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(A) H1 binding to His-tagged H1 chaperones. Bound H1 monitored by CBB staining. (B) In vitro transcription assay with H1 chaperones. Additions as indicated with H1− (upper) and H1+ (lower) chromatin templates. (C) HAT assays with NAP1. Additions as indicated with acetylation monitored by autoradiography or immunoblot. (D) NAP1 binding to H1+ chromatin. After incubation of indicated components, the NAP1-chromatin interaction was monitored by EtBr staining of DNA bound to Ni-NTA agarose-bound His:NAP1. (E) p300-chaperone binding assays. M2-agarose beads without or with bound FLAG:p300 were incubated with His-tagged chaperones that were detected with anti-His antibody. (F) Immobilized template assay for H1 release. After incubation of H1− and H1+ chromatin templates with indicated components (at 300 mM KCl) and capture on streptavidin beads, template-bound (pellet) and unbound (sup) fractions were probed for H1 by immunoblot and for pellet DNA by EtBr. (G) In vitro ChIP assay for H1. H1+ chromatin templates were incubated with the indicated components, captured on streptavidin beads, and subjected to ChIP-qPCR with promoter or array region primers (Table S2). Signals were normalized to input. n=4, mean±SD. *p<0.001. act: Gal4-VP16; <t>CoA:</t> <t>acetyl-CoA.</t> p300DY: HAT activity defective p300 (Fig. S2D-F). (H) In vitro ChIP assay for H1. Analysis as in (G) but with mutations (Fig. S2G) in all four core histones. n=3, mean±SD. (I) In vitro ChIP for H1. Analysis as in (G) but with mutations (Fig. S2G) in individual histones (indicated in red). Reactions contained Gal4-VP16, p300, and acetyl-CoA without (blue bars) or with (red bars) NAP1. n=5, mean±SD. Fractional reduction of H1 retention with addition of NAP1 (NAP1+/−) is shown for each mutant histone chromatin at the top of the graph. (J) In vitro ChIP assay for H1, H2B and H3 release. Analysis as in (G). n=3, mean±SD, *p<0.03 (K) Immobilized template assay for H1 and H2B release. Analysis as in (F). (L) In vitro ChIP assay for H1, H2B and H3 release. Analysis as in (G) but with ACF and ATP addition. n=4, mean±SD, *p<0.005, **p<0.001. See also Figs. S2 and S3.
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Journal: eLife

Article Title: Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

doi: 10.7554/eLife.67952

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , [ 3 H]-acetyl-CoA , American Radiolabeled Chemicals , ART0213B , Radioactive co-factor for p300.

Techniques: Transfection, Affinity Purification, Magnetic Beads, Recombinant, Protease Inhibitor, Cell Culture, Western Blot, Imaging, Solvent, Purification, Stable Transfection, Plasmid Preparation, Sequencing, Generated

(A) H1 binding to His-tagged H1 chaperones. Bound H1 monitored by CBB staining. (B) In vitro transcription assay with H1 chaperones. Additions as indicated with H1− (upper) and H1+ (lower) chromatin templates. (C) HAT assays with NAP1. Additions as indicated with acetylation monitored by autoradiography or immunoblot. (D) NAP1 binding to H1+ chromatin. After incubation of indicated components, the NAP1-chromatin interaction was monitored by EtBr staining of DNA bound to Ni-NTA agarose-bound His:NAP1. (E) p300-chaperone binding assays. M2-agarose beads without or with bound FLAG:p300 were incubated with His-tagged chaperones that were detected with anti-His antibody. (F) Immobilized template assay for H1 release. After incubation of H1− and H1+ chromatin templates with indicated components (at 300 mM KCl) and capture on streptavidin beads, template-bound (pellet) and unbound (sup) fractions were probed for H1 by immunoblot and for pellet DNA by EtBr. (G) In vitro ChIP assay for H1. H1+ chromatin templates were incubated with the indicated components, captured on streptavidin beads, and subjected to ChIP-qPCR with promoter or array region primers (Table S2). Signals were normalized to input. n=4, mean±SD. *p<0.001. act: Gal4-VP16; CoA: acetyl-CoA. p300DY: HAT activity defective p300 (Fig. S2D-F). (H) In vitro ChIP assay for H1. Analysis as in (G) but with mutations (Fig. S2G) in all four core histones. n=3, mean±SD. (I) In vitro ChIP for H1. Analysis as in (G) but with mutations (Fig. S2G) in individual histones (indicated in red). Reactions contained Gal4-VP16, p300, and acetyl-CoA without (blue bars) or with (red bars) NAP1. n=5, mean±SD. Fractional reduction of H1 retention with addition of NAP1 (NAP1+/−) is shown for each mutant histone chromatin at the top of the graph. (J) In vitro ChIP assay for H1, H2B and H3 release. Analysis as in (G). n=3, mean±SD, *p<0.03 (K) Immobilized template assay for H1 and H2B release. Analysis as in (F). (L) In vitro ChIP assay for H1, H2B and H3 release. Analysis as in (G) but with ACF and ATP addition. n=4, mean±SD, *p<0.005, **p<0.001. See also Figs. S2 and S3.

Journal: Molecular cell

Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway

doi: 10.1016/j.molcel.2019.02.016

Figure Lengend Snippet: (A) H1 binding to His-tagged H1 chaperones. Bound H1 monitored by CBB staining. (B) In vitro transcription assay with H1 chaperones. Additions as indicated with H1− (upper) and H1+ (lower) chromatin templates. (C) HAT assays with NAP1. Additions as indicated with acetylation monitored by autoradiography or immunoblot. (D) NAP1 binding to H1+ chromatin. After incubation of indicated components, the NAP1-chromatin interaction was monitored by EtBr staining of DNA bound to Ni-NTA agarose-bound His:NAP1. (E) p300-chaperone binding assays. M2-agarose beads without or with bound FLAG:p300 were incubated with His-tagged chaperones that were detected with anti-His antibody. (F) Immobilized template assay for H1 release. After incubation of H1− and H1+ chromatin templates with indicated components (at 300 mM KCl) and capture on streptavidin beads, template-bound (pellet) and unbound (sup) fractions were probed for H1 by immunoblot and for pellet DNA by EtBr. (G) In vitro ChIP assay for H1. H1+ chromatin templates were incubated with the indicated components, captured on streptavidin beads, and subjected to ChIP-qPCR with promoter or array region primers (Table S2). Signals were normalized to input. n=4, mean±SD. *p<0.001. act: Gal4-VP16; CoA: acetyl-CoA. p300DY: HAT activity defective p300 (Fig. S2D-F). (H) In vitro ChIP assay for H1. Analysis as in (G) but with mutations (Fig. S2G) in all four core histones. n=3, mean±SD. (I) In vitro ChIP for H1. Analysis as in (G) but with mutations (Fig. S2G) in individual histones (indicated in red). Reactions contained Gal4-VP16, p300, and acetyl-CoA without (blue bars) or with (red bars) NAP1. n=5, mean±SD. Fractional reduction of H1 retention with addition of NAP1 (NAP1+/−) is shown for each mutant histone chromatin at the top of the graph. (J) In vitro ChIP assay for H1, H2B and H3 release. Analysis as in (G). n=3, mean±SD, *p<0.03 (K) Immobilized template assay for H1 and H2B release. Analysis as in (F). (L) In vitro ChIP assay for H1, H2B and H3 release. Analysis as in (G) but with ACF and ATP addition. n=4, mean±SD, *p<0.005, **p<0.001. See also Figs. S2 and S3.

Article Snippet: Acetylated histones were detected either by autoradiography or by immunoblotting with antibodies to specific acetylated histone marks (see ). table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-FLAG M2 antibody Sigma F1804; RRID: AB_262044 anti-His tag antibody Qiagen 34660; RRID: AB_2619735 anti-HA tag antibody Abcam ab9110; RRID: AB_307019 anti-H1 antibody Millipore 05-457; RRID:AB_310843 anti-H1.2 antibody Abcam ab4086; RRID:AB_2117983 anti-H2B antibody Santa Cruz Biotechnology sc-8650; RRID:AB_2118153 anti-Histone H3 antibody Abcam ab1791; RRID: AB_302613 anti-H3K9ac antibody Abcam ab4441; RRID:AB_2118292 anti-H3K14ac antibody Millipore 06-911; RRID:AB_310294 anti-H3K18ac antibody Abcam ab1191; RRID:AB_298692 anti-H4ac antibody Santa Cruz Biotechnology sc-377521 anti-H4K5ac antibody Abcam ab51997; RRID:AB_2264109 anti-H4K8ac antibody Millipore 07-328; RRID:AB_11213282 anti-H4K16ac antibody Upstate 07-329; RRID:AB_310525 anti-p65 antibody Abcam ab7970; RRID:AB_306184 anti-p300 antibody Santa Cruz Biotechnology sc-585; RRID: AB_2231120 anti-RNAP1I Santa Cruz Biotechnology sc-899; RRID:AB_632359 anti-CTCF Millipore 17-10044;RRID:AB_10732951 Bacterial and Virus Strains Biological Samples Chemicals, Peptides, and Recombinant Proteins TPA (12-O-Tetradecanoylphorbol 13-acetate) Sigma P8139 Acetyl-CoA Sigma A2056 3x FLAG peptide Sigma F4799 Glutathione Sepharose 4B GE Healthcare 17075601 Bio-gel HT hydroxyapatite Bio-Rad 130-0520 Anti-FLAG M2 affinity gel Sigma A2220 TALON-beads Clontech 635501 Dynabeads M-280 streptavidin Invitrogen 11205D Dynabeads Protein A Invitrogen 10001D SP sepharose GE healthcare 17108703 HiTrap SP HP GE healthcare 17115201 HiTrap Q HP GE healthcare 17115401 ATP Sigma A2383 ATP (for transcription) GE healthcare 27205601 UTP (for transcription) GE healthcare 27208601 CTP (for transcription) GE healthcare 27206601 3’-O-methyl GTP (for transcription) TriLink N1058 32 P-CTP (for transcription) Perkin Elmer Blu008H001MC 3 H-acetyl-CoA Perkin Elmer NET290050UC MNase TAKARA Bio 2910A Critical Commercial Assays ChIP assay kit Millipore 17-295 QuantiTect SYBR Green PCR kit Qiagen 28106 RNeasy Mini Kit Qiagen 74104 qScript cDNA SuperMix Quant BioSciences 95048 Deposited Data ChIP-seq RNA-seq datasets and files This study {"type":"entrez-geo","attrs":{"text":"GSE126083","term_id":"126083"}} GSE126083 Raw Data Mendeley http://dx.doi.org/10.17632/9r3grs3xfn.1 Experimental Models: Cell Lines Sf9 cells ATCC CRL-1711 High Five cells Thermo Fisher Sc.

Techniques: Binding Assay, Staining, In Vitro, Transcription Assay, Autoradiography, Western Blot, Incubation, Activity Assay, Mutagenesis

KEY RESOURCES TABLE

Journal: Molecular cell

Article Title: Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway

doi: 10.1016/j.molcel.2019.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Acetylated histones were detected either by autoradiography or by immunoblotting with antibodies to specific acetylated histone marks (see ). table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-FLAG M2 antibody Sigma F1804; RRID: AB_262044 anti-His tag antibody Qiagen 34660; RRID: AB_2619735 anti-HA tag antibody Abcam ab9110; RRID: AB_307019 anti-H1 antibody Millipore 05-457; RRID:AB_310843 anti-H1.2 antibody Abcam ab4086; RRID:AB_2117983 anti-H2B antibody Santa Cruz Biotechnology sc-8650; RRID:AB_2118153 anti-Histone H3 antibody Abcam ab1791; RRID: AB_302613 anti-H3K9ac antibody Abcam ab4441; RRID:AB_2118292 anti-H3K14ac antibody Millipore 06-911; RRID:AB_310294 anti-H3K18ac antibody Abcam ab1191; RRID:AB_298692 anti-H4ac antibody Santa Cruz Biotechnology sc-377521 anti-H4K5ac antibody Abcam ab51997; RRID:AB_2264109 anti-H4K8ac antibody Millipore 07-328; RRID:AB_11213282 anti-H4K16ac antibody Upstate 07-329; RRID:AB_310525 anti-p65 antibody Abcam ab7970; RRID:AB_306184 anti-p300 antibody Santa Cruz Biotechnology sc-585; RRID: AB_2231120 anti-RNAP1I Santa Cruz Biotechnology sc-899; RRID:AB_632359 anti-CTCF Millipore 17-10044;RRID:AB_10732951 Bacterial and Virus Strains Biological Samples Chemicals, Peptides, and Recombinant Proteins TPA (12-O-Tetradecanoylphorbol 13-acetate) Sigma P8139 Acetyl-CoA Sigma A2056 3x FLAG peptide Sigma F4799 Glutathione Sepharose 4B GE Healthcare 17075601 Bio-gel HT hydroxyapatite Bio-Rad 130-0520 Anti-FLAG M2 affinity gel Sigma A2220 TALON-beads Clontech 635501 Dynabeads M-280 streptavidin Invitrogen 11205D Dynabeads Protein A Invitrogen 10001D SP sepharose GE healthcare 17108703 HiTrap SP HP GE healthcare 17115201 HiTrap Q HP GE healthcare 17115401 ATP Sigma A2383 ATP (for transcription) GE healthcare 27205601 UTP (for transcription) GE healthcare 27208601 CTP (for transcription) GE healthcare 27206601 3’-O-methyl GTP (for transcription) TriLink N1058 32 P-CTP (for transcription) Perkin Elmer Blu008H001MC 3 H-acetyl-CoA Perkin Elmer NET290050UC MNase TAKARA Bio 2910A Critical Commercial Assays ChIP assay kit Millipore 17-295 QuantiTect SYBR Green PCR kit Qiagen 28106 RNeasy Mini Kit Qiagen 74104 qScript cDNA SuperMix Quant BioSciences 95048 Deposited Data ChIP-seq RNA-seq datasets and files This study {"type":"entrez-geo","attrs":{"text":"GSE126083","term_id":"126083"}} GSE126083 Raw Data Mendeley http://dx.doi.org/10.17632/9r3grs3xfn.1 Experimental Models: Cell Lines Sf9 cells ATCC CRL-1711 High Five cells Thermo Fisher Sc.

Techniques: Virus, Recombinant, SYBR Green Assay, Software