Journal: The Journal of Biological Chemistry

Article Title: De Novo DNA Methyltransferase DNMT3b Interacts with NEDD8-modified Proteins

doi: 10.1074/jbc.M110.155721

Figure Lengend Snippet: DNMT3b interacts with cullins. A, DNMT3b interacts with endogenous CUL4A and CUL5. Extracts of HA-DNMT3b-transfected HEK 293T cells were immunoprecipitated (IP) with anti-HA-conjugated beads and subjected to SDS-PAGE and Western blot analysis (WB). Specific antibodies against NEDDylated proteins (top panel), CUL5 (upper middle panel), and CUL4A (lower middle panel) were used to detect endogenous proteins in the inputs (lanes 3 and 4) and in the immunoprecipitates (lanes 1 and 2). The presence of HA-DNMT3b in the immunoprecipitates was detected with anti-DNMT3b antibody (bottom panel). B, DNMT3b interacts with CUL1, CUL2, CUL3, and CUL5. FLAG-DNMT3b and HA-tagged CUL1, CUL2, CUL3, and CUL5 were co-transfected into HEK 293T cells, and cell extracts were immunoprecipitated with anti-FLAG-conjugated beads. Cullins were detected in the immunoprecipitates (panel) and the input (middle) using anti-HA antibody. Expression of FLAG-DNMT3b was detected with anti-FLAG antibody (bottom).

Article Snippet: HA-CUL1, -2, -3, and -5 ( 31 ), Myc-CUL4A ( 32 ), CUL4A-DN (Addgene plasmid 15821) ( 33 ), CUL4A Δ N100 (Addgene plasmid 19953) ( 34 ), mouse DNMT3b deletion mutants ( 35 ), 5xGAL4TKCAT ( 36 ), and pREP8-RTA ( 37 ) were described previously.

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: De Novo DNA Methyltransferase DNMT3b Interacts with NEDD8-modified Proteins

doi: 10.1074/jbc.M110.155721

Figure Lengend Snippet: DNMT3b associates preferentially with NEDDylated CUL4A and inactive CUL4A attenuates DNA methylation. A, DNMT3b interacts preferentially with NEDDylated CUL4A. HEK 293T cells were transfected with HA-DNMT3b, and 2 days after transfection, cells were collected into two tubes; in one tube protein extract was prepared and immunoprecipitated (IP) with NEM, and the second immunoprecipitation was done without NEM. Anti-CUL4A and Anti-NEDD8 antibodies were used to detect endogenous CUL4A and NEDDylated proteins that were co-immunoprecipitated with DNMT3b, respectively. WB, Western blot. B, HEK 293T cells were transfected with FLAG-DNMT3b and Myc-CUL4A, and immunoprecipitations were performed with anti-FLAG-conjugated beads. CUL4A was detected in the immunoprecipitates (top) and the input (middle) using anti-Myc antibody. The presence of DNMT3b in the immunoprecipitation was detected with anti-FLAG antibody (bottom). C, as in B but with transfected Myc-CUL4A Δ N100 (deletion of aa 1–100). D, HEK 293T cells were transfected with HA-DNMT3b and CUL4A or CUL4A-DN (aa 1–440, deletion of the C terminus), and immunoprecipitations were performed with anti-HA-conjugated beads. CUL4A and CUL4A-DN were detected in the immunoprecipitates (top) and the input (bottom) using anti-CUL4A antibody. E, an inactive CUL4A mutant attenuates DNMT3b-dependent DNA methylation. pREP8-RTA target DNA was transfected into HEK 293T cells with or without CUL4A Δ N100 or DNMT3b. Methylation of the plasmid DNA was analyzed as in Fig. 5B.

Article Snippet: HA-CUL1, -2, -3, and -5 ( 31 ), Myc-CUL4A ( 32 ), CUL4A-DN (Addgene plasmid 15821) ( 33 ), CUL4A Δ N100 (Addgene plasmid 19953) ( 34 ), mouse DNMT3b deletion mutants ( 35 ), 5xGAL4TKCAT ( 36 ), and pREP8-RTA ( 37 ) were described previously.

Techniques: DNA Methylation Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Methylation, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: De Novo DNA Methyltransferase DNMT3b Interacts with NEDD8-modified Proteins

doi: 10.1074/jbc.M110.155721

Figure Lengend Snippet: DNMT3b recruits CUL4A and NEDD8 to chromatin of a repressed promoter. DNMT3b recruits CUL4A and NEDD8 to chromatin. ChIP assays were performed on HEK 293T cells that were transfected with DNMT3b fused to GAL4 DNA binding domain (GAL4-DNMT3b) together with a plasmid that contains 5xGAL4 consensus binding sites (5xGAL4TKCAT). Endogenous NEDD8 (A) and CUL4A (B) were immunoprecipitated, and their association with the plasmid DNA as detected by real-time PCR relative to serum IgG (C) is presented. D, ChIP assay performed on HCT-116 or HCT 3b KO cell lines. The immunoprecipitated DNA was subjected to real-time PCR using primers for the endogenous PCDH10 promoter. The results are presented as -fold enrichment relative to control IgG. Error bars, S.D.

Article Snippet: HA-CUL1, -2, -3, and -5 ( 31 ), Myc-CUL4A ( 32 ), CUL4A-DN (Addgene plasmid 15821) ( 33 ), CUL4A Δ N100 (Addgene plasmid 19953) ( 34 ), mouse DNMT3b deletion mutants ( 35 ), 5xGAL4TKCAT ( 36 ), and pREP8-RTA ( 37 ) were described previously.

Techniques: Transfection, Binding Assay, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: The actin-MRTF-SRF transcriptional circuit controls tubulin acetylation via α-TAT1 gene expression

doi: 10.1083/jcb.201702157

Figure Lengend Snippet: Expression of active MRTF-A restores tubulin acetylation and α- TAT mRNA levels in INF2 KD RPE-1 cells. (A and B) Control and INF2 KD RPE-1 cells were treated with 0.5 µM cytochalasin D or not for 48 h. Cells were then stained for Ac-tub and endogenous MRTF-A (A). (B) The percentage of nuclear MRTF-A and that of cells with acetylated MT arrays were quantified ( n = 300–450 cells per experimental point). (C–F) INF2 KD cells expressing the active MRTF-A ΔN100 mutant or not were stained for MRTF-A and Ac-tub (C). (D) The percentage of transfected cells with acetylated MT arrays was quantified and compared with that of untransfected cells. (E) The activity of the MRTF-SRF complex was measured in INF2 KD cells expressing MRTF-A ΔN100 and expressed relative to that in untransfected INF2 KD cells. (F) The level of α-TAT1 mRNA was quantified in MRTF-A ΔN100-transfected INF2 KD cells and expressed relative to those in untransfected cells. Nuclei were visualized with TO-PRO in A and C. Bars, 10 µm. Data in B, D, and E represent the mean and SEM of four independent experiments; those in F represent the mean and SEM of three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Nachury (Stanford University, Stanford, CA); GFP-Smoothened (plasmid 25395; Addgene; ), and the MRTF-A ΔN100 mutant (plasmid 19848; Addgene; ) were gifts from P. Beachy (Johns Hopkins University, Baltimore, MD) and Ron Prywes (Columbia University, New York, NY), respectively; mCherry-ER-3 (plasmid 55041; Addgene) was a gift from M. Davidson (The Florida State University, Tallahassee, FL).

Techniques: Expressing, Staining, Mutagenesis, Transfection, Activity Assay

Journal: The Journal of Cell Biology

Article Title: The actin-MRTF-SRF transcriptional circuit controls tubulin acetylation via α-TAT1 gene expression

doi: 10.1083/jcb.201702157

Figure Lengend Snippet: The α- TAT1 gene contains functional CArG boxes. (A) Schematic representation of the α-TAT1 gene with an indication of the putative CArG boxes (CArG elements 1–4) present in the region examined and the fragments (Prom1, Prom2, and Int1) used in our analysis. (B) The indicated constructs were transfected in control and INF2 KD RPE-1 cells. The luciferase activity was measured and was expressed relative to that of cells transfected with minP. (C) The four CArG boxes present in the constructs Prom1, Prom2, and Int1 were placed in tandem upstream from a minimal promoter (α-TAT1 CArGs1–4). A similar construct was made in which the four central nucleotides of each box were mutated in such a way that all of them were C or G (α-TAT1 CArGs1–4m). Control cells were transfected with the indicated constructs in the presence or absence of the MRTF-A ΔN100 construct, and the luciferase activity of the cells was assayed. The histogram represents the luciferase activity obtained in each case relative to that of cells transfected with minP. Data in B and C represent the mean and SEM of seven and four independent experiments, respectively; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Nachury (Stanford University, Stanford, CA); GFP-Smoothened (plasmid 25395; Addgene; ), and the MRTF-A ΔN100 mutant (plasmid 19848; Addgene; ) were gifts from P. Beachy (Johns Hopkins University, Baltimore, MD) and Ron Prywes (Columbia University, New York, NY), respectively; mCherry-ER-3 (plasmid 55041; Addgene) was a gift from M. Davidson (The Florida State University, Tallahassee, FL).

Techniques: Functional Assay, Construct, Transfection, Luciferase, Activity Assay