h3k9ac antibody  (Proteintech)

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Heatmap showing calculated STARS scores for SAGA/ATAC complex genes from a chromatin-focused CRISPR dropout screen, with red color correlating with stronger genetic dependencies and asterisks marking statistical significance (*p<0.05). b, Schematic of SAGA/ATAC chromatin-regulatory complexes illustrating the H3K4me2/3 reader function of SGF29, KAT2A/2B-dependent histone acetylation, modulation of H2A/H2B de-ubiquitination by the DUB module, and WDR5-dependent regulation of H3K4me3. c, Protein expression from the CPTAC proteomics database of primary brain tumors revealing increased expression of selected SAGA/ATAC-associated proteins in high grade (HGG) versus low grade glioma (LGG). d-f, Kaplan-Meier survival analyses comparing overall survival (OS) in pediatric brain tumor patients grouped according to low and high SGF29 and TRRAP protein expression in their primary tumors. Panels d-e show survival curves based on SGF29 and TRRAP expression in a pan-brain tumor analysis, and panel f shows a survival analysis based on TRRAP expression in an HGG patient cohort. g, Immunoblots of lysates from control and SU-DIPGXIII+Cas9 cells transduced with control or SGF29 sgRNAs collected 16 days after starting selection showing a reduction in SGF29 protein abundance and SAGA/ATAC-targeted H3K9ac. h-i, Number of live Cas9-expressing H3K27M mutant DMG cells ( h ) or non-transformed H3 wild-type cells ( i ) 12-16 days after starting selection to express either control or SGF29 -targeting sgRNAs. j, Normalized viability of H3K27M mutant DMG cells (purple curves) or H3 wildtype BJ fibroblasts and NHA-hTERT cells (black curves) following seven days of treatment with increasing doses of SGF29-IN-1 to block SGF29 chromatin-binding via its tandem Tudor domains. k-l, Average number of live BT869 ( k ) or SU-DIPGXIII ( l ) DMG cells following four days of treatment with increasing doses of SGF29-IN-1. m, Average number of SU-DIPGXIII cells following four days of treatment with SGF29-IN-1 comparing control and clonal SGF29 KO SU-DIPGXIIIP*+ZsG/luc cells confirming on-target inhibition of SGF29. n-o, Representative immunofluorescence images ( n ), and quantification of mean H3K9ac staining intensity ( o ) from BT869 cells treated with vehicle or 25 µM SGF29-IN-1 for 24 hours.

Article Snippet: H3K9ac primary antibody stock (cat# 9649, Cell Signaling Technology) was prepared at 1:3,000 in primary antibody dilution buffer (0.3% triton-X 100, 1% BSA, 1xPBS) and allowed to incubate overnight at room temperature.

Techniques: CRISPR, Ubiquitin Proteomics, Expressing, Western Blot, Control, Transduction, Selection, Quantitative Proteomics, Mutagenesis, Transformation Assay, Blocking Assay, Binding Assay, Inhibition, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Growth curves of SU-DIPGXIII cells transduced with shRNAs to knockdown both KAT2A and KAT2B, showing significantly reduced growth compared to control shRNA-transduced cells. b, Number of live SU-DIPGXIII+Cas9 cells expressing a control plasmid (HA/flag-GFP) or sgRNAs to KO SAGA-associated acetyltransferase activity ( TADA2B sgRNAs), or de-ubiquitinase activity ( ENY2 sgRNA). c, Schematic illustrating inhibition of histone-modifying modules of SAGA/ATAC complexes using chemical probes to target SAGA/ATAC-dependent histone acetylation (GSK4027, CPTH2, garcinol), SAGA-dependent H2A/H2B de-ubiquitination (USP22si-02), or WDR5-mediated H3K4me3 methylation (OICR-9529, WM856, WDR5-IN-4, and WDR5-IN-6). d, Average normalized cell viability from CellTiter-Glo assays conducted seven days after starting treatment of H3K27M mutant cells (teal curves) or non-transformed, H3 wildtype, control cells (black curves) with increasing doses of the KAT2A/2B bromodomain-targeting acetyltransferase inhibitor, GSK4027. e, Immunoblots confirming that treatment of BT245 cells with GSK4027 reduced H3K9ac in a dose-dependent manner, but not total H3 or SGF29 protein abundance. f-h, Dose response curves showing inhibition of H3K27M mutant DMG cell viability, but no effect on control H3 wild-type cells (black curves) following seven days of treatment with the DUB inhibitor, USP22i-S02 ( f, yellow curves), the WDR5 WIN site inhibitor, WDR5-IN-4 ( g, blue curves), or the WDR5 WBM site inhibitor, WDR5-IN-6 ( h, blue curves). i-j Average number of live SU-DIPGXIIIP*+ZsG/luc cells ( i ), and average BT245 ( j ) cell viability (CellTiter-Glo) following treatment with combinations of GSK4027 and USP22si-02 to simultaneously target both SAGA/ATAC-dependent histone acetyltransferase activity and SAGA-dependent H2A/H2B de-ubiquitination. k-l, BLISS synergy plots showing a combined effect of GSK4027 and WDR5-IN-6 treatment in reducing SU-DIPGXIIIP*+ZsG/luc growth ( k, **p<1.74×10 -3 ), and a combined effect of GSK4027 and WDR5-IN-4 treatment in reducing BT245 cell growth ( l , ****p<2.5×10 -10 ).

Article Snippet: H3K9ac primary antibody stock (cat# 9649, Cell Signaling Technology) was prepared at 1:3,000 in primary antibody dilution buffer (0.3% triton-X 100, 1% BSA, 1xPBS) and allowed to incubate overnight at room temperature.

Techniques: Transduction, Knockdown, Control, shRNA, Expressing, Plasmid Preparation, Activity Assay, Inhibition, Ubiquitin Proteomics, Methylation, Mutagenesis, Transformation Assay, Western Blot, Quantitative Proteomics

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Heatmap showing log 2 fold change (log 2 FC) for various histone post-translational modifications in SGF29 KO HSJD-DIPG007 and SU-DIPGXIIIP* cells compared to control cells, revealing a consistent reduction in H3K9ac and H3K14ac associated with gene activation and a reduction in the phosphorylation of H310 and H3S28 associated with mitosis as determined by mass spectrometry. b, Heatmap (top panel) and average profile plots (bottom panel) of CUT&RUN tracks centered on significant H3K9ac-down peaks (2,870 regions, *p<0.01), in SGF29 KO versus control SU-DIPGXIII cells. This analysis suggests that the H3K9ac-down peaks are marked by strong H3K4me3 binding, low H3K27M association, and a moderate levels of H3K27ac and H3K4me1 enhancer marks. c, Heatmap and average profile plots of CUT&RUN tracks centered on the significant H3K4me3-down peaks (8,596 regions, *p<0.01), revealing strong H3K27M oncohistone binding and H3K27ac/H3K4me1 enrichment, but low H3K9ac signal. d, Genome browser snapshots of the DCTN5 and PLK1 loci showing representative H3K9ac-down peaks observed in the SGF29 KO cells (marked with boxes). e, Genome browser snapshot showing H3K4me3-down peaks (marked with boxes) in the promoter and gene body of the DAB1 gene, which co-localized with H3K27M, H3K27ac, and H3K4me1 peaks. In panels b-e, H3K27M CUT&RUN was performed in H3.1KO and H3K27M KO SU-DIPGXIII cells to control for antibody specificity. f, Pie charts showing the overlap between H3K9ac-down and H3K4me3-down peaks and various genomic elements indicating that SGF29 KO leads to reduced H3K9ac at gene promoters, whereas H3K4me3 was reduced primarily at introns and intergenic regions. g-h, Enriched consensus sequences for transcription factor-biding sites within the H3K9ac- ( g ) and H3K4me3-down ( h ) peak sets identified using HOMER. Interferon-responsive transcription factor binding sites (ISRE and IRF2) were enriched in the H3K9ac-down peaks, whereas SOX-family transcription factor binding sites were enriched in the H3K4me3-down peaks. i-j, Fold enrichment of known transcription factor and chromatin-regulator binding sites from the ChIP ATLAS database within the H3K9ac- ( i ) and H3K4me3-down ( j ) peak sets, suggesting an overlap between these SGF29-regulated regions and known binding sites for SWI/SNF chromatin remodelers and developmental transcription factors like SOX9/10, ASCL1, and OLIG2.

Article Snippet: H3K9ac primary antibody stock (cat# 9649, Cell Signaling Technology) was prepared at 1:3,000 in primary antibody dilution buffer (0.3% triton-X 100, 1% BSA, 1xPBS) and allowed to incubate overnight at room temperature.

Techniques: Control, Activation Assay, Phospho-proteomics, Mass Spectrometry, Binding Assay

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Heatmap and average profile plots of CUT&RUN tracks centered on significant H3K9ac-down peaks (2,085 regions, *p<0.05) in SGF29 KO versus control SU-DIPGXIII cells, revealing moderate H3K27M enrichment and an overlap between these peaks and active enhancer marks (H3K4me1/H3K27ac enriched). b, Genome browser snapshot of the LMNA gene body showing H3K9ac-up peaks co-localizing with active chromatin marks. c, Heatmap and average profile plots of CUT&RUN tracks from SGF29 KO versus control SU-DIPGXIII cells centered around the significant H3K4me3-up peaks (861 regions, *p<0.05) revealing lower H3K27M binding and a poised enhancer chromatin signature (increased H3K4me1/H3K27ac ratio). d, Genome browser snapshot of the SLC44A3 gene body showing SGF29 KO-increased H3K4me3 peaks that co-localized with H3K9ac, H3K27ac, and H3K4me1. e, Pie charts showing the overlap between the H3K9ac- and H3K4me3-up peaks and various genomic elements suggesting SGF29KO results leads to increased H3K9ac primarily at intronic and intergenic regions and increased H3K4me3 at promoters. f-g, Consensus transcription factor-binding sites in the H3K9ac-up ( f ) and H3K4me3-up ( g ) peaks from HOMER motif analysis. AP-1 family transcription factor binding sites were overrepresented in the H3K9ac-up peaks, whereas binding sites for interferon-responsive transcription factors (ISRE and IRF2) were enriched in the H3K4me3-up peaks. h-I, Fold enrichment of transcription factor and chromatin regulator binding sites, as catalogued in the ChIP Atlas database, within the H3K9ac- ( h ) and H3K4me3-up ( i ) peak sets. These analyses suggest an overlap between the H3K9ac-up peaks and OLIG2 and SWI/SNF binding sites and an overlap between the H3K4me3-up peaks and known PRC2 binding sites.

Article Snippet: H3K9ac primary antibody stock (cat# 9649, Cell Signaling Technology) was prepared at 1:3,000 in primary antibody dilution buffer (0.3% triton-X 100, 1% BSA, 1xPBS) and allowed to incubate overnight at room temperature.

Techniques: Control, Binding Assay

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Heatmap of RNAseq Z-scores summarizing differentially expressed genes (DEGs) in SGF29 KO SU-DIPGXIII and BT245 DMG cells and in SU-DIPGXIII cells following vehicle (DMSO) or HAT inhibitor treatment (GSK4027 and CPTH2). b-c, Venn diagrams illustrating the overlap between the H3K9ac- and H3K4me3-down peaks from the CUT&RUN analyses (see Fig. 4b -c ) and the significantly downregulated transcripts from the RNAseq analysis of SGF29 KO versus control cells ( b ) or HATi-versus vehicle-treated cells ( c ). d-e, GO analysis of genes located within ±30 kb of the H3K9ac-down peaks ( d ) and associated with the HATi-downregulated transcripts ( e ). f-g, GSEA on the significant DEGs in the SGF29 KO versus control cells ( f ) and in the HATi-treated versus vehicle-treated cells ( g ), suggesting a downregulation of MYC and mTOR target genes and metabolic genes associated with malignancy. In panels d-e , downregulated gene sets associated with metabolic regulation are shown in green. h-i, GO analysis of genes located within ±30 kb of the H3K4me3-down peaks ( h ), or showing decreased expression upon SGF29 KO in the RNAseq study ( i ), indicating reduced H3K4me3 binding and transcription of genes involved in neuronal and glial differentiation (shown in purple). j-k, GO analysis of genes located within ±30 kb of H3K9ac-up peaks ( j ) or showing increased expression in the RNAseq analysis of SGF29 KO cells ( k ), indicating increased H3K9ac binding and transcription of genes related to the extracellular matrix (ECM) and metabolic regulation (shown in blue and green, respectively). m-o, Kaplan-Meier survival curves grouping pediatric brain tumors by high and low protein expression of SAGA/ATAC gene targets suggesting that increased expression of the SAGA/ATAC-induced gene, KDM1A, correlates with worse clinical outcomes ( m ), whereas the SAGA/ATAC-repressed genes, STX3 and TMBIM1, correlate with improved survival, n-o .

Article Snippet: H3K9ac primary antibody stock (cat# 9649, Cell Signaling Technology) was prepared at 1:3,000 in primary antibody dilution buffer (0.3% triton-X 100, 1% BSA, 1xPBS) and allowed to incubate overnight at room temperature.

Techniques: Control, Expressing, Binding Assay