Journal: Nucleic Acids Research

Article Title: DBC1 is a key positive regulator of enhancer epigenomic writers KMT2D and p300

doi: 10.1093/nar/gkac585

Figure Lengend Snippet: DBC1 binds to and enhances the histone-modifying activities of p300 and KMT2D. ( A, B ) In vitro HAT ( A ) and HMT ( B ) assays using recombinant nucleosomes and immunopurified FLAG-DBC1 from SW480 cells. HAT and HMT reactions were analyzed by immunoblot using the indicated antibodies. ( C ) Association of ectopically expressed FLAG-DBC1 with the endogenous p300 and KMT2D complex in SW480 cells. Immunoprecipitation (IP) and immunoblotting were performed using the indicated antibodies. ( D ) Endogenous CoIP between DBC1 and KMT2D or p300 in SW480 WT and D1KO cells. Immunoprecipitation and immunoblotting were performed using the indicated antibodies. ( E ) Schematic representation of the KMT2D fragments used in GST pull-down assays and their binding abilities to GST–DBC1 (see also ). PHD, plant homeotic domain; HMG, high mobility group; FYR, FY-rich domain. ( F ) 293T cells were transfected with FLAG-H3 along with HA-p300-Br-HAT and/or FLAG-DBC1, and cell lysates were analyzed by immunoblot with the indicated antibodies. ( G ) In vitro HMT assays using recombinant nucleosomes with immunopurified HA-KMT2D from 293T cells transfected with HA-KMT2D and FLAG-DBC1 as indicated. HMT reactions were analyzed by immunoblot using the indicated antibodies. ( H ) 293T cells were transfected as indicated, and FLAG-KMT2D immunoprecipitates were analyzed by immunoblot with the indicated antibodies. ( I ) Endogenous CoIP of the indicated core components, DBC1 and p300 with KMT2D in 293T WT and D1KO cells. Immunoprecipitation and immunoblotting were performed using the indicated antibodies. ( J ) In vitro autoacetylation assays of p300-Br-HAT with or without DBC1 as indicated. Acetylation (Ac) levels of p300-Br-HAT were determined by immunoblot using pan-acetyl-lysine (AcK) antibody. ( K ) Endogenous p300 was immunoprecipitated from SW480 WT and D1KO cells with p300 antibodies, and p300 acetylation levels and CoIPed DBC1 and KMT2D were detected by immunoblot using the indicated antibodies.

Article Snippet: HA-KMT2D protein was incubated with recombinant nucleosomes (BPS Bioscience) in the presence or absence of FLAG-DBC1 in HMT buffer (50 mM Tris–HCl pH 8.5, 50 mM NaCl, 1 mM DTT, 5% glycerol) supplemented with 30 μM S -adenosylmethionine (SAM, Sigma-Aldrich) at 30°C for 4 h. For HAT assays, bacterially expressed and purified GST–p300-Br-HAT or GST–CBP-Br-HAT protein was incubated with recombinant nucleosomes with or without GST–DBC1 in HAT buffer [50 mM Tris–HCl pH 8.0, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium butyrate, 5% glycerol] with 30 μM acetyl-CoA (Sigma-Aldrich) at 30°C for 10–180 min. For HAT and HMT assays using immunopurified DBC1, the reaction mixture was incubated for 12 h. For HAT–HMT combined in vitro assays, GST–p300-Br-HAT and HA–KMT2D or FLAG-KMT2D SET proteins were incubated with or without GST–DBC1 in HMT buffer supplemented with 30 μM acetyl-CoA and 30 μM SAM at 30°C for 1–4 h. For the inhibition of HAT or HMT activity, 10 μM SGC-CBP30 (Selleckchem) or OICR-9429 (Tocris), respectively, was added to the reaction.

Techniques: In Vitro, Recombinant, Western Blot, Immunoprecipitation, Binding Assay, Transfection

Journal: Nucleic Acids Research

Article Title: DBC1 is a key positive regulator of enhancer epigenomic writers KMT2D and p300

doi: 10.1093/nar/gkac585

Figure Lengend Snippet: DBC1 facilitates SE-associated histone modifications by coordinating the interplay of p300 and KMT2D. ( A ) Protein levels in SW480 cells depleted of KMT2D or treated with SGC-CBP30 were monitored by immunoblot using the indicated antibodies. ( B ) ChIP-qPCR assays. Sheared chromatin from SW480 cells infected with lentiviruses expressing either a control (NS) or KMT2D shRNA or SW480 WT cells treated with 5 μM SGC-CBP30 for 1 h was immunoprecipitated with the indicated histone modification antibodies. qPCR analyses were performed using primers specific for SEs of the PROX1, ASB4, FSCN1 and NTSR1 genes. ChIP signals were normalized with inputs and shown as fold change relative to control IgG signal, and are means ± SD ( n = 3). * P < 0.05, ** P < 0.01. ( C, D ) In vitro HMT assays using recombinant nucleosomes, immunopurified HA-KMT2D, GST–p300-Br-HAT and GST–DBC1 as indicated ( C ) and in vitro HAT assays using recombinant nucleosomes, immunopurified FLAG-KMT2D SET, GST–p300-Br-HAT and GST–DBC1 as indicated ( D ). The reactions were analyzed by immunoblot using the indicated antibodies. ( E ) 293T cells were transfected with the indicated constructs, and HA-p300 immunoprecipitates were analyzed by immunoblots with the indicated antibodies. ( F ) In vitro translated HA-KMT2D SET, HA-DBC1 or both were incubated with GST–p300-Br-HAT bound to beads as indicated. Bound proteins were analyzed by immunoblot with anti-HA antibody. ( G ) Proximity biotinylation assays. SW480 WT and D1KO cells transfected with 3×FLAG-TurboID-KMT2D SET or 3×FLAG-TurboID were treated with biotin as indicated. Biotinylated endogenous proteins were pulled down using streptavidin beads and analyzed by immunoblot with the indicated antibodies. ( H ) The PROX1 locus is schematically depicted with locations of H3K27ac peaks in SW480 WT and D1KO, sgRNAs (arrow) and ChIP-PCR primers (arrowhead). ( I ) SW480 D1KO cells were infected with lentiviruses encoding the dCas9–DBC1 fusion protein and sgRNAs targeting the PROX1 SE. ChIP-qPCR assays using SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were performed with the indicated antibodies. ** P < 0.01. ( J ) Relative levels of DBC1 mRNA and PROX1 seRNA in SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were determined by qRT–PCR. ** P < 0.001. (K) Protein levels of PROX1 and DBC1 in SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were monitored by immunoblot using the indicated antibodies.

Article Snippet: HA-KMT2D protein was incubated with recombinant nucleosomes (BPS Bioscience) in the presence or absence of FLAG-DBC1 in HMT buffer (50 mM Tris–HCl pH 8.5, 50 mM NaCl, 1 mM DTT, 5% glycerol) supplemented with 30 μM S -adenosylmethionine (SAM, Sigma-Aldrich) at 30°C for 4 h. For HAT assays, bacterially expressed and purified GST–p300-Br-HAT or GST–CBP-Br-HAT protein was incubated with recombinant nucleosomes with or without GST–DBC1 in HAT buffer [50 mM Tris–HCl pH 8.0, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium butyrate, 5% glycerol] with 30 μM acetyl-CoA (Sigma-Aldrich) at 30°C for 10–180 min. For HAT and HMT assays using immunopurified DBC1, the reaction mixture was incubated for 12 h. For HAT–HMT combined in vitro assays, GST–p300-Br-HAT and HA–KMT2D or FLAG-KMT2D SET proteins were incubated with or without GST–DBC1 in HMT buffer supplemented with 30 μM acetyl-CoA and 30 μM SAM at 30°C for 1–4 h. For the inhibition of HAT or HMT activity, 10 μM SGC-CBP30 (Selleckchem) or OICR-9429 (Tocris), respectively, was added to the reaction.

Techniques: Western Blot, ChIP-qPCR, Infection, Expressing, Control, shRNA, Immunoprecipitation, Modification, In Vitro, Recombinant, Transfection, Construct, Incubation, Quantitative RT-PCR