Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: PLoS ONE

Article Title: MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

doi: 10.1371/journal.pone.0023747

Figure Lengend Snippet: A. Full length MAGE-A3, full length MAGE-C2, or corresponding MAGE homology domain constructs, bind to full length KAP1 and RBCC-KAP1 detected by a Mammalian Two-Hybrid Assay. CHO cells transfected with the indicated plasmids were assayed for SEAP (secreted alkaline phosphatase) activity 48 h after transfection, using a chemiluminescent substrate CSPD. pM3, pM53 and pVP16-T are positive control vectors. pM and pVP16 are negative control vectors. pVP16-KAP1 alone shows minimal light units. MHD = MAGE homology domain. B. MAGE-A3 and MAGE-C2 decrease KAP1 repression of a ZNF382 responsive reporter gene. Luciferase activities were measured in CHO-5xGal4-UAS-TK-Luc-2p cells transfected with (+) control vector (Gal4-DBD), with vector expressing full length (FL) MAGE-A3 or C2, or with their corresponding MAGE homology domains (MHDs). Maximum repression occurs in the presence of ZNF382 alone. Addition of MAGE expression plasmids causes dose dependent release of repression with increased luciferase activity. C. MAGE-C2 mutant L152A, L153A and MAGE-A3 mutant L120A, L121A, which cannot bind KAP1, fail to relieve repression mediated by ZNF382. D. MAGE-A3 and C2 mediated KAP1 repression of a ZNF382 responsive reporter gene is reversible. Addition of ZNF382 expression plasmids initiate dose dependent regaining of repression with decreased luciferase activity.

Article Snippet: For human target validation studies and immunoprecipitation we used: anti–MAGE-C2 (polyclonal, Santa Cruz Biotechnology), anti-human KAP1 (polyclonal, Novus Biologicals, recognizing the NH 2 -terminal region 1–50 amino acids of KAP1), anti-KAP1 monoclonal 20C1 (ChIP grade, Abcam), anti-FLAG monoclonal M2 (Sigma) and anti-human H3me3K9 (monoclonal, E6204, SABiosciences), and anti-human Ki67 (polyclonal, ab15580, Abcam).

Techniques: Construct, Two Hybrid Assay, Transfection, Activity Assay, Positive Control, Negative Control, Luciferase, Control, Plasmid Preparation, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

doi: 10.1371/journal.pone.0023747

Figure Lengend Snippet: MAGE I expression decreases binding of KAP1, H3me3K9, and repression of the ID1 tumor suppressor gene (A, B, D). In contrast, MAGE I expression increases binding of KAP1, H3me3K9, and repression of mRNA and protein levels of the Ki67 gene (E, F, H, I, J, L). Note MAGE I binds to Ki67 gene sites but not ID1 gene sites (C, G, K). “M” denotes Mock transfection control. “A3” and “C2” denote MAGE-A3 and MAGE-C2, respectively.

Article Snippet: For human target validation studies and immunoprecipitation we used: anti–MAGE-C2 (polyclonal, Santa Cruz Biotechnology), anti-human KAP1 (polyclonal, Novus Biologicals, recognizing the NH 2 -terminal region 1–50 amino acids of KAP1), anti-KAP1 monoclonal 20C1 (ChIP grade, Abcam), anti-FLAG monoclonal M2 (Sigma) and anti-human H3me3K9 (monoclonal, E6204, SABiosciences), and anti-human Ki67 (polyclonal, ab15580, Abcam).

Techniques: Expressing, Binding Assay, Transfection, Control

Journal: PLoS ONE

Article Title: MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

doi: 10.1371/journal.pone.0023747

Figure Lengend Snippet: MAGE-C2 L152A L153A expression does not affect binding of KAP1, H3me3K9, and repression of the ID1 tumor suppressor gene (A, B, D). MAGE-C2 L152A L153A enrichment is seen at ID1 sites compared to MAGE-C2 and mock negative control (C). MAGE-C2 L152A L153A expression dose not increases binding of KAP1, H3me3K9, and repression of mRNA level of the Ki67 gene (E, F, H, I, J). Note MAGE-C2 L152A L153A fails to bind to the Ki67 gene sites but ID1 gene sites (C, G, K). “M” denotes Mock transfection control. “C2” denotes MAGE-C2. “MC” represents MAGE-C2 L152A L153A .

Article Snippet: For human target validation studies and immunoprecipitation we used: anti–MAGE-C2 (polyclonal, Santa Cruz Biotechnology), anti-human KAP1 (polyclonal, Novus Biologicals, recognizing the NH 2 -terminal region 1–50 amino acids of KAP1), anti-KAP1 monoclonal 20C1 (ChIP grade, Abcam), anti-FLAG monoclonal M2 (Sigma) and anti-human H3me3K9 (monoclonal, E6204, SABiosciences), and anti-human Ki67 (polyclonal, ab15580, Abcam).

Techniques: Expressing, Binding Assay, Negative Control, Transfection, Control

Journal: PLoS ONE

Article Title: MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

doi: 10.1371/journal.pone.0023747

Figure Lengend Snippet: HEK293T cells were transiently transfected with His tagged wild-type MAGE-C2 (WT), mutant (non-binding) MAGE-C2 L152A L153A , or empty vectors (Mock) and co-transfected with ZNF382, KAP1, and HA tagged ubiquitin. Cells were incubated for 5 hours in the presence of 25 µM MG132 (C2211, SIGMA). Top two panels: ZNF382 (around 55 kDa), MAGE-C2 (around 50 KD) and KAP1 (110 KD) were detected in whole lysates by immunoblotting with anti-KAP1 (upper panel) and anti-FLAG antibodies (lower panel). Third panel: His tagged ubiquitinated proteins were immunoprecipitated with anti-His and detected with anti-HA. High-molecular-weight ubiquitinated species were seen only in blots of cells transfected with both ZNF382 and MAGE-C2. Note that no ubiquitination occurred when wild type MAGE-C2 was replaced with MAGE-C2 L152A L153A which does not bind to KAP1, indicating MAGE–KAP1 binding is required for ZNF382 ubiquitination. Also please note an ubiquitinated ZNF382 degradation product of lesser molecular weight in the presence of wild type MAGE-C2 and KAP1. Lowest panel: immunoprecipitation and immunoblotting confirms expression of ZNF382.

Article Snippet: For human target validation studies and immunoprecipitation we used: anti–MAGE-C2 (polyclonal, Santa Cruz Biotechnology), anti-human KAP1 (polyclonal, Novus Biologicals, recognizing the NH 2 -terminal region 1–50 amino acids of KAP1), anti-KAP1 monoclonal 20C1 (ChIP grade, Abcam), anti-FLAG monoclonal M2 (Sigma) and anti-human H3me3K9 (monoclonal, E6204, SABiosciences), and anti-human Ki67 (polyclonal, ab15580, Abcam).

Techniques: Transfection, Mutagenesis, Binding Assay, Ubiquitin Proteomics, Incubation, Western Blot, Immunoprecipitation, High Molecular Weight, Molecular Weight, Expressing

Journal: PLoS ONE

Article Title: MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

doi: 10.1371/journal.pone.0023747

Figure Lengend Snippet: KAP1 performs diverse functions by serving as a molecular scaffold that binds multiple proteins which allow it to regulate chromatin environments. KAP1 has an N terminal RING-B-box coiled-coil (RBCC) domain that binds to the KRAB domains of KZNFs, which target KAP1 to specific DNA sequences through their zinc finger DNA binding motifs. KAP1 mediates localized compaction of euchromatin to heterochromatin that is necessary for suppression of specific gene transcription, and that is associated with chromatin modifications including histone de-acetylation, histone 3 tri-methylation on K9, and HP1 binding to both DNA and histones. In some cases, MAGE expression enhances KAP1 E3 ubiquitin ligase activity, resulting in KZNF ubiquitination and degradation, thereby de-repressing KZNF mediated gene repression, shown as (-). In other cases, MAGE enhances KZNF and KAP1 localization to specific gene loci, shown as (+) (After A. Ivanov ).

Article Snippet: For human target validation studies and immunoprecipitation we used: anti–MAGE-C2 (polyclonal, Santa Cruz Biotechnology), anti-human KAP1 (polyclonal, Novus Biologicals, recognizing the NH 2 -terminal region 1–50 amino acids of KAP1), anti-KAP1 monoclonal 20C1 (ChIP grade, Abcam), anti-FLAG monoclonal M2 (Sigma) and anti-human H3me3K9 (monoclonal, E6204, SABiosciences), and anti-human Ki67 (polyclonal, ab15580, Abcam).

Techniques: Binding Assay, Methylation, Expressing, Ubiquitin Proteomics, Activity Assay

Journal: PLoS ONE

Article Title: MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

doi: 10.1371/journal.pone.0023747

Figure Lengend Snippet: Real time PCR primers for target gene screening.

Article Snippet: For human target validation studies and immunoprecipitation we used: anti–MAGE-C2 (polyclonal, Santa Cruz Biotechnology), anti-human KAP1 (polyclonal, Novus Biologicals, recognizing the NH 2 -terminal region 1–50 amino acids of KAP1), anti-KAP1 monoclonal 20C1 (ChIP grade, Abcam), anti-FLAG monoclonal M2 (Sigma) and anti-human H3me3K9 (monoclonal, E6204, SABiosciences), and anti-human Ki67 (polyclonal, ab15580, Abcam).

Techniques: Real-time Polymerase Chain Reaction, Binding Assay