Journal: bioRxiv

Article Title: Leveraging the BAF chromatin remodeling complex for targeted transcriptional rewiring in cancer

doi: 10.64898/2026.03.30.715217

Figure Lengend Snippet: a, CUT&RUN binding profiles at BCL6 peaks. Binding profiles of BCL6 (left) and SMARCA4 (right) at BCL6 peaks for DMSO/TRIP1 (2 µM) treated cells after 4 and 8 hours. BCL6 peaks were called from DMSO-treated KARPAS-422 samples. Profiles were centered on peaks and extended 5 kb upstream and downstream of the peak location. b , Correlation between BCL6 and SMARCA4 (top), BCL6 and H3K27ac (middle), or SMARCA4 and H3K27ac (bottom) signal changes on gene bodies upon TRIP1 (8h, 2 µM) treatment compared to DMSO. Single dots represent hg38 genes. c, Relationship between TRIP1-induced BCL6/SMARCA4 binding change and H3K27 acetylation or SMARCA4 binding. Genes were ranked based on their differential binding of BCL6 (top) and SMARCA4 (bottom) after TRIP1 treatment (8h, 2 µM) and segmented into respective 20% quantiles. SMARCA4 (top) and H3K27ac (middle) changes at genes per BCL6 quintile. H3K27ac change (bottom) at genes segmented by SMARCA4 quintiles. After overall association was assessed by Kruskal-Wallis was successfully, pairwise comparisons were made by Dunn’s post-hoc test with Benjamini-Hochberg FDR correction. d , Relationship of TRIP1-induced gene expression changes with BCL6/SMARCA4 binding changes after 4 and 8 hours of TRIP1 treatment. Pearson correlation coefficient and R 2 were calculated. Dots represent single genes colored by their gene expression change upon TRIP1 treatment (2 µM after 16 hours vs. DMSO). Selected differentially expressed genes and BCL6 are highlighted. For b-d , Normalized CUT&RUN signal on gene bodies ±3 kb up and downstream to include regulatory regions was calculated for TRIP1 and DMSO. The scores were subtracted to calculate differential binding. e , CUT&RUN binding profiles at BCL6 peaks. Binding profiles of BCL6 at BCL6 peaks after short, low-dose TRIP1 (1 µM) treatment up to 2h. BCL6 peaks were called from DMSO-treated KARPAS-422 CUT&RUN samples. Profiles were centered on peaks and extended 5 kb upstream and downstream of the peak location. f-h , Genome tracks of the ARID3A gene locus. Time-resolved ( f ) BCL6 and SMARCA4 or ( g ) RNA Pol II serine 2/5 phosphorylation signal is computed along the gene locus to infer transcriptional dynamics. For g , Below, RNA-seq reads are mapped to the gene locus. h , BAF ATPase-dependent BCL6 eviction after 1 hour of DMSO or TRIP1 (1 µM) co-treatment with BRM-014 (1 µM). BCL6signal is computed along the ARID3A gene locus. Exon position and genome location are indicated below the genome tracks. All CUT&RUN data is from two merged independent replicates. i , CaspaseGlo 3/7 apoptosis ATPase pre-inhibition. KARPAS-422 cells were pre-treated with DMSO or SMARCA2/4 ATPase inhibitor (BRM-014) for 8 hours, followed by co-treatment with 1 µM of TRIP1 for 16 hours. Caspase 3/7 activity is normalized to DMSO control without TRIP1 co-treatment; data represent mean ± SD, n = 6 independent replicates. j , BCL6 transcriptional reporter ATPase pre-inhibition. KARPAS-422 cells expressing a BCL6 transcriptional reporter were pre-treated with DMSO or SMARCA2/4 ATPase inhibitor (BRM-014) for 8 hours, followed by co-treatment with 0.5 µM of TRIP1 for 24 hours. Reporter activity is normalized to DMSO vehicle control without TRIP1 co-treatment; data represent mean ± SD, n = 6 independent replicates.

Article Snippet: The following antibodies were used: IgG rabbit isotype control (Cell Signaling Technology, CST3900), BCL6 (Cell Signaling Technology, CST49360), SMARCA4 (Cell Signaling Technology, CST49360), H3K27ac (Cell Signaling Technology, CST8173), RNA Pol II Ser2 phospho (Cell Signaling Technology, CST13499), RNA Pol II Ser5 phospho (Cell Signaling Technology, CST13523).

Techniques: Binding Assay, Gene Expression, Phospho-proteomics, RNA Sequencing, Inhibition, Activity Assay, Control, Expressing

Journal: bioRxiv

Article Title: Leveraging the BAF chromatin remodeling complex for targeted transcriptional rewiring in cancer

doi: 10.64898/2026.03.30.715217

Figure Lengend Snippet: a, CUT&RUN binding profiles at BCL6 peaks. Binding profiles of BCL6 (left) and SMARCA4 (right) at BCL6 peaks for DMSO/TRIP1 (2 µM) treated cells after 4 and 8 hours. BCL6 peaks were called from DMSO-treated KARPAS-422 samples. Profiles were centered on peaks and extended 5 kb upstream and downstream of the peak location. b , Correlation between BCL6 and SMARCA4 (top), BCL6 and H3K27ac (middle), or SMARCA4 and H3K27ac (bottom) signal changes on gene bodies upon TRIP1 (8h, 2 µM) treatment compared to DMSO. Single dots represent hg38 genes. c, Relationship between TRIP1-induced BCL6/SMARCA4 binding change and H3K27 acetylation or SMARCA4 binding. Genes were ranked based on their differential binding of BCL6 (top) and SMARCA4 (bottom) after TRIP1 treatment (8h, 2 µM) and segmented into respective 20% quantiles. SMARCA4 (top) and H3K27ac (middle) changes at genes per BCL6 quintile. H3K27ac change (bottom) at genes segmented by SMARCA4 quintiles. After overall association was assessed by Kruskal-Wallis was successfully, pairwise comparisons were made by Dunn’s post-hoc test with Benjamini-Hochberg FDR correction. d , Relationship of TRIP1-induced gene expression changes with BCL6/SMARCA4 binding changes after 4 and 8 hours of TRIP1 treatment. Pearson correlation coefficient and R 2 were calculated. Dots represent single genes colored by their gene expression change upon TRIP1 treatment (2 µM after 16 hours vs. DMSO). Selected differentially expressed genes and BCL6 are highlighted. For b-d , Normalized CUT&RUN signal on gene bodies ±3 kb up and downstream to include regulatory regions was calculated for TRIP1 and DMSO. The scores were subtracted to calculate differential binding. e , CUT&RUN binding profiles at BCL6 peaks. Binding profiles of BCL6 at BCL6 peaks after short, low-dose TRIP1 (1 µM) treatment up to 2h. BCL6 peaks were called from DMSO-treated KARPAS-422 CUT&RUN samples. Profiles were centered on peaks and extended 5 kb upstream and downstream of the peak location. f-h , Genome tracks of the ARID3A gene locus. Time-resolved ( f ) BCL6 and SMARCA4 or ( g ) RNA Pol II serine 2/5 phosphorylation signal is computed along the gene locus to infer transcriptional dynamics. For g , Below, RNA-seq reads are mapped to the gene locus. h , BAF ATPase-dependent BCL6 eviction after 1 hour of DMSO or TRIP1 (1 µM) co-treatment with BRM-014 (1 µM). BCL6signal is computed along the ARID3A gene locus. Exon position and genome location are indicated below the genome tracks. All CUT&RUN data is from two merged independent replicates. i , CaspaseGlo 3/7 apoptosis ATPase pre-inhibition. KARPAS-422 cells were pre-treated with DMSO or SMARCA2/4 ATPase inhibitor (BRM-014) for 8 hours, followed by co-treatment with 1 µM of TRIP1 for 16 hours. Caspase 3/7 activity is normalized to DMSO control without TRIP1 co-treatment; data represent mean ± SD, n = 6 independent replicates. j , BCL6 transcriptional reporter ATPase pre-inhibition. KARPAS-422 cells expressing a BCL6 transcriptional reporter were pre-treated with DMSO or SMARCA2/4 ATPase inhibitor (BRM-014) for 8 hours, followed by co-treatment with 0.5 µM of TRIP1 for 24 hours. Reporter activity is normalized to DMSO vehicle control without TRIP1 co-treatment; data represent mean ± SD, n = 6 independent replicates.

Article Snippet: The following antibodies were used: IgG rabbit isotype control (Cell Signaling Technology, CST3900), BCL6 (Cell Signaling Technology, CST49360), SMARCA4 (Cell Signaling Technology, CST49360), H3K27ac (Cell Signaling Technology, CST8173), RNA Pol II Ser2 phospho (Cell Signaling Technology, CST13499), RNA Pol II Ser5 phospho (Cell Signaling Technology, CST13523).

Techniques: Binding Assay, Gene Expression, Phospho-proteomics, RNA Sequencing, Inhibition, Activity Assay, Control, Expressing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of Pol II transcription cycle in normal (N) tissue and tumor (T) of the large intestine. RNA expression level of Pol II transcription cycle-associated genes in GTEx and TCGA databases were analyzed. I initiation, E elongation, T termination. ( B ) Volcano plot for fold change of T vs N of 104 Pol II transcription-associated genes in COAD. NELFCD and SUPT4H1 genes are indicated with an arrow. NELFCD gene is highlighted in red. Statistical test: differential expression analysis was performed using DESeq2 (negative binomial generalized linear model with Wald test); P values were adjusted for multiple testing using the Benjamini–Hochberg method (adj.P.Val). The numbers of tumor (T) and normal (N) samples for COAD are provided in Table . ( C ) Western blot of NELF-C-AID DLD-1 whole-cell extract (WCE) using the indicated antibodies. The treatment time (h) of IAA is also indicated. Western blot image of NELF-C is reused in ( G ). ( D ) Western blot of SPT4-AID DLD-1 WCE using the indicated antibodies. The treatment time (h) of IAA is also indicated. ( E ) Western blot of NELF-C-AID DLD-1 cell nuclear fraction using the indicated antibodies. The treatment time (h) of IAA is indicated. Western blot images of NELF-C and Pol I are reused in Fig. . ( F ) Cell count ratios of 12, 24, and 36 h IAA to 0 h IAA are shown. Parental, NELF-C-AID, and SPT4-AID DLD-1 cells are displayed in light gray, red, and dark gray, respectively. Error bars represented the mean ± SEM (biological replicates, n = 4). Statistical test: paired two-sample t test (Student’s t test), two-sided, performed within each Group comparing the same Dish across adjacent timepoints (0 h vs 12 h, 12 h vs 24 h, 24 h vs 36 h) on the 0h-normalized ratios. P values are shown. n.s. (not significant, P > 0.05). ( G ) Western blot of NELF-C-AID DLD-1 whole-cell extract (WCE) using the indicated antibodies. The treatment time (h) of IAA is indicated. Western blot image of NELF-C is reused in ( C ). ( H ) Cell cycle (%) of parental and NELF-C-AID DLD-1 cells following the indicated treatment time (h) of IAA. Error bars represented the mean ± SEM (biological replicates, n = 3). .

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: RNA Expression, Quantitative Proteomics, Western Blot, Cell Characterization

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Log 2 fold change of T vs N of the Pol II transcription-associated genes. Upregulated (>1) and down-regulated (<-1) genes are highlighted in red and blue, respectively. NELFCD gene is indicated with an arrow. ( B ) Volcano plot of the log2 fold change of Tumors (T) vs Normal tissues (N) on 835 cell cycle-related genes in COAD. CDKN1A , CDKN1B , and CDKN1C genes are indicated in red. The numbers of tumor (T) and normal (N) samples for COAD are provided in Table . Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. ( C ) Log2 of the RNA expression levels of NELFCD , SUPT4H1 , CDKN1A , and CDKN1C genes in N and T of the indicated tissues and tumors are compared. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Statistical test: Wilcoxon signed-rank test. P values are shown. The numbers of tumor (T) and normal (N) samples for each tissue types are provided in Table . ( D ) Log 2 fold change of T vs N of NELFCD across indicated tissue types. ( E ) Comparative proteomic analysis of NELF subunits and SPT4 in human primary colon cancers and its adjacent tissues. Statistical test: negative binomial distribution-based Wald test. P values are shown. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( F ) Schematic model of transcription addicted by NELF in tumor.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: RNA Expression

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of POINT-seq strategy. Chromatin of NELF-C-AID or SPT4-AID DLD-1 cells was stringently isolated using Urea and Empigen detergent. Pol II intact nascent transcript (POINT) was precipitated with Pol II CTD antibody from DNA-digested chromatin fraction. ( B ) Example view of POINT-seq on RPS23 gene in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. ( C ) Metagene analysis of POINT-seq on scaled transcription unit −/+2.5 kb in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. ( D ) Violin plots of Termination Index (TI) in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median. ( E ) Example view of SIRV-normalized POINT-seq on RPS23 and HELLS genes in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). ( F ) Metagene analysis of POINT-seq on scaled transcription unit −/+2.5 kb in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). ( G ) Violin plots of TI in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. ** P = 0.0021, **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Isolation

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Example view of TT-seq on PTPN13 gene in the indicated cell line treated for 4 h with DMSO or IAA, followed by NVP-2 treatment for 0, 0.5, or 1 h. ( B ) Profiles of TT-seq mean signal in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA, followed by NVP-2 treatment for 0, 0.5, or 1 h. Data for 2 biological replicates with ≥300 kb genes are shown. ( C ) Box plots of elongation activity (EA) derived from TT-seq. Data for short and long genes are shown. n = 65. Statistical test: Wilcoxon rank-sum test. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). P value is 0.029. ( D ) Scatter plot showing the correlation between EA and TI. The regression line (red) and the 95% confidence interval (green) are also shown. n = 129. Statistical method: Spearman correlation. P value is 0.036. ( E ) Metagene of POINT-seq, TT-seq, Pol II EI (TT-seq/POINT-seq) for non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( F ) Box plots of Pol II EI (TT-seq/POINT-seq and TT-seq/PRO-seq) for non-overlapping pc genes (n = 3459) in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Statistical test: Kruskal–Wallis test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( G ) Pol II EI for indicated three gene-length classes of normalized non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( H ) INTS3 ChIP-seq profile across normalized transcription units of non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( I ) Box plots of the log2 of the Dox/Ctrl termination index, two biological replicates in all genes, genes with transcription readthrough which is based on previous study (Dasilva et al, ), and NELF-C KD affected genes (top 25%, highlighted in orange box). 0: no change in TI index upon shCtrl or shINTS11 induction by Dox. The number of genes in each category is indicated on the figure. Statistical test: Kruskal–Wallis test. * P = 0.039, ** P = 0.0019, **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%).

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Activity Assay, Derivative Assay, ChIP-sequencing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of Pol II elongation index (EI) analysis. Pol II EI is determined as TT/POINT. 4sU (Green circle), Pol II (blue oval). ( B ) Pol II EI for all expressed pc genes across normalized transcription units in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Last exon (LE)-0.5 kb ~ +2.5 kb window is highlighted in gray. ( C ) Pol II EI for non-overlapping pc genes across LE. ( D ) Metagene of CSTF64 and XRN2 ChIP-seq for non-overlapping pc genes across normalized transcription units (top) and LE (bottom) in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Last exon (LE)-0.5 kb ~ +2.5 kb window is highlighted in gray. ( E ) Quantification of CSTF64 and XRN2 ChIP-seq signals (IAA/DMSO) in 2.5 kb DoG regions of non-overlapping pc genes. Statistical test: Kruskal–Wallis test. **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: ChIP-sequencing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Example view of SIRV-normalized POINT-seq on CD58 gene adjacent to a RI zone in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). DNA replication initiation (RI) zone is highlighted in gray. RI score: 54.6579. Re-analyzed Pu-seq data shows RI zones in green. ( B ) Metagene analysis of POINT-seq on scaled RI zones −/+2.5 kb in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). ( C ) Box plots of SIRV-normalized POINT-seq signals in RI zones in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( D ) Scatter plots of SIRV-normalized POINT-seq signals and RI score in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). Cut-off value is Log 2 (1.25) in SIRV-normalized POINT-seq. Pol II-affected (P-A) and not affected (P-NA) zones are classified as higher and lower than the cut-off value, respectively. RI zones that are above the cut-off in the three timepoints (4, 12, 24 h IAA) are indicated in red. ( E ) Example view of SIRV-normalized POINT-seq on CD2AP gene adjacent to a P-NA zone in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). DNA RI zone is highlighted in gray. RI score: 42.8401. ( F ) Box plots of SIRV-normalized POINT-seq signals in P-A ( n = 5878) and P-NA ( n = 6651) RI zones in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( G ) Size in bp of P-A ( n = 5878) or P-NA ( n = 6651) RI zones. Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( H ) Distance in bp between the closest gene and the P-A ( n = 5878) and P-NA ( n = 6651) RI zones. Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%).

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques:

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Representative images of PLA with the indicated antibodies after 4 h DMSO and IAA in NELF-C-AID DLD-1 cells. Blue: Hoechst, Red: PLA. Scale bar size is 5 µm. ( B ) Box plots of the PLA foci per nucleus after 4 h DMSO and IAA in NELF-C-AID DLD-1 cells. The PLA was performed with indicated antibodies. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Dots represent individual nuclei. Statistical test: Brunner–Munzel test. Statistical test: Wilcoxon rank-sum test. P values are shown. not significant (n.s.). ( C ) Western blot of chromatin fraction of NELF-C-AID DLD-1 cells (0, 4, and 24 h IAA) using the indicated antibodies. ( D ) Cell cycle (%) of 24 h DMSO or MLN-4924 treated NELF-C-AID DLD-1 cells. The cells were pre-treated with IAA or DMSO for 24 h. Error bars represented the mean ± SEM (biological replicates, n = 3). ( E ) BrdU dot blot assay to evaluate global DNA synthesis. ( F ) BrdU-IP-seq of two biological replicates (R1 and R2) for FOCAD gene regions in parental and NELF-C-AID DLD-1 cells treated with IAA for 4 h. IP efficiencies (IP/input) are shown. BrdU plus (magenta) and minus (green) zones in Parental minus NELF-C-AID (Δ). Normalized POINT-seq profiles for (+) and (−) strands in NELF-C-AID DLD-1 cells treated with IAA for 0 h (DMSO) or 4 h (IAA) are shown. Pol II transcription termination defect are indicated by arrows. FOCAD DoG region is highlighted by gray. Early and Late S phase are separated by dashed line. ( G ) BrdU-IP-qPCR analysis to assess local DNA synthesis rates in region exhibiting termination defect. The top panel shows the locations of primer sets used for qPCR. The bottom panel displays the IP/input values for each locus, normalized to the ELAVL2 locus, a late-replicating region that is not expected to replicate at the onset of the S phase (ELAVL2 locus = 1). Error bars represented the mean ± SEM (biological replicates, n = 3). P values are indicated.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Western Blot, Dot Blot, DNA Synthesis

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Representative images of PLA with the indicated antibodies after 4 h DMSO and IAA in MCM2 (right) or PCNA (left) expressing NELF-C-AID DLD-1 cells. Blue: Hoechst, Green: MCM2 or PCNA, Red: PLA. Scale bar size is 5 µm. ( B ) Box plots of the PLA foci per nucleus after 4 h DMSO and IAA in MCM2 or PCNA expressing NELF-C-AID DLD-1 cells. The PLA was performed with the indicated antibodies. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Dots represent individual nuclei. Statistical test: Brunner–Munzel test. P values are shown. n.s. (not significant). ( C ) Schematic representation of the BrdU-IP assay performed following release from M phase. ( D ) BrdU-IP-seq of two biological replicates (R1 and R2) for the indicated chromosome 3 window in parental and NELF-C-AID DLD-1 cells treated with IAA for 4 h. IP efficiencies (IP/input) are shown. BrdU plus (magenta) and minus (green) zones in Parental minus NELF-C-AID (Δ). Dashed lines indicate 1.25 in BrdU-IP (%). Normalized POINT-seq profiles for (+) and (−) strands in NELF-C-AID DLD-1 cells treated with IAA for 0 h (DMSO) or 4 h (IAA) are shown. Pol II transcription termination defect are indicated by arrows. ( E ) Quantification of the POINT-seq (IAA 4 h – DMSO) signal over minus and plus BrdU zones located downstream of expressed pc genes (from TES + 2.5 to TES + 5 kb). Violin plots show the distribution (kernel density) of log10 POINT-seq read coverage mean values. Embedded box plots indicate the median (center; white dot/line) and the interquartile range (box; 25th–75th percentiles). Whiskers extend to 1.5×IQR; whisker endpoints represent the minimum and maximum values within this range. Statistical test: Brunner–Munzel test. **** P < 0.0001. ( F ) Model of NELF-mediated transcription termination and DNA replication initiation or elongation. With NELF, Pol II transcription is terminated at proximal PAS before Pol II reaches DNA RI zone. Without NELF, Pol II transcription is extended to distal PAS and impair replication in the RI zone. This perturbs DNA RI and/or causes T-R conflict (HO, Head-ON), resulting in cell quiescence.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Expressing, Whisker Assay

Journal: eLife

Article Title: Rtf1-dependent transcriptional pausing regulates cardiogenesis

doi: 10.7554/eLife.94524

Figure Lengend Snippet: ( a ) Schematic of CRISPR/Cas9 mutagenesis of the rtf1 gene. Two mutant alleles, rtf1 LA2678 and rtf1 LA2679 , were recovered after targeting of rtf1 exon 3. Both alleles are predicted to disrupt translation of the Rtf1 protein and eliminate the histone modification domain (HMD), Plus3, polymerase II (Pol II) interaction, and Polymerase Associated Factor 1 Complex (PAF1C) interaction domains. ( b ) Agarose gel electrophoresis results of genotyping rtf1 mutants by PCR. Deletions in rtf1 LA2678 and rtf1 LA2679 alleles can be distinguished from wild-type allele using primers rtf1-e3-F and rtf1-e3-R. ( c ) Western blot detecting Rtf1 and β-actin (loading control) proteins in lysates of wild-type and rtf1 mutant embryos. The image is representative of two independent experiments. Figure 1—source data 1. PDF file containing original agarose gel image for , indicating the relevant bands and genotypes. Figure 1—source data 2. Original file for agarose gel image displayed in . Figure 1—source data 3. PDF file containing original western blots for , indicating the relevant bands and genotypes. Figure 1—source data 4. Original files for western blots displayed in .

Article Snippet: Immunoprecipitations were carried out as previously described ( ) on approximately 5 μg of chromatin (minimum 2.5 μg, maximum 8.8 μg) using 5 μL of total RNA Pol II antibody (Cell Signaling D8L4Y).

Techniques: CRISPR, Mutagenesis, Modification, Agarose Gel Electrophoresis, Western Blot, Control

Journal: eLife

Article Title: Rtf1-dependent transcriptional pausing regulates cardiogenesis

doi: 10.7554/eLife.94524

Figure Lengend Snippet: ( a ) Schematics of Rtf1 wild-type (wt*) and Rtf1 mutant constructs (ΔHMD and ΔPlus3). Domains are indicated by colored boxes (histone modification domain [HMD]: red, Plus3: blue, polymerase II [Pol II], and Polymerase Associated Factor 1 Complex [PAF1C] interaction: gray). Deleted regions are represented by lines. Numbers refer to the amino acid positions in wild-type Rtf1. Rtf1 wt*, ΔHMD, and ΔPlus3 also harbor 8 silent mutations that alter the rtf1 translation blocking morpholino binding site. ( b ) Projections of confocal z-stacks of whole-mount immunostaining detecting N-terminal FLAG-tagged Rtf1 constructs (red) expressed in 75% epiboly zebrafish embryos. Nuclei are labeled by DAPI staining (blue). Scale bars represent 20 µm. ( c ) Representative images of RNA in situ hybridization detecting nkx2.5 expression in 10 somite stage (10S) zebrafish embryos co-injected with rtf1 morpholino and mRNA encoding Rtf1 wild-type (wt*) or mutant (ΔHMD and ΔPlus3) proteins. Scale bars represent 0.1 mm.

Article Snippet: Immunoprecipitations were carried out as previously described ( ) on approximately 5 μg of chromatin (minimum 2.5 μg, maximum 8.8 μg) using 5 μL of total RNA Pol II antibody (Cell Signaling D8L4Y).

Techniques: Mutagenesis, Construct, Modification, Blocking Assay, Binding Assay, Immunostaining, Labeling, Staining, RNA In Situ Hybridization, Expressing, Injection

Journal: eLife

Article Title: Rtf1-dependent transcriptional pausing regulates cardiogenesis

doi: 10.7554/eLife.94524

Figure Lengend Snippet: Representative ChIP-seq tracks displaying RNA Polymerase II (RNA Pol II) read densities (y-axis) for control (red), rtf1 morphant (blue), and flavopiridol-treated rtf1 morphant (light blue) embryos at cardiac mesoderm-related genes, including hand2 ( a ), gata5 ( b ), aplnrb ( c ), bmp4 ( d ), nkx2.7 ( e ), and rbfox1l ( f ).

Article Snippet: Immunoprecipitations were carried out as previously described ( ) on approximately 5 μg of chromatin (minimum 2.5 μg, maximum 8.8 μg) using 5 μL of total RNA Pol II antibody (Cell Signaling D8L4Y).

Techniques: ChIP-sequencing, Control

Journal: eLife

Article Title: Rtf1-dependent transcriptional pausing regulates cardiogenesis

doi: 10.7554/eLife.94524

Figure Lengend Snippet: ( a ) Cumulative frequency plot of pause release ratios (PRRs) of 6078 genes with substantial RNA Polymerase II (RNA Pol II) signal. Both control (red) and flavopiridol-treated rtf1 morphant (light blue) samples displayed significantly different PRR frequencies compared to untreated rtf1 morphants (blue). ***: p<2.2 × 10 –16 ; Welch’s paired two-tailed t -test. The median PRR of control samples is indicated by a vertical gray dashed line. ( b ) Dot plot comparing PRRs in controls and rtf1 morphants. Each dot represents the PRR values for a single gene that differ significantly (colored point) or are not significantly different (gray point) between controls and rtf1 morphants. ( c ) Dot plot comparing PRRs in rtf1 morphants and flavopiridol-treated rtf1 morphants. Each dot represents the PRR values for a single gene that differ significantly (colored point) or are not significantly different (gray point) between rtf1 morphants and flavopiridol-treated rtf1 morphants. Dot colors in ( b ) and ( c ) are based on the density of points, with lighter colors indicating more dense points. ( d ) Representative images of RNA in situ hybridization detecting myl7 expression in 24 hpf control and Rtf1-deficient (±flavopiridol) zebrafish embryos. ( e ) Quantification of myl7 signal intensity in control and Rtf1-deficient (±flavopiridol) embryos at 24 hpf. Numbers on bars indicate the number of embryos analyzed. ***: p<0.001. ( f ) Representative images of RNA in situ hybridization detecting myl7 expression in 24 hpf control and Rtf1-deficient (± cdk9 morpholino) zebrafish embryos. ( g ) Quantification of myl7 signal intensity in control and Rtf1-deficient (± cdk9 morpholino) embryos at 24 hpf. Numbers on bars indicate the number of embryos analyzed. ***: p<0.001. ( h ) Venn diagram of significantly altered gene expression in 8–9 somite stage rtf1 morphant embryos (±flavopiridol treatment). Shaded circles represent genes that are significantly: downregulated in rtf1 morphants vs. uninjected controls (blue), upregulated in rtf1 morphants vs. uninjected controls (yellow), downregulated in flavopiridol-treated rtf1 morphants vs. vehicle-treated (DMSO) rtf1 morphants (green), and upregulated in flavopiridol-treated rtf1 morphants vs. vehicle-treated rtf1 morphants (pink). Overlapping regions with thick black outlines represent the 1447 genes (*) with expression levels that were significantly rescued in 8–9 somite stage rtf1 morphants by flavopiridol treatment. ( i ) Plot of enriched Biological Process gene ontology terms for the set of genes that were significantly rescued in rtf1 morphants by flavopiridol treatment. Dot sizes indicate the number of genes associated with a given ontology term. Dot color indicates the significance level (adjusted p-value). ( j ) Selected list of genes that are critical to cardiac development and were significantly rescued in rtf1 morphants by flavopiridol treatment. Fold-changes and adjusted p-values were calculated with DESeq2. Scale bars in panels d and f represent 0.1 mm.

Article Snippet: Immunoprecipitations were carried out as previously described ( ) on approximately 5 μg of chromatin (minimum 2.5 μg, maximum 8.8 μg) using 5 μL of total RNA Pol II antibody (Cell Signaling D8L4Y).

Techniques: Control, Two Tailed Test, RNA In Situ Hybridization, Expressing, Gene Expression

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Lamin A/C promotes chromatin compaction suppressing EMT. (A) Schematic representation of chromatin immunoprecipitation (ChIP) experiments performed in MCF7 cells overexpressing TWIST1-GFP, following knockdown (KD) of Lamin A/C or overexpression (OE) of full-length Lamin A or phospho-mutant Lamin A (S22A: phospho-deficient; S22D: phospho-mimetic). ChIP was performed using antibodies against Lamin A/C, EZH2, H3K27me3 [−1 kb of transcription start sites (TSS)], and RNA Polymerase II (Ser2P) and H3K36me3 (+1 kb of TSS) at the promoters of key mesenchymal transcription factors SNAI1, TWIST1, and ZEB1. (B) Schematic representation of ChIP analysis in MCF7 cells with EZH2 knockdown or overexpression of full-length or phospho-mutant EZH2 (T345A: phospho-deficient; T345D: phospho-mimetic) in the context of TWIST1-GFP induction. Promoter occupancy of Lamin A/C, EZH2, and associated histone modifications (H3K27me3 and H3K36me3), as well as RNA Pol II (Ser2P), was examined at the TSS of SNAI1, TWIST1, and ZEB1. Green and red arrows indicate primers for the qPCR experiment. ( C–E ) ChIP PCR analysis in MCF7 cells stably transduced with full-length Lamin A, phosphodeficient (S22A), or phosphomimetic (S22D) variants, followed by transient transfection with pEGFP-N1 (control) or TWIST1-GFP for 48 h. ChIP occupancy of (C) Lamin A, (D) EZH2 at the −1 kb regions, and (E) H3K36me3 at the +1 kb regions of SNAI1, TWIST1, and ZEB1 were assessed. DEFA3 (C) , MYT1 (D) , and GAPDH (E) served as positive controls, while Jun (C) , GAPDH +1 kb (D) , and MYT1 (E) were negative controls for Lamin A/C, EZH2, and H3K36me3 respectively. ( F–H ) ChIP PCR analysis in MCF7 cells stably transduced with full-length EZH2, phosphodeficient (T345A), or phosphomimetic (T345D) variants, followed by transient transfection with pEGFP-N1 or TWIST1-GFP for 48 h. ChIP occupancy of (F) Lamin A, (G) EZH2 at the −1 kb regions, and (H) H3K36me3 at the +1 kb regions of SNAI1, TWIST1, and ZEB1 was assessed. DEFA3 (F) , MYT1 (G) , and GAPDH (H) served as positive controls, while Jun (F) , GAPDH +1 kb (G) , and MYT1 (H) were negative controls for Lamin A/C, EZH2, and H3K36me3 respectively. Data represent mean ± SD from three independent experiments.

Article Snippet: The following antibodies were used: Lamin A/C (Abcam #ab108595), EZH2 (CST #5246), H3K27me3 (Millipore #07-449), H3K36me3 (Abcam #ab9050), RNA Pol II (Ser2p; Covance #MMS-129R), and control IgG (Invitrogen #10500C).

Techniques: Chromatin Immunoprecipitation, Knockdown, Over Expression, Mutagenesis, Stable Transfection, Transduction, Transfection, Control

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Lamin A/C-EZH2 interaction modulates EM plasticity. This schematic illustrates the dynamic regulation of EMT and MET by Lamin A/C–EZH2 signaling. In epithelial cells (top), high Lamin A/C levels promote the sequestration of EZH2 at the nuclear lamina, diminishing the transcriptional activation and expression of key EMT transcription factors (SNAI1, TWIST1, ZEB1) by enforcing H3K27me3-mediated repression at their promoters. Reduced CDK1 activity leads to reduced phosphorylation on both Lamin A/C (Ser22) and EZH2 (Thr345), limiting EZH2 degradation and further stabilizing chromatin repression. During EMT, increased CDK1 activity and phosphorylation of Lamin A/C (Ser22) and EZH2 (Thr345), results in proteasomal degradation of EZH2 which releases the repressive chromatin state, recruits active RNA polymerase II to EMT gene promoters, and upregulates EMT transcription factors, promoting mesenchymal properties. Created in BioRender. Sengupta, K. ( Schematic illustration of EMT and MET regulation by Lamin A/C-EZH2 ).

Article Snippet: The following antibodies were used: Lamin A/C (Abcam #ab108595), EZH2 (CST #5246), H3K27me3 (Millipore #07-449), H3K36me3 (Abcam #ab9050), RNA Pol II (Ser2p; Covance #MMS-129R), and control IgG (Invitrogen #10500C).

Techniques: Activation Assay, Expressing, Activity Assay, Phospho-proteomics

Journal: Nature Cardiovascular Research

Article Title: Pathogenetic mechanisms of muscle-specific ribosomes in dilated cardiomyopathy

doi: 10.1038/s44161-025-00761-8

Figure Lengend Snippet: a , No difference in transcription activity in RPL3 promoter assayed by RNA Pol II ChIP-qPCR. Multiple primer pairs were used for each region. An intergenic region upstream of RPL3L was used as a negative control of non-transcribed region (N = 3 biological replicates of immunoprecipitated chromatin). Data shown as mean ± s.d. Statistical significance determined by two-sided t-test between G27D and R161W. b , R161W does not induce higher RPL3 intronic RNA as a proxy of transcription levels. Total RNA was isolated and cDNA was synthesized using random primers. Primers targeting intron 3 (not overlapping with the NMD exon) and intron 9 of RPL3 were used for amplification (N = 3 biological replicates). Analyzed by two-sided t-test between G27D and R161W. Error bars represent standard deviation. ns, not significant. c , RNA-seq coverage and junction reads in control HEK293 cells and SMG6-KD/SMG7-KO cells from a previous study . N = 3 biological replicates from that study. Data shown as mean ± s.d. d , RNA-seq coverage and junction reads in mouse hearts (WT and KO) and C2C12 cells (OE, overexpression) from three previous studies: Grimes et al. , Milenkovic et al. , and Shiraishi et al. Replicates were pooled. e , Wild type AC16 cells expressing dox-inducible RPL3L (without HA) for 96 h were analyzed with RT-PCR for RPL3 NMD exon activation in the absence of shRPL3. The numbers below the gel are relative band intensity (NMD/Canonical) normalized to lane 2 (No Dox). Related to Fig. .

Article Snippet: Chromatin was incubated with 5 μg of anti-RNA polymerase II (clone F-12, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Activity Assay, ChIP-qPCR, Negative Control, Immunoprecipitation, Isolation, Synthesized, Amplification, Standard Deviation, RNA Sequencing, Control, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay