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BOD1L loss decreases SETD1A on chromatin. ( A ) Browser view of ChIP-seq results against SETD1A, NELFE, <t>RNAP2</t> NTD, Ser5P, <t>Ser2P,</t> H3K4me3 and H3K36me3 at INTS2 loci in sgBOD1L-expressing leukemia cells. ( B ) Average ChIP-seq signals of SETD1A, NELFE, NTD and Ser5P along DR (SETD1A-target) genes and flanking regions from sgAAVS and sgBOD1L-expressing cells are shown. ( C ) Violin plot indicating signal intensity of SETD1A at DR genes. ( D ) Pausing index (Log 2 ) of all genes and BOD1L/SETD1A targets. ( E ) Browser view of ChIP-seq results against SETD1A and BOD1L (top); 8307 peaks were overlapped (bottom). ( F ) Distributions of SETD1A, NTD and Ser5P at 9290 peaks ± 2kb harboring BOD1L binding 1 h after BOD1L degradation. Violin plot indicating signal intensities of each peak. ( G ) Distributions and signal intensities of H3K4me3 at BOD1L-binding regions 24 h after BOD1L degradation. In (C), (D), (F) and (G), data are presented as mean ± SD. ** P < 0.01. * P < 0.05. ns, no significance.

Rnap2 Ser2p, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "BOD1L mediates chromatin binding and non-canonical function of H3K4 methyltransferase SETD1A"

Article Title: BOD1L mediates chromatin binding and non-canonical function of H3K4 methyltransferase SETD1A

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkae605

BOD1L loss decreases SETD1A on chromatin. ( A ) Browser view of ChIP-seq results against SETD1A, NELFE, RNAP2 NTD, Ser5P, Ser2P, H3K4me3 and H3K36me3 at INTS2 loci in sgBOD1L-expressing leukemia cells. ( B ) Average ChIP-seq signals of SETD1A, NELFE, NTD and Ser5P along DR (SETD1A-target) genes and flanking regions from sgAAVS and sgBOD1L-expressing cells are shown. ( C ) Violin plot indicating signal intensity of SETD1A at DR genes. ( D ) Pausing index (Log 2 ) of all genes and BOD1L/SETD1A targets. ( E ) Browser view of ChIP-seq results against SETD1A and BOD1L (top); 8307 peaks were overlapped (bottom). ( F ) Distributions of SETD1A, NTD and Ser5P at 9290 peaks ± 2kb harboring BOD1L binding 1 h after BOD1L degradation. Violin plot indicating signal intensities of each peak. ( G ) Distributions and signal intensities of H3K4me3 at BOD1L-binding regions 24 h after BOD1L degradation. In (C), (D), (F) and (G), data are presented as mean ± SD. ** P < 0.01. * P < 0.05. ns, no significance.

Figure Legend Snippet: BOD1L loss decreases SETD1A on chromatin. ( A ) Browser view of ChIP-seq results against SETD1A, NELFE, RNAP2 NTD, Ser5P, Ser2P, H3K4me3 and H3K36me3 at INTS2 loci in sgBOD1L-expressing leukemia cells. ( B ) Average ChIP-seq signals of SETD1A, NELFE, NTD and Ser5P along DR (SETD1A-target) genes and flanking regions from sgAAVS and sgBOD1L-expressing cells are shown. ( C ) Violin plot indicating signal intensity of SETD1A at DR genes. ( D ) Pausing index (Log 2 ) of all genes and BOD1L/SETD1A targets. ( E ) Browser view of ChIP-seq results against SETD1A and BOD1L (top); 8307 peaks were overlapped (bottom). ( F ) Distributions of SETD1A, NTD and Ser5P at 9290 peaks ± 2kb harboring BOD1L binding 1 h after BOD1L degradation. Violin plot indicating signal intensities of each peak. ( G ) Distributions and signal intensities of H3K4me3 at BOD1L-binding regions 24 h after BOD1L degradation. In (C), (D), (F) and (G), data are presented as mean ± SD. ** P < 0.01. * P < 0.05. ns, no significance.

Techniques Used: ChIP-sequencing, Expressing, Binding Assay

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Journal: Nucleic Acids Research

Article Title: BOD1L mediates chromatin binding and non-canonical function of H3K4 methyltransferase SETD1A

doi: 10.1093/nar/gkae605

Figure Lengend Snippet: BOD1L loss decreases SETD1A on chromatin. ( A ) Browser view of ChIP-seq results against SETD1A, NELFE, RNAP2 NTD, Ser5P, Ser2P, H3K4me3 and H3K36me3 at INTS2 loci in sgBOD1L-expressing leukemia cells. ( B ) Average ChIP-seq signals of SETD1A, NELFE, NTD and Ser5P along DR (SETD1A-target) genes and flanking regions from sgAAVS and sgBOD1L-expressing cells are shown. ( C ) Violin plot indicating signal intensity of SETD1A at DR genes. ( D ) Pausing index (Log 2 ) of all genes and BOD1L/SETD1A targets. ( E ) Browser view of ChIP-seq results against SETD1A and BOD1L (top); 8307 peaks were overlapped (bottom). ( F ) Distributions of SETD1A, NTD and Ser5P at 9290 peaks ± 2kb harboring BOD1L binding 1 h after BOD1L degradation. Violin plot indicating signal intensities of each peak. ( G ) Distributions and signal intensities of H3K4me3 at BOD1L-binding regions 24 h after BOD1L degradation. In (C), (D), (F) and (G), data are presented as mean ± SD. ** P < 0.01. * P < 0.05. ns, no significance.

Article Snippet: Primary antibodies against HA-tag (#3724, Cell Signaling), SETD1A (#61702, Cell Signaling), RNAP2-NTD (#14958, Cell Signaling), RNAP2-Ser5P (#13523, Cell Signaling), RNAP2-Ser2P (#13499, Cell Signaling), NELFE (#ab170104, Abcam), H3K4me3 (#ab8580, Abcam), H3K36me3 (#9050, Abcam), RPA2 (#ab10359, Abcam), and RBBP5 (A300-109A, Bethyl Laboratories) were used.

Techniques: ChIP-sequencing, Expressing, Binding Assay

Figure 1. Citrullination of R1810 at RNAP2-CTD, Related to Figure S1 (A) Top: The epitope within repeats 31/32 of the CTD domain of RNAP2 used to generate a-Cit1810. Bottom: Duplicated western blot of T47D nuclear extract with a-Cit1810 and a-total-RNAP2 is shown. Line on the left mark is the migration of the 250-kDa size marker. (B) Extracts from T47D cells expressing a-amanitin-resistant WTr or R1810Ar mutant of RNAP2 were precipitated with a-HA antibody and probed with a-Cit1810 or a-RNAP2. (C) Representative super-resolution images of T47D cells immunostained with a-Cit1810 (red) in combination with a-total-RNAP2 (green), a-S2P-RNAP2 (green), and a-S5P-RNAP2 (green). (D) Plot representing the mean Pearson correlation coefﬁcient of individual cells for a-Cit1810-RNAP2 with a-total-RNAP2 (n = 22), a-S2P-RNAP2 (n = 24), and a-S5P-RNAP2 (n = 24); values presented as the mean ± SEM. **p value < 0.005; p value > 0.05.

Journal: Molecular cell

Article Title: Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

doi: 10.1016/j.molcel.2018.10.016

Figure Lengend Snippet: Figure 1. Citrullination of R1810 at RNAP2-CTD, Related to Figure S1 (A) Top: The epitope within repeats 31/32 of the CTD domain of RNAP2 used to generate a-Cit1810. Bottom: Duplicated western blot of T47D nuclear extract with a-Cit1810 and a-total-RNAP2 is shown. Line on the left mark is the migration of the 250-kDa size marker. (B) Extracts from T47D cells expressing a-amanitin-resistant WTr or R1810Ar mutant of RNAP2 were precipitated with a-HA antibody and probed with a-Cit1810 or a-RNAP2. (C) Representative super-resolution images of T47D cells immunostained with a-Cit1810 (red) in combination with a-total-RNAP2 (green), a-S2P-RNAP2 (green), and a-S5P-RNAP2 (green). (D) Plot representing the mean Pearson correlation coefﬁcient of individual cells for a-Cit1810-RNAP2 with a-total-RNAP2 (n = 22), a-S2P-RNAP2 (n = 24), and a-S5P-RNAP2 (n = 24); values presented as the mean ± SEM. **p value < 0.005; p value > 0.05.

Article Snippet: SC-8338 Rabbit polyclonal anti-CCNT1 Bethyl Labs A303-499A IgG mouse Millipore 12-371 IgG Rabbit Cell Signaling 2729S Mouse monoclonal anti-Ser2P RNAP2 Hiroshi Kimura, MBL Life science CMA602 Mouse monoclonal anti-Ser5P RNAP2 Hiroshi Kimura, MBL Life science CMA603 Chemicals, Peptides, and Recombinant Proteins Lipofectamine 3000 Invitrogen L3000008 Cl amidine Calbiochem 506282 a-amanitin Sigma Aldrich A2263 G418 Sigma Aldrich 000000004727878001 Doxycycline Sigma Aldrich D9891 SMARTpool On-target plus siRNAs for human PADI2 Dharmacon (Thermo Scientific) M-019485-01 SMARTpool On-target plus siRNAs for human PADI3 Dharmacon (Thermo Scientific) M-021051-01 CARM1 siRNA (h) Santa Cruz Biot. sc-44875 PRMT5 siRNA (h) Santa Cruz Biot. sc-41073 PADI4 siRNA (h) Santa Cruz Biot. sc-61283 Proteinase K ThermoFisher Scientific AM2546 TURBO DNase ThermoFisher Scientific AM2239 Propidium iodide Molecular Probe P-1304 DNase I (RNase-free) ThermoFisher Scientific AM2222 Cell proliferation ELISA BrdU Colorimetric assay Roche 11647229001 Dynabeads MyOne Streptavidin T1 ThermoFisher 65601 Dynabeads M-280 Sheep Anti-Mouse IgG ThermoFisher 11201D Protein G Plus / Protein A Agarose Millipore IP05 (Continued on next page) e1 Molecular Cell 73, 1–13.e1–e7, January 3, 2019

Techniques: Western Blot, Migration, Marker, Expressing, Mutagenesis

Figure 4. PADI2 Occupancy on Active Genes, Related to Figure S4 (A) Left: Spie chart showing the distribution of PADI2 ChIP-seq peaks over various genomic regions. A dashed curved line indicates the region from 3 kb upstream of TSS to 3 kb downstream of TTS (or polyadenylation site); the numbers in parentheses show the proportion occupied by each region in the genome. Right: Enrichment of normalized PADI2 reads in various genome regions relative to random distribution is shown (*p value < 102; **p value < 103; ***p value < 104). (B) Distribution of normalized PADI2 reads around the center of RNAP2 peaks in T47D cells. (C) RNAP2 and PADI2 occupancy across genes classiﬁed with increasing levels of expression. p value was calculated by Wilcoxon-Mann-Whitney test in comparison to silent genes as indicated (**p value < 103; ***p value < 105).

Journal: Molecular cell

Article Title: Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

doi: 10.1016/j.molcel.2018.10.016

Figure Lengend Snippet: Figure 4. PADI2 Occupancy on Active Genes, Related to Figure S4 (A) Left: Spie chart showing the distribution of PADI2 ChIP-seq peaks over various genomic regions. A dashed curved line indicates the region from 3 kb upstream of TSS to 3 kb downstream of TTS (or polyadenylation site); the numbers in parentheses show the proportion occupied by each region in the genome. Right: Enrichment of normalized PADI2 reads in various genome regions relative to random distribution is shown (*p value < 102; **p value < 103; ***p value < 104). (B) Distribution of normalized PADI2 reads around the center of RNAP2 peaks in T47D cells. (C) RNAP2 and PADI2 occupancy across genes classiﬁed with increasing levels of expression. p value was calculated by Wilcoxon-Mann-Whitney test in comparison to silent genes as indicated (**p value < 103; ***p value < 105).

Techniques: ChIP-sequencing, Expressing, MANN-WHITNEY, Comparison

Figure 5. Cit1810 at CTD-RNAP2 Regulates Pausing in Breast Cancer Cells, Related to Figure S5 (A) RNAP2 ChIP qPCR assay performed in T47D cells expressing only the HA-tagged wild-type (WTr) or R1810Ar mutant of RNAP2 with the HA antibody. Non-immune IgG was used as negative control. y axis: fold change over the input samples. Data represent mean ± SEM of at least three biological experiments, **p value % 0.01; p value > 0.05. (B and C) Difference in RNAP2 density in T47D cells expressing HA-tagged R1810Ar mutant versus WTr RNAP2 (B) across genes classiﬁed by expression. (C) Pausing index of RNAP2 as indicated. p value was calculated by Wilcoxon-Mann-Whitney test in comparison to silent genes (**p value < 103; ***p value < 105). (D and E) PADI2-dependent genes (n = 2,186) showing (D; left) average proﬁle of difference in RNAP2 density (R1810Ar WTr) around TSS. (Right) Heatmap at TSS of genes ranked from highest to lowest RNAP2 density is shown (R1810Ar WTr). (E) Higher pausing index in cell expressing R1810Ar mutant as compared to WTr form of RNAP2. (F) Browser snapshots showing RNAP2 occupancy for HMGN1 and control gene LRRC39 in cells expressing HA-tagged WTr or R1810Ar form of RNAP2.

Journal: Molecular cell

Article Title: Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

doi: 10.1016/j.molcel.2018.10.016

Figure Lengend Snippet: Figure 5. Cit1810 at CTD-RNAP2 Regulates Pausing in Breast Cancer Cells, Related to Figure S5 (A) RNAP2 ChIP qPCR assay performed in T47D cells expressing only the HA-tagged wild-type (WTr) or R1810Ar mutant of RNAP2 with the HA antibody. Non-immune IgG was used as negative control. y axis: fold change over the input samples. Data represent mean ± SEM of at least three biological experiments, **p value % 0.01; p value > 0.05. (B and C) Difference in RNAP2 density in T47D cells expressing HA-tagged R1810Ar mutant versus WTr RNAP2 (B) across genes classiﬁed by expression. (C) Pausing index of RNAP2 as indicated. p value was calculated by Wilcoxon-Mann-Whitney test in comparison to silent genes (**p value < 103; ***p value < 105). (D and E) PADI2-dependent genes (n = 2,186) showing (D; left) average proﬁle of difference in RNAP2 density (R1810Ar WTr) around TSS. (Right) Heatmap at TSS of genes ranked from highest to lowest RNAP2 density is shown (R1810Ar WTr). (E) Higher pausing index in cell expressing R1810Ar mutant as compared to WTr form of RNAP2. (F) Browser snapshots showing RNAP2 occupancy for HMGN1 and control gene LRRC39 in cells expressing HA-tagged WTr or R1810Ar form of RNAP2.

Techniques: ChIP-qPCR, Expressing, Mutagenesis, Negative Control, MANN-WHITNEY, Comparison, Control

Figure 6. Cit1810 at RNAP2-CTD Is Recognized by P-TEFb (A) Immunoprecipitation with a-CDK9 (left) and a-PADI2 (right) or non-immune rabbit IgG of T47D extracts followed by western blot with the indicated antibodies. (B) Extracts from T47D cells in the presence (siCtrl) or absence (siPADI2) of PADI2 were immunoprecipitated with a-total-RNAP2 followed by western blot for the indicated antibodies along with relative quantiﬁcations underneath. (C and D) Immunoprecipitation of extracts from (C) T47D and (D) Raji cells expressing only the HA-tagged a-amanitin-resistant WTr or R1810Ar mutant of RNAP2 were precipitated with a-HA antibody and probed with the indicated antibodies. The relative quantiﬁcation is shown underneath each gel. (E) (Top) Schematic representation of the pull-down assays with T47D nuclear extracts and RNAP2-CTD biotinylated peptides (wild-type R1810 or cit1810). (Bottom) Results of the pull-down experiments with wild-type (R1810) or cit1810 biotinylated RNAP2-CTD peptides shown as western blots probed with the indicated antibodies against CDK9 and CCNT1 are shown. Quantiﬁcation of the increase with the Cit1810 relative to the wild-type R1810 is shown underneath.

Journal: Molecular cell

Article Title: Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

doi: 10.1016/j.molcel.2018.10.016

Figure Lengend Snippet: Figure 6. Cit1810 at RNAP2-CTD Is Recognized by P-TEFb (A) Immunoprecipitation with a-CDK9 (left) and a-PADI2 (right) or non-immune rabbit IgG of T47D extracts followed by western blot with the indicated antibodies. (B) Extracts from T47D cells in the presence (siCtrl) or absence (siPADI2) of PADI2 were immunoprecipitated with a-total-RNAP2 followed by western blot for the indicated antibodies along with relative quantiﬁcations underneath. (C and D) Immunoprecipitation of extracts from (C) T47D and (D) Raji cells expressing only the HA-tagged a-amanitin-resistant WTr or R1810Ar mutant of RNAP2 were precipitated with a-HA antibody and probed with the indicated antibodies. The relative quantiﬁcation is shown underneath each gel. (E) (Top) Schematic representation of the pull-down assays with T47D nuclear extracts and RNAP2-CTD biotinylated peptides (wild-type R1810 or cit1810). (Bottom) Results of the pull-down experiments with wild-type (R1810) or cit1810 biotinylated RNAP2-CTD peptides shown as western blots probed with the indicated antibodies against CDK9 and CCNT1 are shown. Quantiﬁcation of the increase with the Cit1810 relative to the wild-type R1810 is shown underneath.

Techniques: Immunoprecipitation, Western Blot, Expressing, Mutagenesis

Figure 7. Illustration of Structural Model of PADI2 with R1810 at RNAP2-CTD, Related to Figures S6 and S7 For a Figure360 author presentation of Figure 7, see https://doi.org/10.1016/j.molcel.2018.10.016. (A) Close-up of the peptide binding site of PADI2 shown in cartoon and surface representation (green) and R1810 CTD-RNAP2 peptide in ribbon (magenta) and stick representation of side chains (colored by atom type: oxygen, red; nitrogen, blue; C, gray) of amino acid selected for mutation studies: non-conserved (ARG580 and LEU642, shown in orange) and conserved (ASP374 and SER401, shown in blue). (B) Box plot showing the distribution of Rosetta scores for the top 200 structural models per- formed for PADI2 and PADI3 proteins complexed with R1810 peptide. Central horizontal lines in the box mark the median and box edges of the ﬁrst (Q1) and third (Q3) quartiles; top and bottom errors bars mark the Q1 and Q3 + 1.53 inter- quartile range, respectively; outliers are shown as empty circles. (C) In vitro citrullination immunoblot using the C-terminal CTD half containing R1810 as sub- strate, in absence (lane 2) or presence of recom- binant PADI2 as WT (lane 2) and PADI2 mutants of conserved residues (D374K and S401A, lanes 3 and 4) and of PADI2 unique residues (R580E and L642T, lanes 5 and 6). (D) Proposed model of Cit1810 function in tran- scription. PADI2 catalyzed R1810 to the Cit1810 at RNAP2-CTD, facilitate association with P-TEFb (CDK9-CCNT1) complex, and hence overcome RNAP2 accumulation and lead to an increase in transcription and cell proliferation.

Journal: Molecular cell

Article Title: Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

doi: 10.1016/j.molcel.2018.10.016

Figure Lengend Snippet: Figure 7. Illustration of Structural Model of PADI2 with R1810 at RNAP2-CTD, Related to Figures S6 and S7 For a Figure360 author presentation of Figure 7, see https://doi.org/10.1016/j.molcel.2018.10.016. (A) Close-up of the peptide binding site of PADI2 shown in cartoon and surface representation (green) and R1810 CTD-RNAP2 peptide in ribbon (magenta) and stick representation of side chains (colored by atom type: oxygen, red; nitrogen, blue; C, gray) of amino acid selected for mutation studies: non-conserved (ARG580 and LEU642, shown in orange) and conserved (ASP374 and SER401, shown in blue). (B) Box plot showing the distribution of Rosetta scores for the top 200 structural models per- formed for PADI2 and PADI3 proteins complexed with R1810 peptide. Central horizontal lines in the box mark the median and box edges of the ﬁrst (Q1) and third (Q3) quartiles; top and bottom errors bars mark the Q1 and Q3 + 1.53 inter- quartile range, respectively; outliers are shown as empty circles. (C) In vitro citrullination immunoblot using the C-terminal CTD half containing R1810 as sub- strate, in absence (lane 2) or presence of recom- binant PADI2 as WT (lane 2) and PADI2 mutants of conserved residues (D374K and S401A, lanes 3 and 4) and of PADI2 unique residues (R580E and L642T, lanes 5 and 6). (D) Proposed model of Cit1810 function in tran- scription. PADI2 catalyzed R1810 to the Cit1810 at RNAP2-CTD, facilitate association with P-TEFb (CDK9-CCNT1) complex, and hence overcome RNAP2 accumulation and lead to an increase in transcription and cell proliferation.

Techniques: Binding Assay, Mutagenesis, In Vitro, Western Blot

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1) Product Images from "BOD1L mediates chromatin binding and non-canonical function of H3K4 methyltransferase SETD1A"

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