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$SPT6 And IWS1 chromatin binding and transcriptional roles. ( A ) A schematic representation of SPT6 N-terminal (1–283 amino acids), core (284–1287 amino acids), and C-terminal (1288–1726 amino acids) regions. The illustration denotes N to C terminal protein domains. ( B ) Overexpressed Flag-tagged SPT6 from HEK293T total protein extracts was immunoprecipitated using an anti-Flag antibody and analyzed by western blotting using indicated antibodies. ( C ) SPT6 and IWS1 mean ChIP-seq signal intensities plotted at RefSeq-annotated genes. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( D ) Heatmap representing the Pearson correlation coefficients calculated for IWS1, SPT6, and RNAPII merged ChIP-seq signals, on RefSeq-annotated genes (±1 kb). ( E ) SPT6/RNAPII and IWS1/RNAPII mean ChIP-seq signal ratios calculated on RefSeq-annotated genes in HeLa cells. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( F ) Total protein extracts from HeLa cells were separated using a 5%–60% glycerol gradient. A total of 12 fractions were recovered after ultracentrifugation and analyzed by western blotting. ( G ) Western blot showing SPT6 and IWS1 depletions upon <t>siRNA</t> transfection in <t>RNA-seq</t> experiments. Lower panel shows protein quantification relative to GAPDH and to the siCT condition ( n = 3). ( H ) SPT6 and IWS1 target genes in HeLa cells. Positive targets correspond to genes having a |FC| > |±1.5| and a padj < 0.05. ( I ) Density plot showing the gene count of SPT6 and IWS1 readthrough targets, highlighting the distribution of their log 2 fold-change (FC) values. ( J ) SEC22B gene locus featuring RNAPII, SPT6, and IWS1 ChIP-seq profiles, as well as RNA-seq profiles upon depletion of SPT6 and IWS1 (negative strand). The arrow highlights readthrough transcription ( K ) Western blot showing SPT6 and IWS1 depletion upon siRNA transfection (see the “Materials and methods” section for details). ( L ) SEC22B readthrough and mRNA levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( M ) SEC22B readthrough levels were assayed by nuclear run-on experiments. The input represents total nuclear RNAs. The siSPT6 (-BrdU) condition is used to control the specificity of the anti-BrdU immunoprecipitation. Values were normalized to the siCT condition arbitrarily set to 1, and to the KDSR and 18S housekeeping genes.$
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1) Product Images from "Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination"

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkaf179

$SPT6 And IWS1 chromatin binding and transcriptional roles. ( A ) A schematic representation of SPT6 N-terminal (1–283 amino acids), core (284–1287 amino acids), and C-terminal (1288–1726 amino acids) regions. The illustration denotes N to C terminal protein domains. ( B ) Overexpressed Flag-tagged SPT6 from HEK293T total protein extracts was immunoprecipitated using an anti-Flag antibody and analyzed by western blotting using indicated antibodies. ( C ) SPT6 and IWS1 mean ChIP-seq signal intensities plotted at RefSeq-annotated genes. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( D ) Heatmap representing the Pearson correlation coefficients calculated for IWS1, SPT6, and RNAPII merged ChIP-seq signals, on RefSeq-annotated genes (±1 kb). ( E ) SPT6/RNAPII and IWS1/RNAPII mean ChIP-seq signal ratios calculated on RefSeq-annotated genes in HeLa cells. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( F ) Total protein extracts from HeLa cells were separated using a 5%–60% glycerol gradient. A total of 12 fractions were recovered after ultracentrifugation and analyzed by western blotting. ( G ) Western blot showing SPT6 and IWS1 depletions upon siRNA transfection in RNA-seq experiments. Lower panel shows protein quantification relative to GAPDH and to the siCT condition ( n = 3). ( H ) SPT6 and IWS1 target genes in HeLa cells. Positive targets correspond to genes having a |FC| > |±1.5| and a padj < 0.05. ( I ) Density plot showing the gene count of SPT6 and IWS1 readthrough targets, highlighting the distribution of their log 2 fold-change (FC) values. ( J ) SEC22B gene locus featuring RNAPII, SPT6, and IWS1 ChIP-seq profiles, as well as RNA-seq profiles upon depletion of SPT6 and IWS1 (negative strand). The arrow highlights readthrough transcription ( K ) Western blot showing SPT6 and IWS1 depletion upon siRNA transfection (see the “Materials and methods” section for details). ( L ) SEC22B readthrough and mRNA levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( M ) SEC22B readthrough levels were assayed by nuclear run-on experiments. The input represents total nuclear RNAs. The siSPT6 (-BrdU) condition is used to control the specificity of the anti-BrdU immunoprecipitation. Values were normalized to the siCT condition arbitrarily set to 1, and to the KDSR and 18S housekeeping genes.$
Figure Legend Snippet: SPT6 And IWS1 chromatin binding and transcriptional roles. ( A ) A schematic representation of SPT6 N-terminal (1–283 amino acids), core (284–1287 amino acids), and C-terminal (1288–1726 amino acids) regions. The illustration denotes N to C terminal protein domains. ( B ) Overexpressed Flag-tagged SPT6 from HEK293T total protein extracts was immunoprecipitated using an anti-Flag antibody and analyzed by western blotting using indicated antibodies. ( C ) SPT6 and IWS1 mean ChIP-seq signal intensities plotted at RefSeq-annotated genes. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( D ) Heatmap representing the Pearson correlation coefficients calculated for IWS1, SPT6, and RNAPII merged ChIP-seq signals, on RefSeq-annotated genes (±1 kb). ( E ) SPT6/RNAPII and IWS1/RNAPII mean ChIP-seq signal ratios calculated on RefSeq-annotated genes in HeLa cells. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( F ) Total protein extracts from HeLa cells were separated using a 5%–60% glycerol gradient. A total of 12 fractions were recovered after ultracentrifugation and analyzed by western blotting. ( G ) Western blot showing SPT6 and IWS1 depletions upon siRNA transfection in RNA-seq experiments. Lower panel shows protein quantification relative to GAPDH and to the siCT condition ( n = 3). ( H ) SPT6 and IWS1 target genes in HeLa cells. Positive targets correspond to genes having a |FC| > |±1.5| and a padj < 0.05. ( I ) Density plot showing the gene count of SPT6 and IWS1 readthrough targets, highlighting the distribution of their log 2 fold-change (FC) values. ( J ) SEC22B gene locus featuring RNAPII, SPT6, and IWS1 ChIP-seq profiles, as well as RNA-seq profiles upon depletion of SPT6 and IWS1 (negative strand). The arrow highlights readthrough transcription ( K ) Western blot showing SPT6 and IWS1 depletion upon siRNA transfection (see the “Materials and methods” section for details). ( L ) SEC22B readthrough and mRNA levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( M ) SEC22B readthrough levels were assayed by nuclear run-on experiments. The input represents total nuclear RNAs. The siSPT6 (-BrdU) condition is used to control the specificity of the anti-BrdU immunoprecipitation. Values were normalized to the siCT condition arbitrarily set to 1, and to the KDSR and 18S housekeeping genes.

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, ChIP-sequencing, Transfection, RNA Sequencing, Quantitative RT-PCR, Control

$SPT6 interacts with IWS1, PNUTS, and PCF11. ( A ) Upper panel: Diagram depicting PNUTS domains: the TND (1–147 amino acids), the PP1-binding domain (348–418 amino acids) and the RNA-binding domain (674–750 amino acids). Lower panel: Diagram depicting PCF11 domains: the CTD-interacting domain (CID) (14–142 amino acids), and the RNA-binding zinc fingers (between 1343 and 1478 amino acids). The illustration denotes N-to-C terminal protein domains. ( B ) Nuclear protein extracts from WT or IWS1-knockout (IWS1-/-) J-Lat A1 cells were used to immunoprecipitate endogenous SPT6. Co-immunoprecipitation of IWS1, PCF11 and PNUTS was assessed ( n = 3). ( C ) Overexpressed Flag-tagged WT PNUTS (WT) or Flag-tagged PNUTS–W401A (W401A), a mutant for PP1 interaction, were immunoprecipitated using an anti-Flag antibody from HEK293T total protein extracts. SPT6 co-immunoprecipitation was examined for both WT and mutant PNUTS ( n = 3). ( D ) HeLa cells infected with lentiviruses containing either a control shRNA (shCT) or an shRNA targeting PNUTS (shPNUTS). Cells nuclear fractions were used to immunoprecipitate endogenous SPT6 ( n = 2). “Cyto” stands for Cytoplasmic fraction, and “Nuc” stands for Nuclear fraction used as input.$
Figure Legend Snippet: SPT6 interacts with IWS1, PNUTS, and PCF11. ( A ) Upper panel: Diagram depicting PNUTS domains: the TND (1–147 amino acids), the PP1-binding domain (348–418 amino acids) and the RNA-binding domain (674–750 amino acids). Lower panel: Diagram depicting PCF11 domains: the CTD-interacting domain (CID) (14–142 amino acids), and the RNA-binding zinc fingers (between 1343 and 1478 amino acids). The illustration denotes N-to-C terminal protein domains. ( B ) Nuclear protein extracts from WT or IWS1-knockout (IWS1-/-) J-Lat A1 cells were used to immunoprecipitate endogenous SPT6. Co-immunoprecipitation of IWS1, PCF11 and PNUTS was assessed ( n = 3). ( C ) Overexpressed Flag-tagged WT PNUTS (WT) or Flag-tagged PNUTS–W401A (W401A), a mutant for PP1 interaction, were immunoprecipitated using an anti-Flag antibody from HEK293T total protein extracts. SPT6 co-immunoprecipitation was examined for both WT and mutant PNUTS ( n = 3). ( D ) HeLa cells infected with lentiviruses containing either a control shRNA (shCT) or an shRNA targeting PNUTS (shPNUTS). Cells nuclear fractions were used to immunoprecipitate endogenous SPT6 ( n = 2). “Cyto” stands for Cytoplasmic fraction, and “Nuc” stands for Nuclear fraction used as input.

Techniques Used: Binding Assay, RNA Binding Assay, Zinc-Fingers, Knock-Out, Immunoprecipitation, Mutagenesis, Infection, Control, shRNA

Differential readthrough transcripts upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 readthrough targets (TES + 5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed readthrough transcripts in HeLa cells following SPT6 and/or PNUTS depletion. Readthrough transcripts with a log 2 FC >1 in the siSPT6 condition compared to the control condition were considered positive. The readthrough regions were identified as described in . The heatmap plots the average log2FC scores (siRNA/siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and/or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of readthrough transcripts relative to the control condition for the three clusters shown in panel (B). ( D – F ) Same as in panels (A)–(C) but for SPT6 and/or PCF11 depletion. ( G ) CTNND1 gene locus featuring RNA-seq data (positive strand) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. . The arrow highlights readthrough transcription. The scale is shown at the top right corner of the figure.

Figure Legend Snippet: Differential readthrough transcripts upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 readthrough targets (TES + 5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed readthrough transcripts in HeLa cells following SPT6 and/or PNUTS depletion. Readthrough transcripts with a log 2 FC >1 in the siSPT6 condition compared to the control condition were considered positive. The readthrough regions were identified as described in . The heatmap plots the average log2FC scores (siRNA/siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and/or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of readthrough transcripts relative to the control condition for the three clusters shown in panel (B). ( D – F ) Same as in panels (A)–(C) but for SPT6 and/or PCF11 depletion. ( G ) CTNND1 gene locus featuring RNA-seq data (positive strand) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. . The arrow highlights readthrough transcription. The scale is shown at the top right corner of the figure.

Techniques Used: RNA Sequencing, Control

Differential PROMPTs regulation upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 PROMPT targets (TSS, -5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed PROMPTs in HeLa cells following SPT6 and/or PNUTS depletion. PROMPTs with a log 2 FC >1 in the siSPT6 condition compared to the control condition were considered positive. PROMPT regions were identified as described in . The heatmap plots the average log 2 FC scores (siRNA/siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and/or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of PROMPTs relative to the control condition for the three clusters shown in panel (B). ( D ) PXDN PROMPT levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( E – G ) Same as in panels (A)–(C) but for SPT6 and/or PCF11 depletion. ( H ) Same as in panel (D) but for the GGCT gene. ( I ) PVT1 gene locus featuring RNA-seq data (positive strand is in positive values and negative strand in negative values) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. . The arrow highlights PROMPTs. The scale is shown at the top right corner of the figure.

Figure Legend Snippet: Differential PROMPTs regulation upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 PROMPT targets (TSS, -5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed PROMPTs in HeLa cells following SPT6 and/or PNUTS depletion. PROMPTs with a log 2 FC >1 in the siSPT6 condition compared to the control condition were considered positive. PROMPT regions were identified as described in . The heatmap plots the average log 2 FC scores (siRNA/siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and/or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of PROMPTs relative to the control condition for the three clusters shown in panel (B). ( D ) PXDN PROMPT levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( E – G ) Same as in panels (A)–(C) but for SPT6 and/or PCF11 depletion. ( H ) Same as in panel (D) but for the GGCT gene. ( I ) PVT1 gene locus featuring RNA-seq data (positive strand is in positive values and negative strand in negative values) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. . The arrow highlights PROMPTs. The scale is shown at the top right corner of the figure.

Techniques Used: RNA Sequencing, Control, Quantitative RT-PCR

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Journal: Nucleic Acids Research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: SPT6 And IWS1 chromatin binding and transcriptional roles. ( A ) A schematic representation of SPT6 N-terminal (1–283 amino acids), core (284–1287 amino acids), and C-terminal (1288–1726 amino acids) regions. The illustration denotes N to C terminal protein domains. ( B ) Overexpressed Flag-tagged SPT6 from HEK293T total protein extracts was immunoprecipitated using an anti-Flag antibody and analyzed by western blotting using indicated antibodies. ( C ) SPT6 and IWS1 mean ChIP-seq signal intensities plotted at RefSeq-annotated genes. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( D ) Heatmap representing the Pearson correlation coefficients calculated for IWS1, SPT6, and RNAPII merged ChIP-seq signals, on RefSeq-annotated genes (±1 kb). ( E ) SPT6/RNAPII and IWS1/RNAPII mean ChIP-seq signal ratios calculated on RefSeq-annotated genes in HeLa cells. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( F ) Total protein extracts from HeLa cells were separated using a 5%–60% glycerol gradient. A total of 12 fractions were recovered after ultracentrifugation and analyzed by western blotting. ( G ) Western blot showing SPT6 and IWS1 depletions upon siRNA transfection in RNA-seq experiments. Lower panel shows protein quantification relative to GAPDH and to the siCT condition ( n = 3). ( H ) SPT6 and IWS1 target genes in HeLa cells. Positive targets correspond to genes having a |FC| > |±1.5| and a padj < 0.05. ( I ) Density plot showing the gene count of SPT6 and IWS1 readthrough targets, highlighting the distribution of their log 2 fold-change (FC) values. ( J ) SEC22B gene locus featuring RNAPII, SPT6, and IWS1 ChIP-seq profiles, as well as RNA-seq profiles upon depletion of SPT6 and IWS1 (negative strand). The arrow highlights readthrough transcription ( K ) Western blot showing SPT6 and IWS1 depletion upon siRNA transfection (see the “Materials and methods” section for details). ( L ) SEC22B readthrough and mRNA levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( M ) SEC22B readthrough levels were assayed by nuclear run-on experiments. The input represents total nuclear RNAs. The siSPT6 (-BrdU) condition is used to control the specificity of the anti-BrdU immunoprecipitation. Values were normalized to the siCT condition arbitrarily set to 1, and to the KDSR and 18S housekeeping genes.

Article Snippet: Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in ) were cloned into the pLKO.1-TRC cloning vector (gift from David Root, Addgene plasmid #10878; http://n2t.net/addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, ChIP-sequencing, Transfection, RNA Sequencing, Quantitative RT-PCR, Control

Journal: Nucleic Acids Research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: SPT6 interacts with IWS1, PNUTS, and PCF11. ( A ) Upper panel: Diagram depicting PNUTS domains: the TND (1–147 amino acids), the PP1-binding domain (348–418 amino acids) and the RNA-binding domain (674–750 amino acids). Lower panel: Diagram depicting PCF11 domains: the CTD-interacting domain (CID) (14–142 amino acids), and the RNA-binding zinc fingers (between 1343 and 1478 amino acids). The illustration denotes N-to-C terminal protein domains. ( B ) Nuclear protein extracts from WT or IWS1-knockout (IWS1-/-) J-Lat A1 cells were used to immunoprecipitate endogenous SPT6. Co-immunoprecipitation of IWS1, PCF11 and PNUTS was assessed ( n = 3). ( C ) Overexpressed Flag-tagged WT PNUTS (WT) or Flag-tagged PNUTS–W401A (W401A), a mutant for PP1 interaction, were immunoprecipitated using an anti-Flag antibody from HEK293T total protein extracts. SPT6 co-immunoprecipitation was examined for both WT and mutant PNUTS ( n = 3). ( D ) HeLa cells infected with lentiviruses containing either a control shRNA (shCT) or an shRNA targeting PNUTS (shPNUTS). Cells nuclear fractions were used to immunoprecipitate endogenous SPT6 ( n = 2). “Cyto” stands for Cytoplasmic fraction, and “Nuc” stands for Nuclear fraction used as input.

Techniques: Binding Assay, RNA Binding Assay, Zinc-Fingers, Knock-Out, Immunoprecipitation, Mutagenesis, Infection, Control, shRNA

Journal: Nucleic Acids Research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Differential readthrough transcripts upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 readthrough targets (TES + 5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed readthrough transcripts in HeLa cells following SPT6 and/or PNUTS depletion. Readthrough transcripts with a log 2 FC >1 in the siSPT6 condition compared to the control condition were considered positive. The readthrough regions were identified as described in . The heatmap plots the average log2FC scores (siRNA/siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and/or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of readthrough transcripts relative to the control condition for the three clusters shown in panel (B). ( D – F ) Same as in panels (A)–(C) but for SPT6 and/or PCF11 depletion. ( G ) CTNND1 gene locus featuring RNA-seq data (positive strand) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. . The arrow highlights readthrough transcription. The scale is shown at the top right corner of the figure.

Techniques: RNA Sequencing, Control

Journal: Nucleic Acids Research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Differential PROMPTs regulation upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 PROMPT targets (TSS, -5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed PROMPTs in HeLa cells following SPT6 and/or PNUTS depletion. PROMPTs with a log 2 FC >1 in the siSPT6 condition compared to the control condition were considered positive. PROMPT regions were identified as described in . The heatmap plots the average log 2 FC scores (siRNA/siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and/or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of PROMPTs relative to the control condition for the three clusters shown in panel (B). ( D ) PXDN PROMPT levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( E – G ) Same as in panels (A)–(C) but for SPT6 and/or PCF11 depletion. ( H ) Same as in panel (D) but for the GGCT gene. ( I ) PVT1 gene locus featuring RNA-seq data (positive strand is in positive values and negative strand in negative values) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. . The arrow highlights PROMPTs. The scale is shown at the top right corner of the figure.

Techniques: RNA Sequencing, Control, Quantitative RT-PCR