Journal: PLoS ONE

Article Title: Identification of Druggable Cancer Driver Genes Amplified across TCGA Datasets

doi: 10.1371/journal.pone.0098293

Figure Lengend Snippet: Identification of cancer amplified genes with high copy number versus expression correlation.

Article Snippet: For NSD3 knockdown studies, we used On-Targetplus SMARTpool siRNA targeting human Nsd3 (Thermo Scientific).

Techniques: Amplification, Expressing, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Identification of Druggable Cancer Driver Genes Amplified across TCGA Datasets

doi: 10.1371/journal.pone.0098293

Figure Lengend Snippet: (A) Copy number (x-axis) and mRNA expression (y-axis) for NSD3 and SETD1 in breast cancers and melanomas, respectively. Correlation coefficient for copy number and mRNA expression are listed in the top right (r value). (B) BRD4 and YEATS4 shRNA activity in a panel of cancer cell lines (Project Achilles). shRNA score denotes the log2 based decrease in the representative shRNA compared to pooled shRNA in cancer cell lines after several rounds of proliferation post-shRNA . Yellow bars indicate cell lines with BRD4 or YEATS4 copy number >4 and black bars indicate cell lines with BRD4 or YEATS4 copy number <4. (C) Frequency of amplification (red bar), mutation (green bar), and deletion (blue bar) for NSD3 , SETDB1 , YEATS4 , and BRD4 in various cancers. The percentages shown reflect the overall rate of gene amplification, mutation and/or deletion in each cancer type. Vertical aligned bars reflect samples from the same patient. (D) Relative NSD3 protein level (y-axis, normalized to b-actin protein levels) compared with NSD3 copy number (x-axis) in SW48, H1581, SW837, and H1703 cells. (E) Relative proliferation (y-axis) and (F) relative apoptosis levels of cancer cell lines H1581, H1703, SW48, and SW837 cells 3 days after transfection with NSD3 siRNA, as measured by Cell Titer Glo and Caspase Glo assays, respectively. (G) Cell cycle profile of H1703 cells 24 or 48 hours after transfection with NSD3 siRNA compared to non-transfected controls. (H) Relative changes of cells in apoptosis, G1 or G2 phases (y-axis) in cell lines 48 hours-post NSD3 siRNA transfection compared to uninfected controls.

Article Snippet: For NSD3 knockdown studies, we used On-Targetplus SMARTpool siRNA targeting human Nsd3 (Thermo Scientific).

Techniques: Expressing, shRNA, Activity Assay, Amplification, Mutagenesis, Transfection

Journal: PLoS ONE

Article Title: Inhibitor-Sensitive FGFR1 Amplification in Human Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0020351

Figure Lengend Snippet: (A) Copy number estimates at chromosome arm 8p11-12q for 44 NSCLC samples (columns; ordered by amplification of 8p11) having amplification greater than 3.25 copies (log2 ratio of 0.7) from a collection of 732 NSCLC primary samples and cell lines. The horizontal line indicates the region containing FGFR1 , LETM2 and WHSC1L1 genes. The color scale ranges from blue (deletion) to red (amplification) with estimated copy numbers shown. Grey regions represent the absence of SNP copy number data. (B) Bar graph depicting percentages of samples harboring 8p11-12 amplification in lung adenocarcinomas (AC) and squamous cell carcinoma (SCC) demonstrates that FGFR1 amplification is observed in SCC at much higher frequency than AC. (C) FGFR1 expression (upper panel) shown in ten NSCLC cells; eight cell lines harboring FGFR1 amplification—HCC1734, HCC95, NCI-H2444, Calu3, NCI-H2077, NCI-H1703, NCI-H1581 and NCI-H520 (indicated by red horizontal bar below)—one NSCLC cell line harboring deletion of the region HCC15 (indicated by blue horizontal bar below)– and three NSCLC cells with no amplification—A427, NCI-H226, NCI-H2170 (indicated by black horizontal bar below)– using actin as a loading control (shown in lower panel). FGFR1 copy number status and 8p11-12 amplicon length determined by SNP array is indicated below cells harboring amplification. Of note, NCI-H2077 and NCI-1581 were found to be genotypically identical by fingerprinting analysis.

Article Snippet: Antibodies used for immunoblotting were: anti- FGFR1 antibody (# 3472, Cell Signaling Technologies, Danvers, MA, United States), anti- phospho FRS2 Y436 (#3861, Cell Signaling Technologies, Danvers, MA, United States), anti-phospho-FRS2 Y196 (#3864, Cell Signaling Technologies, Danvers, MA, United States), anti-FRS2 (# sc-17841, Santa Cruz Biotechnology, Santa Cruz, CA, United States). anti-WHSC1L1 monoclonal antibody (# sc-130009, Santa Cruz Biotechnology), anti-LETM2 monoclonal antibody (# ab84626, Abcam), and anti-Actin monoclonal antibody (# sc-1615, Santa Cruz Biotechnology).

Techniques: Amplification, Expressing, Control

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Scatterplot of protein interactions for BRD4-NUT determined by mass spectrometry proteomics. Enrichment for each protein in the immunoprecipitate relative to the input is plotted for 293TRex cells (x-axis) and for 797TRex cells (y-axis) induced to express epitope-tagged BRD4-NUT. NSD3 interacts with BRD4-NUT in both naïve (293TRex) and patient-derived (797TRex) cells. ( B ) CUT&RUN profiles for NSD3 (red), histone H3K36me2 (red), BRD4-NUT (purple), histone H3K27ac (green), and IgG (black) from patient-derived 10-15 and TC-797 cells endogenously expressing BRD4-NUT. Black bars indicate BRD4-NUT megadomains. ( C ) Heatmaps of the CUT&RUN signal for NSD3, histone H3K36me2, BRD4-NUT, and histone H3K27ac within each megadomain and at histone H3K27ac peaks outside megadomains in 10-15 and TC-797 cells indicate that NSD3 and histone H3K36me2 are strongly enriched in megadomains. Megadomains were normalized to the same length and 50 kb of flanking DNA is shown next to each normalized megadomain. H3K27ac peak centers were aligned and 10 kb of DNA flanking each H3K27ac peak is shown. IgG signal has been subtracted from each heatmap. ( D ) Pairwise Pearson correlations of CUT&RUN enrichment relative to IgG show that NSD3 and histone H3K36me2 enrichment are more strongly correlated with BRD4-NUT enrichment than histone H3K27ac enrichment in megadomains. ( E ) Immunofluorescence micrographs of histone H3K27ac (green), BRD4-NUT (purple), and NSD3 (red) show that, like BRD4-NUT, NSD3 overlaps with histone H3K27ac within BRD4-NUT condensates. Scale bars, 5 µm. ( F ) Profiles quantifying normalized fluorescence intensity along the yellow lines in the merged images from (E). Fluorescence intensity was normalized by dividing by the total fluorescence intensity across each yellow line. ( G ) Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of Mander’s coefficients of BRD4-NUT with histone H3K27ac and NSD3 with histone H3K27ac. Data points represent individual nuclei. 110 (10-15 cells) and 89 (TC-797 cells) nuclei were used to analyze the overlap of BRD4-NUT with H3K27ac. 106 (10-15 cells) and 95 (TC-797 cells) nuclei were used to analyze the overlap of NSD3 with H3K27ac.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Mass Spectrometry, Derivative Assay, Expressing, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of Mander’s coefficients of histone H3K27ac with BRD4-NUT and histone H3K27ac with NSD3. Data points represent individual nuclei. 110 (10-15 cells) and 89 (TC-797 cells) nuclei were used to analyze the overlap of histone H3K27ac with BRD4-NUT. 106 (10-15 cell) and 95 (TC-797 cell) nuclei were used to analyze the overlap of histone H3K27ac with NSD3.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques:

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Immunoblots indicate that NSD3 (long and short isoforms) levels are reduced, whereas BRD4-NUT, histone H3K36me2, and histone H3K27ac levels in 10-15 cells remain unchanged upon CRISPRi-mediated depletion of NSD3 (sgNSD3). A non-target sgRNA (sgNT) was used as a control. GAPDH and histone H3 were used as internal controls for non-histone proteins and histone modifications, respectively. ( B ) Quantification of the immunoblots from (A). NSD3short and NSD3long were combined to obtain the level of NSD3. Data points represent biological replicates. Bar and error bars represent the mean and standard error of the mean, respectively. p values determined using paired t-tests. ( C ) CUT&RUN profiles for NSD3 (red), histone H3K36me2 (red), BRD4-NUT (purple), histone H3K27ac (green), and IgG (black) from control (sgNT) and NSD3-depleted (sgNSD3) 10-15 cells. Chromatin occupancy of histone H3K36me2 and BRD4-NUT within megadomains (black bars) is reduced upon depletion of NSD3. ( D ) Heatmaps of the CUT&RUN signal for NSD3, histone H3K36me2, BRD4-NUT, and histone H3K27ac within megadomains in control (sgNT) and NSD3-depleted (sgNSD3) 10-15 cells. Megadomains were normalized to the same length and 50 kb of flanking DNA is shown next to each normalized megadomain. IgG signal has been subtracted from each heatmap. ( E ) Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) indicate that NSD3, histone H3K36me2, and BRD4-NUT, but not histone H3K27ac are reduced within megadomains in NSD3-depleted (sgNSD3) compared to control (sgNT) 10-15 cells. Data points represent the average IgG-normalized CUT&RUN signals within megadomains from two biological replicates. p values determined using one-sided Mann-Whitney U tests.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Immunoblots indicate NSD3 (long and short isoforms) levels are reduced, but BRD4-NUT, histone H3K36me2, and histone H3K27ac levels in TC-797 cells are unchanged after CRISPRi-mediated depletion of NSD3 (sgNSD3). A non-target sgRNA (sgNT) was used as a control. GAPDH and histone H3 were used as internal controls for non-histone proteins and histone modifications, respectively. ( B ) Quantification of the immunoblots from (A). NSD3short and NSD3long were combined to obtain the level of NSD3. Data points represent biological replicates. Bar and error bars represent the mean and standard error of the mean, respectively, of biological replicates. p value determined using paired t-tests. ( C ) CUT&RUN profiles for NSD3 (red), histone H3K36me2 (red), BRD4-NUT (purple), histone H3K27ac (green), and IgG (black) from control (sgNT) TC-797 cells and NSD3-depleted (sgNSD3) TC-797 cells. Chromatin occupancy of histone H3K36me2 and BRD4-NUT within megadomains (black bars) is reduced upon depletion of NSD3. ( D ) Heatmaps of the CUT&RUN signal for NSD3, histone H3K36me2, BRD4-NUT, and histone H3K27ac within each megadomains in control (sgNT) and NSD3-depleted (sgNSD3) TC-797 cells. Megadomains were normalized to the same length, and 50 kb of flanking DNA is shown next to each normalized megadomain. IgG signal has been subtracted from each heatmap. ( E ) Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) indicate that NSD3, histone H3K36me2, BRD4-NUT, and histone H3K27ac are reduced within megadomains in NSD3-depleted (sgNSD3) compared to control (sgNT) TC-797 cells. Data points represent the average IgG-normalized CUT&RUN signals within megadomains from two biological replicates. p values determined using one-sided Mann-Whitney U tests.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Cell proliferation of control (sgNT) and NSD3-depleted (sgNSD3) 10-15 and TC-797 cells on each day given on the x-axis determined by CellTiter-Glo assay. Data points and error bars represent the mean and standard error of the mean (SEM), respectively, of three biological replicates. p value determined using paired t-test. ( B ) Photomicrographs of control (sgNT) and NSD3-depleted (sgNSD3) 10-15 and TC-797 cells show that the depletion of NSD3 induces differentiated cell morphology as evidenced by enlarged cells and nuclei, flattened cells, and a lower nucleus-to-cytoplasm ratio. Cells were transduced with a lentivirus expressing sgNT or sgNSD3 for 11 days, followed by Wright-Giemsa staining. Scale bars, 20 µm. ( C ) Immunoblots indicate that depletion of NSD3 leads to the upregulation of epithelial-specific differentiation marker keratin 7 (KRT7) in 10-15 and TC-797 cells. Cells were transduced with a lentivirus expressing sgNT (control) or sgNSD3 for 5 days (TC-797 cells) or 8 days (10-15 cells). GAPDH was used as an internal control. Quantification is shown to the right of each immunoblot; data points represent biological replicates; bar and error bars represent mean and standard error of the mean, respectively. p value determined using paired t-test.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Control, Glo Assay, Transduction, Expressing, Staining, Western Blot, Marker

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) (Left) Heatmaps of the CUT&RUN signal for NSD3, histone H3K36me2, BRD4-NUT, and histone H3K27ac centered at histone H3K27ac peaks overlapping BRD4-NUT peaks (H3K27ac-BRD4-NUT peaks) outside of megadomains in control (sgNT) and NSD3-depleted (sgNSD3) 10-15 cells. H3K27ac-BRD4-NUT peak centers outside of megadomains were aligned, and 10 kb of DNA flanking each peak is shown. IgG signal has been subtracted from each heatmap. (Right) Profiles of the mean CUT&RUN signal for NSD3, histone H3K36me2, BRD4-NUT, and histone H3K27ac centered at H3K27ac-BRD4-NUT peaks outside of megadomains in control (sgNT, blue) and NSD3-depleted (sgNSD3, red) 10-15 cells with 10 kb flanking DNA. IgG signal has been subtracted from each profile. ( B ) Same as (A) for TC-797 cells.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Control

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Immunofluorescence for NSD3 (red), BRD4-NUT (purple), and histone H3K27ac (green) in control (sgNT) and NSD3-depleted (sgNSD3) 10-15 and TC-797 cells, respectively, show that the depletion of NSD3 destabilizes BRD4-NUT condensates containing histone H3K27ac. White dashed lines outline nuclei identified by DAPI staining. Scale bars, 10 µm. ( B ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly reduces the mean fluorescence intensity of BRD4-NUT in BRD4-NUT condensates. 5,710 (sgNT, purple) and 4,318 (sgNSD3, pink) condensates were analyzed from 10-15 cell nuclei; 5,594 (sgNT, purple) and 7,511 (sgNSD3, pink) condensates were analyzed from TC-797 cell nuclei. p values determined using one-sided Mann-Whitney U tests. ( C ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly increases the number of BRD4-NUT condensates per nucleus. 216 (sgNT, purple) and 123 (sgNSD3, pink) 10-15 cell nuclei were analyzed; 164 (sgNT, purple) and 168 (sgNSD3, pink) TC-797 cell nuclei were analyzed. p values determined using one-sided Mann-Whitney U tests. ( D ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly reduces the mean fluorescence intensity of histone H3K27ac in condensates. 10,064 (sgNT, dark green) and 9,151 (sgNSD3, light green) condensates were analyzed from 10-15 cell nuclei; 10,691 (sgNT, dark green) and 13,408 (sgNSD3, light green) condensates were analyzed from TC-797 cell nuclei. p values determined using one-sided Mann-Whitney U tests. ( E ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly increases the number of condensates per nucleus. 377 (sgNT, dark green) and 247 (sgNSD3, light green) 10-15 cell nuclei were analyzed; 314 (sgNT, dark green) and 309 (sgNSD3, light green) TC-797 cell nuclei were analyzed. p values determined using one-sided Mann-Whitney U tests.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Immunofluorescence, Control, Staining, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly decreases the total nuclear fluorescence intensity of NSD3. 161 (sgNT, dark red) and 124 (sgNSD3, light red) 10-15 cell nuclei were analyzed; 150 (sgNT, dark red) and 141 (sgNSD3, light red) TC-797 cell nuclei were analyzed. p values determined using one-sided Mann-Whitney U tests. ( B ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) of the total nuclear fluorescence intensity of BRD4-NUT. 216 (sgNT, purple) and 123 (sgNSD3, pink) 10-15 cell nuclei were analyzed; 164 (sgNT, purple) and 168 (sgNSD3, pink) TC-797 cell nuclei were analyzed. NSD3 depletion slightly increases the total nuclear fluorescence intensity of BRD4-NUT in TC-797 cells but does not affect the intensity of BRD4-NUT in 10-15 cells. p values determined using one-sided Mann-Whitney U tests. ( C ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly reduces the total fluorescence intensity of BRD4-NUT condensates. 5,710 (sgNT, purple) and 4,318 (sgNSD3, pink) condensates were analyzed from 10-15 cell nuclei; 5,594 (sgNT, purple) and 7,511 (sgNSD3, pink) condensates were analyzed from TC-797 cell nuclei. p values determined using one-sided Mann-Whitney U tests. ( D ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) of the total nuclear fluorescence intensity of histone H3K27ac. 377 (sgNT, dark green) and 247 (sgNSD3, light green) 10-15 cell nuclei were analyzed; 314 (sgNT, dark green) and 309 (sgNSD3, light green) TC-797 cell nuclei were analyzed. NSD3 depletion slightly increases the total nuclear fluorescence intensity of histone H3K27ac in 10-15 cells but does not affect the intensity of histone H3K27ac in TC-797 cells. p values determined using one-sided Mann-Whitney U tests. ( E ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) indicate that depletion of NSD3 significantly reduces the total fluorescence intensity of histone H3K27ac in condensates. 10,064 (sgNT, dark green) and 9,151 (sgNSD3, light green) condensates were analyzed from 10-15 cell nuclei; 10,691 (sgNT, dark green) and 13,408 (sgNSD3, light green) condensates were analyzed from TC-797 cell nuclei. p values determined using one-sided Mann-Whitney U tests. ( F ) Violin plots (solid lines indicate medians; red dots indicate means; dashed lines indicate 25th and 75th percentiles) of nuclear volume identified by DAPI staining. 455 (sgNT, dark gray) and 273 (sgNSD3, light gray) 10-15 cell nuclei were analyzed; 268 (sgNT, dark gray) and 303 (sgNSD3, light gray) TC-797 cell nuclei were analyzed. NSD3 depletion significantly increases the nuclear volume of 10-15 cells but does not affect the nuclear volume of TC-797 cells. p values determined using one-sided Mann-Whitney U tests.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Fluorescence, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) P ( s ) curves of the average intrachromosomal contact frequency separated by the genomic distance on the x-axis in control (sgNT, black) and NSD3-depleted (sgNSD3, red) 10-15 and TC-797 cells. ( B ) P ( s ) curves of the average intrachromosomal contact frequency for loci from identical amounts of Kc167 Drosophila melanogaster cells spiked-in to identical amounts of control (sgNT, black) and NSD3-depleted (sgNSD3, red) 10-15 and TC-797 cells separated by the genomic distance on the x-axis. Overlap of the Drosophila P ( s ) curves in both conditions at all genomic separations indicates no technical differences in the Hi-C analysis between control and NSD3-depleted cells.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Control, Hi-C

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Intrachromosomal ( cis ) Hi-C contact maps at 100 kb resolution (left bottom) from control (sgNT; below diagonal) and NSD3-depleted 10-15 cells (sgNSD3; above diagonal) show that depletion of NSD3 reduces cis megadomain-megadomain (MD-MD) contacts. Boxed regions displaying individual MD-MD contacts are enlarged to the right and above the contact map at 10 kb resolution. CUT&RUN profiles for NSD3, BRD4-NUT, and histone H3K27ac are aligned with the contact maps (sgNT: vertically; sgNSD3: horizontally for the 100 kb resolution map). Black bars indicate megadomains. ( B ) Interchromosomal ( trans ) Hi-C contact maps at 100 kb resolution from control (sgNT; below diagonal) and NSD3-depleted (sgNSD3; above diagonal) 10-15 cells show that the depletion of NSD3 reduces trans MD-MD contacts. CUT&RUN profiles for NSD3, BRD4-NUT, and histone H3K27ac are aligned with the contact maps (sgNT: vertically; sgNSD3: horizontally for the 100 kb resolution map). Black bars indicate megadomains. ( C ) Genome-wide pileups of mean observed/expected cis Hi-C contact frequencies at MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) 10-15 cells. Values of the central pixel are given at top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( D ) Genome-wide pileups of mean observed/expected trans Hi-C contact frequencies at MD-MD contacts for control (sgNT) and NSD3-depleted 10-15 cells (sgNSD3). Values of the central pixel are given at top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( E ) Genome-wide pileups of the fold-change in mean observed/expected cis Hi-C contact frequencies at MD-MD contacts for NSD3-depleted (sgNSD3) versus control (sgNT) 10-15 cells. The value of the central pixel is given at top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( F ) Genome-wide pileups of the fold-change in mean observed/expected trans Hi-C contact frequencies at MD-MD contacts for NSD3-depleted (sgNSD3) versus control (sgNT) 10-15 cells. The value of the central pixel is given at top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( G ) Box plots (horizontal lines show medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of mean observed/expected cis Hi-C contact frequencies for MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) 10-15 cells show a significant reduction of cis MD-MD contacts upon NSD3 depletion. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 157 cis MD-MD contacts. p value determined using a one-sided Mann-Whitney U test. ( H ) Box plots (horizontal lines show medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of mean observed/expected trans Hi-C contact frequencies for MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) 10-15 cells show a significant reduction of trans MD-MD contacts upon NSD3 depletion. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 1,218 trans MD-MD contacts. p values determined using a one-sided Mann-Whitney U test. ( I ) For each trans MD-MD contact ( n = 1,218), the fold-change in mean observed/expected Hi-C contact frequency versus the observed MD-MD contact frequency in control (sgNT) 10-15 cells. The color of data points reflects the rank of the observed trans MD-MD contact frequency from least to greatest in control (sgNT) 10-15 cells. ( J ) For each cis MD-MD contact ( n = 157), the fold-change in mean observed/expected Hi-C contact frequency versus the observed MD-MD contact frequency in control (sgNT) 10-15 cells. The color of data points reflects the rank of the observed cis MD-MD contact frequency from least to greatest in control (sgNT) 10-15 cells. ( K ) For each cis MD-MD contact ( n = 157), the fold-change in mean observed/expected Hi-C contact frequency versus the genomic distance separating the interacting megadomains. The color of data points reflects the rank of the observed cis MD-MD contact frequency from least to greatest in control (sgNT) 10-15 cells.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Hi-C, Control, Genome Wide, MANN-WHITNEY

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Intrachromosomal ( cis ) Hi-C contact maps at 100 kb resolution (left bottom) from control (sgNT; below diagonal) and NSD3-depleted (sgNSD3; above diagonal) TC-797 cells show that the depletion of NSD3 reduces cis megadomain-megadomain (MD-MD) contacts. Boxed regions displaying individual MD-MD contacts are enlarged to the right and the top of the contact map at 10 kb resolution. CUT&RUN profiles for NSD3, BRD4-NUT, and histone H3K27ac are aligned with the contact maps (sgNT: vertically; sgNSD3: horizontally for the 100 kb resolution map). Black bars indicate megadomains. ( B ) Interchromosomal ( trans ) Hi-C contact maps at 100 kb resolution from control (sgNT; below diagonal) and NSD3-depleted (sgNSD3; above diagonal) TC-797 cells show that the depletion of NSD3 reduces trans MD-MD contacts. CUT&RUN profiles for NSD3, BRD4-NUT, and histone H3K27ac are aligned with the contact maps (sgNT: vertically; sgNSD3: horizontally for the 100 kb resolution map). Black bars indicate megadomains. ( C ) Genome-wide pileups of mean observed/expected cis Hi-C contact frequencies at MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) TC-797 cells. Values of the central pixel are given at top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( D ) Genome-wide pileups of mean observed/expected trans Hi-C contact frequencies at MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) TC-797 cells. Values of the central pixel are given at top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( E ) Genome-wide pileups of the fold-change in mean observed/expected cis Hi-C contact frequencies at MD-MD contacts for NSD3-depleted (sgNSD3) versus control (sgNT) TC-797 cells. The value of the central pixel is given at the top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( F ) Genome-wide pileups of the fold-change in mean observed/expected trans Hi-C contact frequencies at MD-MD contacts for NSD3-depleted (sgNSD3) versus control (sgNT) TC-797 cells. The value of the central pixel is given at the top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( G ) Box plots (horizontal lines show medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of mean observed/expected cis Hi-C contact frequencies for MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) TC-797 cells show a significant reduction of cis MD-MD contacts upon NSD3 depletion. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 78 cis MD-MD contacts. p values determined using a one-sided Mann-Whitney U test. ( H ) Box plots (horizontal lines show medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of mean observed/expected trans Hi-C contact frequencies for MD-MD contacts for control (sgNT) and NSD3-depleted (sgNSD3) TC-797 cells show a significant reduction of trans MD-MD contacts upon NSD3 depletion. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 659 trans MD-MD contacts. p values determined using a one-sided Mann-Whitney U test. ( I ) For each trans MD-MD contact (n = 659), the fold-change in mean observed/expected Hi-C contact frequency versus the observed MD-MD contact frequency in control (sgNT) TC-797 cells. The color of data points reflects the rank of the observed trans MD-MD contact frequency from least to greatest in control (sgNT) TC-797 cells. ( J ) For each cis MD-MD contact ( n = 78), the fold-change in mean observed/expected Hi-C contact frequency versus the observed MD-MD contact frequency in control (sgNT) TC-797 cells. The color of data points reflects the rank of the observed cis MD-MD contact frequency from least to greatest in control (sgNT) TC-797 cells. ( K ) For each cis MD-MD contact ( n = 78), the fold-change in mean observed/expected Hi-C contact frequency versus genomic distance separating the interacting megadomains. The color of data points reflects the rank of the observed cis MD-MD contact frequency from least to greatest in control (sgNT) TC-797 cells.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Hi-C, Control, Genome Wide, MANN-WHITNEY

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Genome-wide pileups of mean observed/expected trans Hi-C contact frequencies at MD-MD contacts categorized by observed trans MD-MD contact frequencies in control (sgNT) cells for control (sgNT) and NSD3-depleted (sgNSD3) 10-15 and TC-797 cells. Below the observed/expected pileups are pileups of the fold-change for NSD3-depleted (sgNSD3) versus control (sgNT) cells. The value of the central pixel is given at the top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( B ) Genome-wide pileups of mean observed/expected cis Hi-C contact frequencies at MD-MD contacts categorized by observed cis MD-MD contact frequencies in control (sgNT) cells for control (sgNT) and NSD3-depleted (sgNSD3) 10-15 and TC-797 cells. Below the observed/expected pileups are pileups of the fold-change for NSD3-depleted (sgNSD3) versus control (sgNT) cells. The value of the central pixel is given at the top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups. ( C ) Genome-wide pileups of mean observed/expected cis Hi-C contact frequencies at MD-MD contacts categorized by genomic distance separating the interacting megadomains for control (sgNT) and NSD3-depleted (sgNSD3) 10-15 and TC-797 cells. Below the observed/expected pileups are pileups of the fold-change for NSD3-depleted (sgNSD3) versus control (sgNT) cells. The value of the central pixel is given at the top left. Windows extending beyond chromosome ends and contacts with zero or undefined observed/expected values in any dataset were excluded from pileups.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Genome Wide, Hi-C, Control

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Volcano plots (significance versus fold change) of differentially expressed genes show that after depletion of NSD3 the majority of genes within megadomains (purple dots) are downregulated in 10-15 cells and TC-797 cells. Horizontal dashed lines indicate a Benjamini-Hochberg adjusted p value of 0.05; dashed vertical lines indicate a 1.5-fold change in expression. ( B ) Gene Set Enrichment Analysis (GSEA) indicates that genes within megadomains are downregulated upon depletion of NSD3 (sgNSD3) compared to control (sgNT) in 10-15 and TC-797 cells. NES, normalized enrichment score.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) indicates that genes within the HALLMARK_MYC_TARGETS_V1 gene set are downregulated upon depletion of NSD3 (sgNSD3) compared to control (sgNT) in 10-15 cells. NES, normalized enrichment score. ( B ) Gene Set Enrichment Analysis (GSEA) indicates that genes within the HALLMARK_MYC_TARGETS_V2 gene set are downregulated upon depletion of NSD3 (sgNSD3) compared to control (sgNT) in 10-15 cells and TC-797 cells. NES, normalized enrichment score. ( C ) Bar chart of the top ten enriched Gene Ontology Biological Processes (GO BP) within upregulated differentially expressed genes upon depletion of NSD3 from 10-15 cells and TC-797 cells. The length of each bar represents the number of enriched genes in each term. The color scale represents the Benjamini-Hochberg adjusted p value. Terms are ranked by statistical significance of enrichment.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Control

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Immunoblots indicate induced expression of HiBiT-tagged NSD3short in MCF10A cells after treatment with 30 ng/mL doxycycline. Water and parental MCF10A cells were used as controls. GAPDH was used as an internal control. ( B ) CUT&RUN profiles for NSD3 (red), HiBiT epitope tag (red), and IgG (black) show NSD3short peaks (black bars) in MCF10A cells induced to express NSD3short versus uninduced MCF10A cells. ( C ) Heatmaps of the CUT&RUN signal for NSD3 and HiBiT epitope tag within each NSD3short peak in uninduced MCF10A cells and cells induced to express NSD3short. NSD3short peak centers were aligned and 100 kb of DNA flanking each NSD3short peak is shown. IgG signal has been subtracted from each heatmap. ( D ) Hi-C contact maps at 5 kb resolution from uninduced MCF10A cells (below diagonal) and cells induced to express NSD3short (above diagonal) show NSD3short-dependent chromatin contacts. Boxed regions displaying interactions separated by >2 Mb are enlarged to the right of the contact map. CUT&RUN profiles for the HiBiT epitope tag are aligned with the contact maps. Black bars indicate NSD3short peaks. ( E ) Genome-wide pileups of mean observed/expected Hi-C contact frequencies at NSD3short-dependent chromatin contacts in uninduced MCF10A cells and cells induced to express NSD3short show that NSD3short promotes chromatin contacts. The mean HiBiT CUT&RUN signal at NSD3short contact sites is shown above the pileups. Values of the central pixel are given at top left. ( F ) Genome-wide pileups of the fold-change in mean observed/expected Hi-C contact frequencies at NSD3short-dependent chromatin contacts in MCF10A cells induced to express NSD3short versus uninduced cells. The value of the central pixel is given at top left. ( G ) Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of observed/expected Hi-C contact frequencies for NSD3short-dependent chromatin contacts in uninduced MCF10A cells and cells induced to express NSD3short. Data points represent individual chromatin contacts. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 771 analyzed contacts. p value determined using a one-sided Mann-Whitney U test. ( H ) Percent of NSD3short-dependent chromatin contacts (red) separated by the distance given on the x-axis and percent of convergent CTCF-CTCF loops (orange) of a given size. The separation between NSD3short contact sites and loop sizes is logarithmically binned. ( I ) Number of NSD3short-dependent chromatin contacts overlapping with AA, BB, or AB Hi-C compartment interactions in uninduced MCF10A cells compared to a random shuffled control set of contacts shows that NSD3short promotes chromatin contacts within the A compartment.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Western Blot, Expressing, Control, Hi-C, Genome Wide, MANN-WHITNEY

Journal: bioRxiv

Article Title: NSD3 stabilizes nuclear compartmentalization and promotes megabase-scale chromatin interactions

doi: 10.64898/2026.02.07.704091

Figure Lengend Snippet: ( A ) Domain organization of NSD3short. A Trp284Ala mutation inactivates the PWWP domain of NSD3short. ( B ) Immunoblots indicate comparable expression levels of HiBiT-tagged NSD3short and NSD3short W284A . MCF10A cells were treated with 30 ng/mL or 25 ng/mL doxycycline to induce expression of NSD3short or NSD3short W284A , respectively. GAPDH was used as an internal control. ( C ) Quantification of immunoblots related to (B). Bar and error bars represent the mean and standard error of the mean, respectively, of five biological replicates. p value determined using paired t-test. ( D ) CUT&RUN profiles for NSD3, HiBiT epitope tag, and IgG from MCF10A cells expressing NSD3short (dark red) and NSD3short W284A (light red). NSD3short W284A chromatin occupancy is reduced within NSD3short peaks (black bars) and redistributes to broad domains (gray shading) with low NSD3short wild-type occupancy. ( E ) Profile (top) and heatmap (bottom) of the Hi-C compartment eigenvector 1 values from uninduced MCF10A cells within NSD3short W284A domains show that NSD3short W284A domains form in regions of negative eigenvector 1 values corresponding to B compartment intervals in uninduced cells. Profile represents the mean of the Hi-C compartment eigenvector 1 value at NSD3short W284A domains. NSD3short W284A domains were normalized to the same length and 200 kb of flanking DNA is shown next to each normalized NSD3short W284A domain. ( F ) Heatmaps of the CUT&RUN signal for NSD3 and HiBiT epitope tag centered at NSD3short peaks in MCF10A cells expressing NSD3short or NSD3short W284A show reduced genome-wide occupancy of NSD3short W284A in NSD3short peaks. NSD3short peak centers were aligned and 100 kb of DNA flanking each NSD3short peak is shown. IgG signal has been subtracted from each heatmap. ( G ) Violin plots (solid lines indicate medians; dashed lines indicate 25th and 75th percentiles) indicate that NSD3 and HiBiT epitope tag CUT&RUN enrichment relative to IgG within NSD3short peaks is decreased for NSD3short W284A compared to NSD3short. p values determined using one-sided Mann-Whitney U tests. ( H ) Heatmaps of the CUT&RUN signal for NSD3 and HiBiT epitope tag within each NSD3short W284A domain in MCF10A cells expressing NSD3short or NSD3short W284A show increased genome-wide occupancy of NSD3short W284A in NSD3short W284A domains. NSD3short W284A domains were normalized to the same length and 100 kb of flanking DNA is shown next to each normalized NSD3short W284A domain. IgG signal has been subtracted from each heatmap. ( I ) Violin plots (solid lines indicate medians; dashed lines indicate 25th and 75th percentiles) indicate that NSD3 and HiBiT epitope tag CUT&RUN enrichment relative to IgG within NSD3short W284A domains is increased for NSD3short W284A compared to NSD3short. p values determined using one-sided Mann-Whitney U tests. ( J ) Hi-C contact maps at 10 kb resolution from MCF10A cells expressing NSD3short (below diagonal) and cells expressing NSD3short W284A (above diagonal) show that NSD3short W284A has a reduced capacity to promote chromatin contacts. Boxed regions displaying interactions separated by >8 Mb are enlarged to the right of the contact map. CUT&RUN profiles for the HiBiT epitope tag are aligned with the contact maps. Black bars indicate NSD3short peaks. ( K ) Genome-wide pileups of mean observed/expected Hi-C contact frequencies at NSD3short-dependent chromatin contacts in MCF10A cells expressing NSD3short and cells expressing NSD3short W284A . The mean HiBiT CUT&RUN signal at NSD3short contact sites is shown above the pileups. Values of the central pixel are given at top left. ( L ) Genome-wide pileups of the fold-change in mean observed/expected Hi-C contact frequencies at NSD3short-dependent chromatin interactions in MCF10A cells expressing NSD3short and cells expressing NSD3short W284A . The value of the central pixel is given at top left. ( M ) Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of observed/expected Hi-C contact frequencies for NSD3short-dependent chromatin contacts in uninduced MCF10A cells, cells expressing NSD3short, and cells expressing NSD3short W284A . Data points represent individual chromatin contacts. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 771 analyzed contacts. p values determined using one-sided Mann-Whitney U tests. Data for uninduced MCF10A cells and cells expressing NSD3short are reproduced from . ( N ) Genome-wide pileups of mean observed/expected Hi-C contact frequencies at all possible NSD3short W284A domain-domain contacts in uninduced MCF10A cells, cells expressing NSD3short, and cells expressing NSD3short W284A show that NSD3short W284A domains do not interact. The mean HiBiT CUT&RUN signal at NSD3short W284A domains is shown above the pileups. Values of the central pixel are given at top left. ( O ) Genome-wide pileups of the fold-change in mean observed/expected Hi-C contact frequencies at all possible NSD3short W284A domain-domain contacts in uninduced MCF10A cells, cells expressing NSD3short, and cells expressing NSD3short W284A . The value of the central pixel is given at top left. ( P ) Box plots (horizontal lines indicate medians; boxes indicate interquartile range; whiskers extend 1.5 times the interquartile range) of observed/expected Hi-C contact frequencies for all possible NSD3short W284A domain-domain contacts in uninduced MCF10A cells, cells expressing NSD3short, and cells expressing NSD3short W284A . Data points represent individual chromatin contacts. Contacts with zero or undefined observed/expected values in any dataset were removed for a total of 1,443 analyzed contacts. p values determined using two-sided Mann-Whitney U tests.

Article Snippet: Primary antibodies were NUT (C52B1) Rabbit mAb (Cell Signaling Technologies #3625, lot 8, RRID: AB_2066833) diluted 1:5,000; Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173S, lot 9, RRID: AB_10949503) diluted 1:1,000; WHSC1L1 rabbit monoclonal antibody (Cell Signaling Technologies #92056, lot 1, RRID: AB_2800178) diluted 1:10,000; Di-Methyl-Histone H3 (Lys36) (C75H12) rabbit monoclonal antibody (Cell Signaling Technologies #2901, lot 5, RRID: AB_1030983) diluted 1:1,000; and anti-HiBiT mouse monoclonal antibody (Promega #N7200, RRID: AB_3665694) diluted 1:10,000.

Techniques: Mutagenesis, Western Blot, Expressing, Control, Hi-C, Genome Wide, MANN-WHITNEY