bleomycin (10 μg/ml, 2 h) and untreated (NT). Total cell lysates were prepared, and HTT complexes were immunoprecipitated using antibodies to total HTT (MCA2050). BRG1, ARID1A, and HTT were detected in the immunoprecipitates (IPs). IgG negative control IPs are shown at the bottom panels. The inputs for HTT, BRG1, ARID1A, and beta-tubulin are shown (right panels; the same lysates were reused for HTT/TCERG1 co-IPs shown in ).

Article Snippet: Other antibodies used were as follows: ATM (2C1) from Genetex (#GTX70103), 1:1,000 dilution; phospho-ATM (Ser1981) from R&D Systems (#AF1655), 1:1,000 dilution; phospho-histone H2A.X (S139) (3F2) from Genetex (#GTX80694), 1:1,000 dilution; histone H2A.X (D17A3) from Cell Signaling Technology (#7631), 1:1,000 dilution; phospho-DNA-PKcs (S2056) from Abcam (#18192), 1:1,000 dilution; DNA-PKcs (E6U3A) from Cell Signaling Technology (#38168), 1:1,000 dilution; TCERG1/CA150 from Bethyl Laboratories (#A300-360A), 1:1,000 dilution; BRG1 (BLR106H) from Bethyl Laboratories (#A700-106), 1:1,000 dilution; ARID1A/BAF250 (BLR279L) from Bethyl Laboratories (#A700-279), 1:1,000 dilution; and b-actin (C4) from Santa Cruz Biotechnology (#sc-47778), 1:2,000 dilution.

Techniques: Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Negative Control

Journal: Life Science Alliance

Article Title: Huntingtin (HTT) interactome in regulation of DNA repair/remodeling and RNA processing pathways

doi: 10.26508/lsa.202503424

Figure Lengend Snippet: (A) Representative images of ISPNs treated with bleomycin (10 μg/ml, 2 h) costained with BRG1 and HTT (MCA2050) antibodies. Orthogonal projections of Z-stacks (shown for red and green merged panels) demonstrate colocalization. Scale bar, 10 μm. (B) Graph shows Pearson’s and Manders’ colocalization coefficients. The data are presented as the mean ± SD. t test with equal variances: * P = 0.009, n = 4 (four images taken from independent wells with 15–20 cells per image). (C) Proximity ligation assay (PLA) in normal (33CAG) and HD (180CAG) ISPNs treated with bleomycin (10 μg/ml, 2 h) and untreated using BRG1 and HTT antibodies. Images shown for 180CAG ISPNs were taken at higher intensity for illustrative purpose, whereas quantitation was done with the same settings as for 33CAG ISPNs. Scale bar, 10 μm. (D) Graphs show quantification of PLA signals using MetaXpress software (Molecular Devices). The data are presented as the mean ± SEM of the number of nuclear PLA sites per cell, sum intensity of nuclear PLA sites per cell, and average nuclear PLA site intensity relative to technical negative control within each experiment. Y-axis labels are displayed at the top of each graph. t test with equal variances: * P = 0.016, ** P = 0.011, *** P = 0.009, # P = 0.005, ## P < 0.001, ^ P = 0.006, ^^ P = 0.002, n = 3 (three images taken from independent wells with 10–20 cells per image).

Article Snippet: Other antibodies used were as follows: ATM (2C1) from Genetex (#GTX70103), 1:1,000 dilution; phospho-ATM (Ser1981) from R&D Systems (#AF1655), 1:1,000 dilution; phospho-histone H2A.X (S139) (3F2) from Genetex (#GTX80694), 1:1,000 dilution; histone H2A.X (D17A3) from Cell Signaling Technology (#7631), 1:1,000 dilution; phospho-DNA-PKcs (S2056) from Abcam (#18192), 1:1,000 dilution; DNA-PKcs (E6U3A) from Cell Signaling Technology (#38168), 1:1,000 dilution; TCERG1/CA150 from Bethyl Laboratories (#A300-360A), 1:1,000 dilution; BRG1 (BLR106H) from Bethyl Laboratories (#A700-106), 1:1,000 dilution; ARID1A/BAF250 (BLR279L) from Bethyl Laboratories (#A700-279), 1:1,000 dilution; and b-actin (C4) from Santa Cruz Biotechnology (#sc-47778), 1:2,000 dilution.

Techniques: Proximity Ligation Assay, Quantitation Assay, Software, Negative Control

Journal: Life Science Alliance

Article Title: Huntingtin (HTT) interactome in regulation of DNA repair/remodeling and RNA processing pathways

doi: 10.26508/lsa.202503424

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids encoding tagged TCERG1 (T7-tag), BRG1 (FLAG tag), or ARID1A (V5-tag). Western blotting was performed with total cell lysates using antibodies to tags or with protein-specific antibodies as indicated. The inputs for beta-tubulin are shown as a loading control. (B) Normal ISPNs were transfected with siRNA to DNA-PKcs or with scrambled control siRNA. Western blot with indicated antibodies of total cell lysates. Graphs shows Western blot quantitation of total and phospho-DNA-PKcs normalized to actin. Data are presented as % of control siRNA. (C) Representative images of ISPNs transfected with siRNA to DNA-PKcs or with scrambled control siRNA and costained with antibodies to pDNA-PKcs and to SC35. Scale bar, 5 μm. The graph shows mean intensity (±SD) of green (pDNA-PKcs) and red(SC35) channels within cell nuclei. *Mann–Whitney rank sum test: P < 0.01, n = 10 (individual cells).

Article Snippet: Other antibodies used were as follows: ATM (2C1) from Genetex (#GTX70103), 1:1,000 dilution; phospho-ATM (Ser1981) from R&D Systems (#AF1655), 1:1,000 dilution; phospho-histone H2A.X (S139) (3F2) from Genetex (#GTX80694), 1:1,000 dilution; histone H2A.X (D17A3) from Cell Signaling Technology (#7631), 1:1,000 dilution; phospho-DNA-PKcs (S2056) from Abcam (#18192), 1:1,000 dilution; DNA-PKcs (E6U3A) from Cell Signaling Technology (#38168), 1:1,000 dilution; TCERG1/CA150 from Bethyl Laboratories (#A300-360A), 1:1,000 dilution; BRG1 (BLR106H) from Bethyl Laboratories (#A700-106), 1:1,000 dilution; ARID1A/BAF250 (BLR279L) from Bethyl Laboratories (#A700-279), 1:1,000 dilution; and b-actin (C4) from Santa Cruz Biotechnology (#sc-47778), 1:2,000 dilution.

Techniques: Transfection, FLAG-tag, Western Blot, Control, Quantitation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet: (a) Pseudo-bulked L2FC plotted against the negative logarithm of the p value (two-tailed Wald test with multiple testing correction by Benjamini-Hochberg method) for genes in VPKO Early versus VKO Early cells. Genes significantly (p < 0.01) and robustly (|L2FC| > 0.5) regulated are colored green. Candidate genes pertaining to lipid and sterol metabolism and epithelial morphogenesis and adhesion are labelled. (b) Gene Ontology terms for biological processes that are significantly over-represented among genes upregulated by Pbrm1 inactivation in Vhl -null cells. GO terms whose over-representation is significant (p < 0.01; one-sided Fisher’s exact test corrected by false discovery rate), and whose member genes together exhibit a net positive average L2FC in VPKO Early versus VKO Early cells are shown. Terms are ordered and tiles are colored by the average L2FC. (c) Pseudo-bulked L2FC in VPKO Early versus VKO Early cells plotted against changes in VPKO Late versus VKO Late cells showing that Pbrm1 -dependent regulation of genes is correlated between the two timepoints. (d) Pseudo-bulked L2FC plotted against the negative logarithm of the p value (two-tailed Wald test with multiple testing correction by Benjamini-Hochberg method) for genes in VPKO Late versus VPKO Early cells, showing that genes significantly regulated after Pbrm1 inactivation at the early timepoint (colored green) are mostly not regulated significantly over time (i.e., have either |L2FC| < 0.5 or p > 0.01 in the VPKO Late versus VPKO Early comparison). (e) Pseudo-bulked L2FC in VPKO Late versus VPKO Early cells plotted against changes in VKO Late versus VKO Early cells showing that time-dependent changes in gene expression are correlated in Vhl- null and Vhl/Pbrm1 -null cells. (f) Expression of sets of genes up- or downregulated over time in Vhl -null cells of any PT identity (‘Adaptive Up’ or ‘Adaptive Down’) in ConKO, VKO, and VPKO cells sequenced either 1-3 weeks (early) or 4-12 months (late) following recombination. Median values and inter-quartile ranges of the data are presented separately for cells from each mouse. Pairwise comparisons have been made between means of the median values for mice of different genotypes using two-tailed two-way ANOVA with multiple testing correction using the Benjamini-Hochberg method. Adaptive upregulation is maintained, and adaptive downregulation is partially accelerated following Pbrm1 inactivation in Vhl -null cells. (g) Pseudo-bulked L2FC in VPKO Early versus VKO Early cells plotted against changes in ConPKO Early versus ConKO Early cells, showing that Pbrm1 -dependent gene regulation is correlated in Vhl -null and Vhl -competent cells. (h) Pseudo-bulked L2FC in VKO Early versus ConKO Early cells plotted against changes in VPKO Early versus ConPKO Early cells, showing that Vhl -dependent gene regulation is correlated between Pbrm1 -competent and Pbrm1 -null cells. (c, e, g, h ) Spearman’s correlation coefficient (ρ) calculated for genes that exhibit significant regulation in either of the depicted comparisons. (i) Oil Red O staining in renal sections from ConKO, ConPKO, VKO, and VPKO mice harvested 4-12 months (late) following recombination, showing the accumulation of lipid droplets (stained red) in PT cells of ConPKO and VPKO mice. Scale bar, 100 μm. 40x magnification.

Article Snippet: Antigens were detected by overnight incubation at 4°C with primary antibodies diluted 1:1000 in Dako Antibody Diluent Solution (Agilent S3022): tdTomato – anti-RFP (Rockland 600-401-379, RRID:AB_2209751); PBRM1 – anti-PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865).

Techniques: Two Tailed Test, Comparison, Gene Expression, Expressing, Staining

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet: (a) Pseudo-bulked L2FC in VPKO Late versus ConKO Late cells plotted against ‘predicted’ changes calculated as addition of the effects of Vhl inactivation (VKO Late vs ConKO Late) and early effects of Pbrm1 inactivation (VPKO Early vs VKO Early). Genes that exhibited significant regulation in either of the depicted comparisons are plotted. Genes whose regulation in VPKO Late versus ConKO Late cells exhibit significant (p < 0.01; two-sided Wald z-test) deviation from the additive effects of Vhl and Pbrm1 inactivation are colored green. Spearman’s correlation coefficient (ρ). (b) Cells from ConKO, VKO, and VPKO mice projected onto UMAP space and clusters defined for VPKO Late cells (left panel), and the proportion of cells in each cluster derived from mice of each of the genotypes and timepoints (right panel), showing differential occupancy of Clusters 12, 9, and 2 by VPKO Late cells. (c-e) Proportion of cells from VPKO Late mice or mice from other genotypes and timepoints in Clusters 12 (c) , 9 (d ), and 2 (e ). Pairwise comparisons by Wilcoxon test. Error bars indicate the median and the inter-quartile range. (f) Expression of candidate genes that are part of the integrated stress response (ISR), and expression score for an ISR gene module derived from Han et al., 2023, in cells from Clusters 9, 12, or other clusters, showing that Clusters 12 and 9 are both characterized by increased expression of ISR genes. (g) Proportion of cells with detected reads for candidate genes downregulated in cells from Cluster 2 compared to those in the remaining clusters, showing that Cluster 2 cells are characterized by reduced expression of several PT differentiation transcription factors and adhesion molecules.

Article Snippet: Antigens were detected by overnight incubation at 4°C with primary antibodies diluted 1:1000 in Dako Antibody Diluent Solution (Agilent S3022): tdTomato – anti-RFP (Rockland 600-401-379, RRID:AB_2209751); PBRM1 – anti-PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) HTT immunocomplexes (ICs) analyzed by western blots (WBs) from nuclear protein extracts (NEs) isolated from 3-month-old wild-type C57BL/6 mouse brain. HTT was immunoprecipitated (IP’d) from NEs with an anti-HTT monoclonal antibody (Ab) (MAB2710, Millipore-Sigma). Immunocomplexes (ICs) tested for BRG1 and TC- NHEJ factors DNA-PKcs, Ku70, PNKP, XRCC4, LIG IV, POLR2A, CSB, and TFIIS in HTT ICs. Lane 1; protein molecular weight marker; lane 2: Input; lane 3: IgG control IP; lane 4: IP of HTT with anti-HTT Ab. (B) mHTT WBs IP’d from NEs isolated from 3-month-old HD homozygous zQ175 transgenic mouse brain with an anti-HTT Ab (MAB2710, Millipore-Sigma). mHTT ICs were analyzed by WBs for BRG1, and TC-NHEJ factors. Lane 1; protein molecular weight marker; lane 2: Input; lane 3: IgG control IP; lane 4: IP of HTT with an anti-HTT Ab. (C-G) Proximity ligation assay (PLA) for HTT and NHEJ factors in wild-type SH-SY5Y and in SH-SY5Y cells after treating the cells with DNA damaging agent etoposide (ET; 15µM, for 30 minutes). Generation of red fluorescence indicates representative positive protein-protein interactions. Nuclei stained with DAPI. (C) HTT interaction with Ku70 changes in response to increased DSBs. PLA was performed on SH-SY5Y cells with anti-HTT rabbit monoclonal Ab (5656; Cell Signaling) and anti-Ku70 mouse monoclonal Ab (SC-5309; Santa Cruz) before and after treating cells with ET (-ET and + ET respectively). (D) HTT and Ku70 relative interactions by PLA signals in control untreated (-ET) and ET- treated (+ ET) cells. Data represent mean ± SD, ****p<0.0001. (E) HTT interactions with XRCC4 in response to increased DSBs. PLA was performed with rabbit monoclonal anti-HTT Ab (5656; Cell Signaling) and anti-XRCC4 mouse monoclonal Ab (SC-271087; Santa Cruz) before and after treating the SH-SY5Y cells with ET (-ET and + ET respectively). (F) HTT and XRCC4 relative interactions assessed by relative PLA signals in control (- ET) cells and ET-treated (+ ET) cells. Data represent mean ± SD, ****p<0.0001. (G) HTT and PNKP interactions in response to increased DNA damage. PLA performed with anti-HTT mouse monoclonal Ab (MAB2170; Millipore-Sigma) and anti-PNKP rabbit polyclonal Ab (MBP-1-A7257; Novus) before and after treating SH-SY5Y cells with ET (-ET and + ET respectively). (H) HTT and PNKP interactions assessed by relative PLA signals in control (- ET) cells and ET-treated (+ ET) cells. Data represent mean ± SD, ****p<0.0001. (I) HTT association with genome. Chromatin immunoprecipitation (ChIP) performed to assess association/interaction of HTT with genome in untreated SH-SY5Y cells in a stress-free condition. After ChIP, DNA fragments purified from the cross-linked protein-DNA entities and the purified genomic DNA fragments analyzed by real-time quantitative PCR (qPCR). Relative ChIP values measured with respect to control IgG. Data represent mean ± SEM, ****p<0.0001. (J) DSBs measured by neutral comet analysis. SH-SY5Y cells were treated with DNA damaging agent bleomycin (BL; 5µg/mL, for 30 minutes), and subjected to neutral comet analysis to detect DSBs before (-BL) and after BL treatments (+ BL). Increased comet tail moments after BL treatments indicate presence of DSBs (shown with arrows; right panel). (K) HTT association with genome before and after inducing DSBs. SH-SY5Y cells treated with BL for 30 minutes to induce DSBs, and ChIP performed on control untreated cells and BL-treated cells. Relative ChIP values measured after normalization to control IgG. Data represent mean ± SEM, ****p<0.0001. ( L-P ) Sequential ChIP (ChIP-re-ChIP) analysis was performed to determine whether HTT and NHEJ factors co-occupy the same genomic DNA loci in vivo . (L) HTT-cross-linked genomic DNA. DNA fragments were isolated from the wildtype C57BL/6 mouse brain, and the DNA-protein complexes IP’d with an anti-HTT Ab (MAB2170; Millipore-Sigma), followed by a second immunoprecipitation (IP) of the HTT ICs with either an anti-Ku70 or an anti-IgG Abs. Genomic DNA fragments were isolated from the final Ku70 ICs and IgG ICs, and genomic DNA segments (∼250 bp) encompassing Neurod1 (ND1) or Tubulin Beta 3 Class III (Tubb3) genomic regions were amplified by quantitative PCR (qPCR) using specific primers and PCR products were quantified by qPCR. Relative ChIP values were measured after normalization to control IgG. Data represent mean ± SEM, ****p<0.0001. (M) Ku70-bound genomic DNA. DNA fragments were IP’d with an anti-Ku70 Ab (SC- 5309; Santa Cruz), followed by a second IP of the Ku70 ICs with an anti-HTT (MAB2170; Millipore-Sigma) or an anti-IgG Abs. DNA isolated from the final HTT ICs and IgG ICs, and genomic DNA segments (∼250 bp) encompassing Neurod1 or Tubb3 genes was amplified using specific primers and the PCR products quantified. Relative ChIP values were measured after normalization to control IgG. Data represent mean ± SEM, ****p<0.0001. (N) HTT-DNA complexes from anti-HTT and anti-XRCC4 Abs. Putative HTT-DNA complexes were IP’d with an anti-HTT Ab (MAB2170; Millipore-Sigma), followed by a second IP of the HTT ICs with either an anti-XRCC4 (SC-271087; Santa Cruz) or an anti-IgG Abs. The DNA fragments were isolated from XRCC4 ICs and IgG ICs, and genomic DNA segments (∼250 bp) encompassing Neurod1 or Tubb3 genes amplified, and PCR products analyzed by qPCR. Relative ChIP values measured after normalization to IgG. Data represent mean ± SEM, ****p<0.0001. (O) HTT-DNA complexes from anti-HTT and anti-DNA ligase IV Abs. Putative HTT- DNA complexes were IP’d with an anti-HTT Ab (MAB2170; Millipore-Sigma), followed by a second IP of the HTT ICs with either an anti-DNA ligase IV (14649; Cell Signaling) or an anti-IgG Abs. DNA isolated from the ligase IV ICs and IgG ICs, and genomic DNA segments encompassing Neurod1 or Tubb3 genes were amplified by qPCR and the PCR products analyzed. Relative ChIP values measured after normalization to control IgG. Data represent mean ± SEM, ****p<0.0001. (P) HTT-DNA complexes from anti-HTT and anti-PNKP Abs Putative HTT-bound DNA fragments were IP’d with an anti-HTT Ab (MAB2170; Millipore-Sigma), followed by a second IP of the HTT ICs with either an anti-PNKP Ab (MBP-1-A7257; Novus) or an anti-IgG Abs. DNA isolated from PNKP ICs and IgG ICs, and genomic segments (∼250 bp) encompassing Neurod1 or Tubb3 genes were amplified using specific primers and the PCR products quantified. Relative ChIP values measured after normalization to control IgG. Data represent mean ± SEM, ****p<0.0001.

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: Western Blot, Isolation, Immunoprecipitation, Molecular Weight, Marker, Control, Transgenic Assay, Proximity Ligation Assay, Fluorescence, Protein-Protein interactions, Staining, Chromatin Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, ChIP-chip, In Vivo, Amplification

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) Nuclear extracts (NEs) isolated from 3-month-old C57BL/6 mouse brain, and Ku70 IP’d from the NEs with an anti-Ku70 mouse Ab (SC-5309; Santa Cruz), and the Ku70 immunocomplex (ICs) analyzed by western blots to detect the endogenous HTT, BRG1 and associated NHEJ components. An anti-ApeI Ab (4128; Cell Signaling) used as negative control in the WBs in panels A, B and C. Lane 1; protein molecular weight marker; lane 2: Input; lane 3: IgG control IP; lane 4: IP of Ku70 with anti-Ku70 Ab. (B) NEs isolated from 3-month-old C57BL/6 mouse brain, DNA-PKcs IP’d with an anti- DNA-PKcs Ab (SC-390849; Santa Cruz), and DNA-PKcs ICs analyzed by WBs to detect the presence of HTT, BRG1 and NHEJ complex components. Lane 1; protein molecular weight marker; lane 2: Input; lane 3: IgG control IP; lane 4: IP of DNA-PKcs with anti-DNA-PKcs Ab. (C) NEs isolated from 3-month-old C57BL/6 mouse brain, and XRCC4 IP’d from the NEs with anti-XRCC4 Ab (SC-271087; Santa Cruz), and XRCC4 ICs analyzed by WBs to detect the presence of HTT, BRG1 and associated NHEJ factors. Lane 1; protein molecular weight marker; lane 2: IP Input; lane 3: IgG control IP; lane 4: IP of XRCC4 with anti-XRCC4 Ab. Proximity ligation assay (PLA) performed to assess interaction of HTT and NHEJ proteins in SH-SY5Y cells, and in the SH-SY5Y cells after inducing DSBs by treating the cells with etoposide (ET; 15µM; 30 minutes). Generation of red fluorescence indicates representative positive protein-protein interactions. Nuclei stained with DAPI (panels 4). (D) PLA performed with anti-HTT mouse Ab (MAB2170; Sigma-Millipore) and anti- Ku80 rabbit Ab (2753; Cell Signaling) before and after treating SH-SY5Y cells with ET to assess interaction between HTT and Ku80 in untreated cells and in cells treated with ET (after inducing DSBs). (E) Relative PLA signals show interaction of HTT and Ku80 in control (-ET) cells and ET- treated cells. Data represents mean ± SE, ****p<0.0001. (F) PLA performed with anti-HTT rabbit Ab, (5656; Cell Signaling) and anti-DNA-PKcs mouse Ab (SC-390849; Santa Cruz) before and after treating the SH-SY5Y cells with ET to assess interaction between HTT and DNA-PKcs in response to increased DSBs. (G) Relative PLA signals show interaction of HTT and DNA-PKcs in control (-ET) cells and ET-treated cells. Data represents mean ± SE, ****p<0.0001. (H) PLA performed with anti-HTT rabbit Ab (5656; Cell Signaling) and anti-CSB mouse Ab (SC-398022; Santa Cruz) before and after treating SH-SY5Y cells with ET to assess interaction of HTT with CSB before and after inducing DSBs. (I) Relative PLA signals show interaction of HTT and CSB in control (-ET) cells and ET- treated cells. Data represents mean ± SE, ****p<0.0001. (J) PLA performed with anti-HTT mouse Ab (2760; Millipore-Sigma) and anti-DNA LIG IV (LIG IV) rabbit Ab (14649; Cell Signaling) before and after treating SH-SY5Y cells with ET to assess interaction of HTT with CSB before and after inducing DSBs. (K) Relative PLA signals show interaction of HTT and LIG IV in control untreated cells (- ET) and in ET-treated (+ET) cells. Data represents mean ± SE, ****p<0.0001. (L) PLA performed with anti-HTT rabbit Ab (5656; Cell Signaling) and anti-PARP1 mouse Ab (14649; Santa Cruz) before and after treating SH-SY5Y cells with ET to assess interaction of HTT with PARP1 before and after inducing DSBs. (M) Relative PLA signals showing interaction of HTT and PARP1 in control (-ET) cells and ET-treated cells. Data represents mean ± SE, ****p<0.0001. (N) SH-SY5Y cells treated with BL for 30 minutes, and ChIP performed to assess association of PNKP with genome in control and BL-treated cells. Relative ChIP values measured after normalization to control IgG. Data represents mean ± SE, ****p<0.0001.

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: Isolation, Western Blot, Negative Control, Molecular Weight, Marker, Control, Proximity Ligation Assay, Fluorescence, Protein-Protein interactions, Staining

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) BRG1 ICs contain HTT and NHEJ components. NEs isolated from 3-month-old wild- type C57BL/6 mouse brain tissue (cortex) and BRG1 were IP’d from the NEs with an anti-BRG1 Ab (NB100-2594; Novus). Resulting BRG1 ICs were analyzed by WBs to detect BRG1, HTT and associated NHEJ components in BRG1 ICs. Lane M; protein molecular weight marker; lane 1: Input; lane 2: IgG control; lane 3: IP of BRG1 with anti-BRG1 Ab. (B) FLAG-HTT and Myc-BRG1 association. Plasmids p-FLAG-HTT-Q24 and p-Myc- BRG1, expressing FLAG-tagged wtHTT-Q24 and Myc-tagged human BRG1, respectively, were co-transfected into SH-SY5Y cells, cells harvested and NEs isolated 48 hours post-transfection. Expressions of FLAG-HTT and Myc-BRG1 were analyzed by WBs with anti-FLAG Ab (F3165; Millipore-Sigma) and anti-Myc 9E10 Ab (SC- 40; Santa Cruz) respectively. (C) Myc-tagged BRG1 and FLAG-tagged human HTT-Q24 association. Plasmids p- FLAG-HTT-Q24 and p-Myc-BRG1, expressing FLAG-tagged human HTT-Q24 and Myc-tagged human BRG1, respectively, were co-transfected into SH-SY5Y cells, NEs isolated from the cells 48 hours post-transfection. FLAG-tagged HTT-Q24 were IP’d from the NEs with an anti-FLAG Ab (F3165; Millipore-Sigma), and FLAG ICs analyzed by WBs with an anti-Myc 9E10 Ab (SC-40; Santa Cruz) to detect Myc-tagged BRG1 in FLAG ICs. (D) HTT and BRG1 proximity before and after ET induced DNA damage. Proximity ligation assays (PLA) were performed to assess possible interaction of HTT and BRG1 before (upper panel) and after (lower panel) inducing DNA damage by treating cells with ET (300 ng/mL, for 30 minutes). Reconstitution of red fluorescence indicates interaction of HTT with BRG1. Nuclei stained with DAPI. (E) Proximity of endogenous HTT with BRG1. Relative signals show PLA interactions of endogenous HTT with BRG1 in control untreated SH-SY5Y cells (Cntl; -ET), and in SH-SY5Y cells treated with ET (+ET; 300 ng/mL for 30 minutes). Data represent means ± SD, ****p<0.0001. (F) HTT and BRG1 co-localize in human brain. Postmortem human brain sections (striatum) analyzed by co-immunostaining with an anti-HTT mouse monoclonal Ab (MAB2170; Millipore-Sigma) and anti-BRG1 rabbit polyclonal Ab (3508; Cell Signaling) and sections analyzed by confocal microscopy. Nuclei stained with DAPI, and co-localization of HTT (green fluorescence) with BRG1 (red fluorescence) appear as yellow fluorescence (shown by arrows). (G) Predicted HTT-mediated protein assembly with BRG1, PNKP-DNA, and Ku70/80. AlphaFold3 (AF3) predicted complex is shown with color-coded confidence levels (left) and labeled protein chains (right). AF3 places HTT (green) in the core of an NHEJ protein complex (right). Ku70/80 (purple/pink, top) has a significant interface with HTT, which is sandwiched between BRG1 (cyan) and PNKP (blue) that neighbors the PolyQ extension (purple). The BRG1 Bromodomain (BRD: brown) fits into the HTT central cleft. HTT interfaces with BRG1 BRD and Ku70/80 have the highest AF3 confidence levels (left).

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: Isolation, Molecular Weight, Marker, Control, Expressing, Transfection, Ligation, Fluorescence, Staining, Immunostaining, Confocal Microscopy, Labeling

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) NEs isolated from SH-SY5Y cells constitutively expressing FLAG-tagged BRG1, and the exogenous FLAG-tagged BRG1 IP’d from the NEs with anti-FLAG Ab (F3165; Millipore-Sigma) and the FLAG IC analyzed by western blotting to detect HTT, and the key NHEJ proteins in the FLAG ICs. Lane 1: protein molecular weight marker; lane 2: Input; lane 3 IgG IP; lane 4: FLAG IP and lane 5: total cell extract (Total CE). The IgG heavy chain (IgG-HC) in the XRCC4 WB shown by arrow. (B) NEs isolated from SH-SY5Y cells constitutively expressing exogenous FLAG-tagged wtHTT carrying 19 glutamines (FLAG-wtHTT-Q19), and the exogenous FLAG- wtHTT-Q19 IP’d from the NEs with anti-FLAG Ab (F3165; Millipore-Sigma), and the FLAG ICs analyzed by western blotting to detect key NHEJ proteins in the FLAG ICs. Lane 1: protein molecular weight marker; lane 2: Input; lane 3 IgG IP; lane 4: FLAG IP and lane 5: total cell extract (Total CE). (C) Proximity ligation assay (PLA) performed to assess possible interaction of BRG1 and NHEJ proteins in SH-SY5Y cells. Generation of green fluorescence indicates representative positive protein-protein interactions. Nuclei stained with DAPI. PLA with anti-BRG1 (mouse) Ab and various NHEJ components e.g., Ku80 (Panel 1), Ku70 (Panel 2), PARP1 (Panel 3), XRCC4 (Panel 4), DNA ligase IV (Panel 5), DNA-PKcs (Panel 6), were performed before and after treating SH-SY5Y cells with ET.

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: Isolation, Expressing, Western Blot, Molecular Weight, Marker, Proximity Ligation Assay, Fluorescence, Protein-Protein interactions, Staining

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) NEs isolated from control SH-SY5Y cells (Cntl; lane 2), expressing human BRG1 cDNA (BRG1-OE; lane 3), expressing BRG1-RNAi (BRG1-KD; lane 4), NEs analyzed by WBs to detect BRG1, HTT and NHEJ protein levels; β-actin used as loading control. Lane 1: Protein molecular weight marker in kDa. (B) BRG1-KD SH-SY5Y cells (upper panel) analyzed by immunostaining with anti-53BP1 Ab (Cat # 2675; Cell Signaling) to detect the presence of DSBs (arrows). Nuclei stained with DAPI (blue). Control SH-SY5Y cells (lower panel) analyzed by immunostaining the cells with anti-53BP1 Ab (Cat # 2675; Cell Signaling) to detect the presence of DSBs in nuclei. Nuclei stained with DAPI (Blue). (C) Genomic DNAs isolated from the control SH-SY5Y cells (Cntl; lanes 1 to 3), SH- SY5Y cells expressing BRG1-RNAi (BRG1-KD; BRG1-knocked-down cells; lanes 4 to 6) or cells expressing human BRG1 cDNA (BRG1-OE cells; lanes 7 to 9), and DNA damage assessed by LA-QPCR. ∼8 to 10 kb regions of genome encompassing NEUROD1, BCL2L2, or BDNF) PCR-amplified, and the PCR products quantified. LA denotes long amplicon (8 to 10 kb); SA denotes short amplicon (0.2 to 0.3 kb); Lane 10: 1-kb DNA ladder. (D) Relative DNA damage in various genomic loci (NEUROD1, BCL2L2, or BDNF) quantified in control cells (Cntl), and cells expressing BRG1-RNAi (BRG1-KD) or human BRG1 cDNA (BRG1-OE). Data represents Mean ± SD. ****p<0.0001. (E) DSBs induced at transcriptionally active locus of chromosome 1 (Chr-1A) and transcriptionally inactive locus (Chr-1B) in control SH-SY5Y cells and BRG1-KD SH- SY5Y cells, cell harvested 135 minutes after adding liposomes, and ChIP performed to assess recruitment of HTT at the DSB sites in BRG1-KD and control SH-SY5Y cells. Data represents mean ± SD. ****p<0.0001; ns= not significant. (F) DSBs induced at transcriptionally active locus of chromosome 1 (Chr-1A) and transcriptionally inactive locus (Chr-1B) in control and HTT-KD SH-SY5Y cells, cell harvested 135 minutes after adding liposomes, and ChIP performed to assess the relative recruitment of BRG1 at the DSB site in HTT-KD and control SH-SY5Y cells. Data represents mean ± SD; ns= not significant.

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: Isolation, Control, Expressing, Molecular Weight, Marker, Immunostaining, Staining, Amplification, Liposomes

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) Association of HTT with transcriptionally active DSB regions. DSBs were introduced within the transcriptionally active gene-rich locus (locus A) and in transcriptionally inactive gene-poor locus (locus B) in chromosome 1 and 17 (Chr-1 and Chr-17 respectively) in cells with and without induction of I-SceI restriction enzyme. Induced DSBs and relative occupancy/recruitment of HTT at the DSB sites within the transcriptionally active and in transcriptionally inactive chromosomal regions before and after inducing DSBs were determined by ChIP analysis. Data represent Mean ± SD. **p<0.005; ****p<0.0001; ns= not significant. (B) RNA polymerase II (POLR2A) levels at the DSB sites before and after CRISPR-CAS9 gRNA-mediated induction of DSB at Chr17A locus in SH-SY5Y cells with and without HTT-shRNA-mediated depletion of HTT. Mean ± SD. ***p<0.001. (C) RNA polymerase II (POLR2A) levels at the DSB sites before and after I-Scel induced DSBs at Chromosome 1 at transcriptionally active locus (Chr-1A) as well as in transcriptionally inactive locus (Chr-1B) were measured in cells with and without depletion of HTT. Data represent Mean ± SD. ***p<0.001. NS= not significant. (D) Robust recruitment of NHEJ factors in STHdhQ7 but not in mutant STHdhQ111 cells. STHdhQ7 and mutant STHdhQ111 cells were tested by liposome-mediated delivery of CRISPR-CAS12 gRNA, cells harvested 135 minutes after adding liposomes, and ChIP performed to assess relative recruitment of NHEJ proteins e.g., PNKP, Ku70, and DNA ligase IV at the DSB site in mutant STHdhQ111 and wildtype STHdhQ7 cells. Data represent Mean ± SD. **p<0.005; ***p<0.001; ****p<0.0001; ns= not significant. (E) DSBs persist in mutant STHdhQ111 cells but not in control STHdhQ7 cells. Mutant STHdhQ111 cells (left panel) and control STHdhQ7 cells (right panel) were analyzed by immunostaining with an anti-p-53BP1Ab (2675; Cell Signaling) to detect the presence of double strand breaks (DSBs) in nuclear genome. The p-53BP1-positive DSBs in genomic DNA (green puncta) within the nuclei (blue) of STHdhQ111 cells (left panel) are shown by arrows. Similar nuclear puncta not detected in control cell nuclei (right panel). Nuclei stained with DAPI (blue). (F) Relative amounts of 53BP1-positive puncta indicating DNA damage in mutant STHdhQ111 cells is high compared with control STHdhQ7 cells. Data represent means ± SD, ****p<0.0001. (G) Lower metaphase aberrations in wildtype STHdhQ7 compared to mutant STHdhQ111 cells. Wildtype STHdhQ7 and mutant STHdhQ111 cells in plateau phase were irradiated with 3 Gy, incubated for 18 hours post-irradiation, and G1-type aberrations examined at metaphase. Categories of asymmetric chromosome aberrations scored included dicentrics, centric rings, interstitial deletions-acentric rings, and terminal deletions. The frequency of chromosomal aberrations in STHdhQ111 cells after IR exposure were compared with control cells. Data represent means ± SD, ****p<0.0001). (H) Similar exponential phase aberrations in wildtype STHdhQ7 compared to mutant STHdhQ111 cells. Wildtype STHdhQ7 and mutant STHdhQ111 cells in exponential phase were exposed to 2 Gy IR, and metaphases harvested 3 hours post-irradiation and examined for chromosomal aberrations. The difference between chromatid and chromosomal aberrations induced by IR is not significantly higher in STHdhQ111 cells compared to control cells. ns= not significant. (I) Wildtype STHdhQ7 and mutant STHdhQ111 cells in exponential phase were irradiated with 1 Gy IR. Metaphases were harvested after 1-hour post-irradiation and analyzed for chromosomal aberrations. The differences in chromosomal aberrations between samples treated with IR are not statistically significant between STHdhQ111 and STHdhQ7 cells. Data represent means ± SD, **p<0.005. (J) Chromosome aberrations measured in control cells, HTT-depleted cells and HTT and BRG1 depleted cells before (0 Gy) and after irradiating the cells with IR (3 Gy). Data represent means ± SD, ****p<0.0001. ns = not significant.

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: CRISPR, shRNA, Mutagenesis, Liposomes, Control, Immunostaining, Staining, Irradiation, Incubation

Journal: bioRxiv

Article Title: Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

doi: 10.1101/2024.09.19.613927

Figure Lengend Snippet: (A) Table of genes identified in RNAseq analysis that have P values of less than 0.001 when Drosophila expressing mHTT (UAS-128Qhtt FL ) were compared to WT control. The table depicts the genes sorted by increasing P value of difference when Drosophila expressing mHTT (UAS-128Qhtt FL ) were compared to WT control with P < 0.0001 noted by **** and P < 0.0001 noted by ***. The color of each table cell is based on the variance value of detected RNA level for that gene (row) and mutant (column) compared to WT control with white as no change, green as increased levels, and red as decreased levels. The deeper the color the more the variance from the WT control levels. The variance value is also included in each cell. (B) The table depicts the weighted difference of the level of the co-overexpression compared to mHTT only expression. The color of each table cell is based on the weighted variance value of difference to WT level for that gene (row) and mutant (column) compared to mHTT only expression, with white as similar difference to WT control levels, green as more like WT control levels, and red as less as the WT control levels. The deeper the color the greater the difference in variance to mHTT only expression compared to the WT control levels. The weighted variance value is also included in each cell, and again higher value means closer to WT mRNA level. If the expression level compared to WT level flipped from greater than WT level to less than WT level or vice versa compared to mHTT expression the cell border is bolded. (C) Proposed schematic mechanisms whereby wild-type HTT (wtHTT) stimulates DNA repair to maintain genome integrity and how mutant HTT (mHTT) synchronously disrupts DNA repair and transcription in HD to trigger early neurotoxicity in HD. wtHTT and BRG1 assemble a DSB repair complex with essential NHEJ factors including PNKP, Ku70, Ku80, DNA-PKcs, XRCC4, DNA ligase IV, CSB and PNKP in neurons. This structure senses DSBs during transcription and orchestrates their repair. wtHTT thus plays a pivotal role in DNA repair and in maintaining genome integrity during transcription. In contrast, HTT polyQ expansions inhibit recruitment of various NHEJ factors at the DSB sites, degrading normal TC- NHEJ function and DSB repair. This leads to persistence of DSBs in genome and chronic activation of the DNA-damage-response in HD. Cumulative accumulation of DSBs within transcriptionally active genome adversely impacts expression of neuronal genes, amplifying pro-degenerative impacts. Mutant HTT thus synchronously impairs DNA repair and transcription, triggering neurotoxicity and functional decline in HD.

Article Snippet: Wildtype C57BL/6 mouse brain NEs were immunoprecipitated with an anti-BRG1 Ab (NB100-2594; Novus) under stringent conditions to control for non-specific protein interactions.

Techniques: Expressing, Control, Mutagenesis, Over Expression, Activation Assay, Functional Assay