Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: Interactions of BACH2 IDR with TBK1 and DNMT1 (A) Upper: a schematic representation of mouse BACH2 and BACH2-IDR. Lower: the immunoblot analysis of the affinity-purified samples using an anti-FLAG, anti-TBK1 or anti-DNMT1 antibody. (B) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, or His-FLAG-TBK1 in the presence or absence of 5 μM or10 μM heme. GST and GST-BACH2-IDR were revealed with an anti-GST antibody. TBK1 was revealed with an anti-TBK1 antibody. (C) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, or His-FLAG-DNMT1 in the presence or absence of 5 μM or 10 μM heme. GST and GST-BACH2-IDR were revealed with an anti-GST antibody. DNMT1 was revealed with an anti-DNMT1antibody. (D) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, and B cell extracts in the presence or absence of 10 μM heme. GST and GST-BACH2-IDR were revealed with anti-GST an antibody. TBK1 was revealed with an anti-TBK1 antibody. DNMT1 was revealed with an anti-DNMT1 antibody.

Article Snippet: Recombinant human His-FLAG DNMT1 was purchased from BPS Bioscience.

Techniques: Western Blot, Affinity Purification, In Vitro, Pull Down Assay, Recombinant

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: The list of proteins identified as BACH2_IDR interacting proteins using ReCLIP

Article Snippet: Recombinant human His-FLAG DNMT1 was purchased from BPS Bioscience.

Techniques: Sequencing

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet:

Article Snippet: Recombinant human His-FLAG DNMT1 was purchased from BPS Bioscience.

Techniques: Virus, Recombinant, Protease Inhibitor, Transfection, Protein Purification, Reporter Assay, Luciferase, Cell Isolation, Plasmid Preparation, Reverse Transcription, SYBR Green Assay, Sequencing, Negative Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Poly(ADP-ribose) Polymerase 1 (PARP1) Associates with E3 Ubiquitin-Protein Ligase UHRF1 and Modulates UHRF1 Biological Functions *

doi: 10.1074/jbc.M113.527424

Figure Lengend Snippet: Selective PAR-dependent association of UHRF1 with PARP1 in mammalian cells. A, selective coprecipitation of Myc-UHRF1 with GST-PARP1. GST (lane 1), GST-PARP1 (lanes 2 and 3), and GST-PARP2 (lanes 4 and 5) were expressed in COS-1 cells together with Myc-tagged UHRF1 (lanes 1–5). Interacting proteins were analyzed by GST pulldown and Western blotting with subsequent anti-Myc and anti-GST antibodies. Input corresponds to 1/60th the amount of cell extract used for GST pulldown. In lanes 3 and 5, the PARP inhibitor Ku-0058948 was added throughout the experiment. B, coimmunoprecipitation of PARP1 and DNMT1 with UHRF1 in mouse 3T3 cells. Wild-type mouse cell extracts were immunoprecipitated with an anti-UHRF1 antibody (lane 4) or a control antibody (Ctl, lane 3) and analyzed by Western blotting. Input (lanes 1 and 2) corresponds to 1/30th the amount of cell extract used for immunoprecipitation. C, coimmunoprecipitation of UHRF1 and DNMT1 with PARP1 in mouse 3T3 cells. Wild-type mouse cell extracts were immunoprecipitated with an anti-PARP1 antibody (lanes 2 and 4) or a control antibody (lanes 1 and 3) and analyzed by Western blotting. To prevent any coprecipitation of either partner through DNA, EtBr (10 μg/ml) was added throughout the immunoprecipitation when indicated (lanes 3 and 4). Input corresponds to 1/30th the amount of cell extract used for immunoprecipitation. The association of PARP1 with UHRF1 is not mediated by DNA.

Article Snippet: After 20 min at 25 °C, PARylated or non-PARylated (PARP assay performed without NAD + ) UHRF1 was subsequently incubated alone or together with purified recombinant GST-DNMT1 (300 ng, BPS Bioscience) as indicated in a standard ubiquitination reaction mixture (15 μl) containing 100 ng of human recombinant ubiquitin-activating enzyme (Boston Biochem), 300 ng of human recombinant UbcH5b (Boston Biochem), and 10 μg of Ha-tagged human recombinant ubiquitin (Boston Biochem) in 25 m m Tris-HCl, pH 7.6, 5 m m MgCl 2 , 1 m m DTT, 2 m m ATP, and 100 m m NaCl.

Techniques: Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Poly(ADP-ribose) Polymerase 1 (PARP1) Associates with E3 Ubiquitin-Protein Ligase UHRF1 and Modulates UHRF1 Biological Functions *

doi: 10.1074/jbc.M113.527424

Figure Lengend Snippet: The absence of PARP1 affects the association of UHRF1 with DNMT1 but not their targeting to replicating heterochromatin or the methylation of CpG repeats. A, the interaction of DNMT1 with UHRF1 is reduced in PARP1−/− cells. Left, equivalent amounts of total protein cell lysates from PARP1+/+ (lane 2) and PARP1−/− cells (lane 3) were immunoprecipitated using an anti-UHRF1 antibody (lanes 2 and 3) or a control antibody (ctl, lane 1) and analyzed for the coimmunoprecipitation of DNMT1 by Western blotting. Inputs correspond to 1/30th of the amount of total cell extract used for immunoprecipitation. Right, the signal intensities of the coprecipitating DNMT1 relative to DNMT1 expression and UHRF1 immunoprecipitation were measured in three independent experiments using ImageJ. The coIP in PARP1+/+ cells (lane 2) was set to 1. Mean values ± S.D. are indicated. B, the absence of PARP1 does not perturb the accumulation of DNMT1 onto pericentric heterochromatin. Shown are representative images of DNMT1 (a, c, e, and g (green)) immunostaining of the typical ring-shaped pericentric duplication bodies from PARP1+/+ (a, b, e, and f) or PARP1−/− (c, d, g, and h) cells either mock-treated (a–d) or treated with the PARP inhibitor Ku-0058948 (e–h). DNA was counterstained with DAPI (b, d, f, and h (blue)). Scale bars: 7 μm. C, the absence of PARP1 does not perturb the accumulation of UHRF1 onto pericentric heterochromatin. Shown are representative images of UHRF1 (a, c, e, and g (green)) immunostaining of the typical ring-shaped pericentric duplication bodies from PARP1+/+ (a, b, e, and f) or PARP1−/− (c, d, g, and h) cells either mock-treated (a–d) or treated with the PARP inhibitor Ku-0058948 (e–h). DNA was counterstained with DAPI (b, d, f, and h (blue)). Scale bars: 7 μm. D, the interaction of DNMT1 with PCNA is maintained in PARP1−/− cells. Equivalent amounts of total protein cell lysates from PARP1+/+ (lanes 1 and 2) and PARP1−/− cells (lane 3) were immunoprecipitated using and anti-PCNA antibody (lanes 2 and 3) or a control antibody (lane 1) and analyzed for the coimmunoprecipitation of DNMT1 by Western blotting. Input corresponds to 1/25th of the amount of total cell extract used for immunoprecipitation. E, the methylation profile of pericentric repeats is normal in PARP1−/− cells. Total DNA was isolated from PARP1+/+ or PARP1−/− cells either mock-treated or treated with Ku-0058948 for 24 h and then bisulfite-treated. Histograms show the methylation percentage at individual CpG sites as measured by pyrosequencing.

Article Snippet: After 20 min at 25 °C, PARylated or non-PARylated (PARP assay performed without NAD + ) UHRF1 was subsequently incubated alone or together with purified recombinant GST-DNMT1 (300 ng, BPS Bioscience) as indicated in a standard ubiquitination reaction mixture (15 μl) containing 100 ng of human recombinant ubiquitin-activating enzyme (Boston Biochem), 300 ng of human recombinant UbcH5b (Boston Biochem), and 10 μg of Ha-tagged human recombinant ubiquitin (Boston Biochem) in 25 m m Tris-HCl, pH 7.6, 5 m m MgCl 2 , 1 m m DTT, 2 m m ATP, and 100 m m NaCl.

Techniques: Methylation, Immunoprecipitation, Western Blot, Expressing, Immunostaining, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Poly(ADP-ribose) Polymerase 1 (PARP1) Associates with E3 Ubiquitin-Protein Ligase UHRF1 and Modulates UHRF1 Biological Functions *

doi: 10.1074/jbc.M113.527424

Figure Lengend Snippet: PARP1 selectively inhibits the UHRF1-driven ubiquitination of DNMT1 in vivo and in vitro. A, in vivo ubiquitination assays. The absence of PARP1 enhances the UHRF1-mediated ubiquitination of DNMT1 but not the autoubiquitination of UHRF1 in vivo. Left, PARP1+/+ and PARP1−/− cells were transfected with either GFP-DNMT1 (lanes 5–8) or GFP (lanes 1–4) together with HA-ubiquitin and treated with 5 μm MG-132 for 12 h to inhibit proteasomal degradation. GFP immunoprecipitates were blotted successively with an anti-HA antibody to detect ubiquitinated proteins (lanes 3, 4, 7, and 8) and an anti-GFP antibody (lanes 1, 2, 5, and 6) to detect immunopurified proteins. Right, PARP1+/+ (lanes 1 and 3) and PARP1−/− cells (lanes 2 and 4) were transfected with Myc-UHRF1 together with HA-ubiquitin and treated as described above. Myc immunoprecipitates were blotted successively with an anti-HA antibody to detect ubiquitinated UHRF1 (lanes 3 and 4) and an anti-Myc antibody to detect immunopurified UHRF1 (lanes 1 and 2). B, in vitro ubiquitination assays. Left, PARP1-catalyzed poly(ADP-ribosyl)ation of UHRF1 selectively inhibits its ubiquitination activity onto DNMT1. Purified UHRF1 was first preincubated alone (lanes 1, 2, and 5) or together with purified PARP1 (lanes 3, 4, 6, and 7) as indicated in the PARP activity buffer. PARP activity is induced by the addition of NAD+. The proteins were subsequently assayed for UHRF1 ubiquitination activity onto itself (lanes 1–4) or onto GST-DNMT1 (lanes 5–7). a, ubiquitinated proteins (UHRF1Ub and GST-DNMT1Ub) were detected by immunoblotting using an anti-HA antibody, and the PARP activity was verified by immunoblotting using an anti-PAR antibody. b, the purified recombinant proteins mixed in the experiment were detected by Western blotting using anti-GST, anti-PARP1, and anti-UHRF1 antibodies. The lower amount of PARP1 detected in lanes 4 and 7 is explained by its automodification, which limits its detection by the monoclonal anti-PARP1 antibody used. As a control, reactions were performed with GST (lanes 8 and 9). A representative experiment of three is shown. Upper right, a schematic diagram of the experiment is shown. Lower right, the relative -fold expression (histogram) represents the ImageJ-quantified ubiquitinated protein levels of the samples containing PARP1 relative to the samples without PARP1 (lanes 1, 3, and 4 versus 2; lanes 6 and 7 versus 5). The values represent the mean ± S.D. of three independent experiments.

Article Snippet: After 20 min at 25 °C, PARylated or non-PARylated (PARP assay performed without NAD + ) UHRF1 was subsequently incubated alone or together with purified recombinant GST-DNMT1 (300 ng, BPS Bioscience) as indicated in a standard ubiquitination reaction mixture (15 μl) containing 100 ng of human recombinant ubiquitin-activating enzyme (Boston Biochem), 300 ng of human recombinant UbcH5b (Boston Biochem), and 10 μg of Ha-tagged human recombinant ubiquitin (Boston Biochem) in 25 m m Tris-HCl, pH 7.6, 5 m m MgCl 2 , 1 m m DTT, 2 m m ATP, and 100 m m NaCl.

Techniques: In Vivo, In Vitro, Transfection, Activity Assay, Purification, Western Blot, Recombinant, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Poly(ADP-ribose) Polymerase 1 (PARP1) Associates with E3 Ubiquitin-Protein Ligase UHRF1 and Modulates UHRF1 Biological Functions *

doi: 10.1074/jbc.M113.527424

Figure Lengend Snippet: DNMT1 abundance is reduced in PARP1−/− cells. Protein expression from nonsynchronized (lanes 1 and 2) and synchronized (lanes 3–8) PARP1+/+ and PARP1−/− cells was analyzed by SDS-PAGE and Western blotting with the appropriate antibodies. To evaluate protein stability, cells were treated with cycloheximide before lysis. Progression of serum-starved cells released into fresh medium through the cell cycle was monitored by flow cytometry analysis (not shown). The time points of release as determined by FACS are indicated in parentheses.

Article Snippet: After 20 min at 25 °C, PARylated or non-PARylated (PARP assay performed without NAD + ) UHRF1 was subsequently incubated alone or together with purified recombinant GST-DNMT1 (300 ng, BPS Bioscience) as indicated in a standard ubiquitination reaction mixture (15 μl) containing 100 ng of human recombinant ubiquitin-activating enzyme (Boston Biochem), 300 ng of human recombinant UbcH5b (Boston Biochem), and 10 μg of Ha-tagged human recombinant ubiquitin (Boston Biochem) in 25 m m Tris-HCl, pH 7.6, 5 m m MgCl 2 , 1 m m DTT, 2 m m ATP, and 100 m m NaCl.

Techniques: Expressing, SDS Page, Western Blot, Lysis, Flow Cytometry