Journal: Genes & Development

Article Title: NFATC2IP is a mediator of SUMO-dependent genome integrity

doi: 10.1101/gad.350914.123

Figure Lengend Snippet: NFATC2IP functions in interphase. ( A ) Schematic of the epitope-tagged NFATC2IP proteins fused to a nuclear export signal (NES) or a mutant form of the NES (NESm). (SLD1) SUMO-like domain 1, (SLD2) SUMO-like domain 2. ( B , C ) Determination of TAK-981 LD 50 values from dose response clonogenic survival assays in parental RPE1-hTERT Cas9 TP53 −/− (WT) or isogenic NFATC2IP -KO cells that either were left untransduced or were transduced with a lentivirus encoding the indicated protein. ( B ) Quantitation of TAK-981 LD 50 in cells expressing NES-fused NFATC2IP protein. Data are shown as the mean ± SEM. ( n = 3). (FL) Full-length, (NES) NES-NFATC2IP. Statistical analyses were performed with two-tailed unpaired t -tests. (****) P < 0.0001, (***) P < 0.001, (*) P < 0.05. ( C ) Quantitation of TAK-981 LD 50 in cells expressing NFATC2IP protein fused with the mutant form of NES. Data are shown as the mean ± SEM. ( n = 3). (FL) Full-length NFATC2IP, (NESm) NESm-NFATC2IP. Statistical analyses were performed with two-tailed unpaired t -tests. (****) P < 0.0001, (***) P < 0.001, (ns) P ≥ 0.05. ( D ) Quantitation of MN formation after treatment with 50 nM TAK-981 for 48 h. RPE1-hTERT Cas9 TP53 −/− NFATC2IP -KO cells were transduced with lentivirus encoding the indicated protein. Data are shown as the number of MNs per cell, and a minimum of 495 nuclei was counted for each condition. Bars represent the mean ± SD. ( n = 3). (FL) Full-length NFATC2IP, (NES) NES-NFATC2IP, (NESm) NESm-NFATC2IP. Data were analyzed by performing multiple unpaired t -tests with Bonferroni–Dunn correction. (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (ns) P ≥ 0.05. ( E ) Schematic of the GFP-NFATC2IP protein fused to an S-phase-restricted (SPR) degron consisting of protein fragments of geminin (GMNN) and stem–loop binding protein (SLBP). (SLD1) SUMO-like domain 1, (SLD2) SUMO-like domain 2. ( F ) Quantitation of GFP-positive nuclei displaying EdU (5-ethynyl-2′-deoxyuridine) incorporation in RPE1-hTERT Cas9 TP53 −/− NFATC2IP -KO cells that were transduced with lentivirus encoding the indicated protein. A minimum of 104 GFP-positive nuclei was analyzed per replicate to assess EdU incorporation. Bars represent the mean ± SD. ( n = 4). (FL) Full-length NFATC2IP, (SPR) NFATC2IP-SPR. Two-way ANOVA testing was performed for statistical comparisons. For comparison of EdU + GFP + nuclei, (****) P < 0.0001. For comparison of EdU − GFP + nuclei, (####) P < 0.0001. ( G ) Determination of TAK-981 LD 50 values from dose response clonogenic survival assays in parental RPE1-hTERT Cas9 TP53 −/− (WT) or isogenic NFATC2IP -KO cells that either were left untransduced or were transduced with a lentivirus encoding the indicated protein. Data are shown as the mean ± SEM. ( n = 3). (FL) Full-length NFATC2IP, (SPR) NFATC2IP-SPR. For statistical analyses, two-tailed unpaired t -tests were performed. (***) P < 0.001, (**) P < 0.01, (ns) P ≥ 0.05. ( H ) Quantitation of MN formation after treatment with 50 nM TAK-981 for 48 h. RPE1-hTERT Cas9 TP53 −/− NFATC2IP -KO cells were transduced with a lentivirus encoding the indicated protein. A minimum of 495 nuclei was counted per replicate. Bars represent the mean ± SD. ( n = 3). (FL) Full-length NFATC2IP, (SPR) NFATC2IP-SPR. Data were analyzed using multiple unpaired t -tests with Bonferroni–Dunn correction. (***) P < 0.001, (**) P < 0.01, (ns) P ≥ 0.05. ( I ) Quantitation of proximity ligation signal with biotin-conjugated EdU (SIRF) in RPE1-hTERT Cas9 TP53 −/− NFATC2IP -KO cells transduced with a lentivirus encoding GFP-NFATC2IP. EdU was pulsed for 8 min at a concentration of 125 µM, and DMSO was added in the no-EdU control. For the thymidine chase condition, a portion of the EdU-pulsed cells was incubated in thymidine-containing media (100 µM) for 6 h. For the conditions that included an EdU pulse, data are shown as mean fluorescence intensity (MFI) of the proximity ligation signal generated between biotin-conjugated EdU and the indicated protein in nuclei displaying Alexa fluor 647/EdU costaining. For the negative control, all DAPI-stained nuclei were analyzed to calculate the proximity ligation MFI values. Bars represent the mean ± SD of MFI data combined from repeated independent experiments (for negative control and EdU pulse-only conditions, n = 3; for EdU pulse + thymidine chase condition, n = 2). Numbers of combined data points in each condition are indicated. (FL) Full-length NFATC2IP, (a.u.) arbitrary units.

Article Snippet: SUMO-conjugated polypeptides in subcellular fractionation samples were purified with biotin SUMO capture reagent (Biotin S-Cap, LifeSensors, Inc. SM-101) following the manufacturer's protocol.

Techniques: Mutagenesis, Transduction, Quantitation Assay, Expressing, Two Tailed Test, Binding Assay, Comparison, Ligation, Concentration Assay, Control, Incubation, Fluorescence, Generated, Negative Control, Staining

Journal: Genes & Development

Article Title: NFATC2IP is a mediator of SUMO-dependent genome integrity

doi: 10.1101/gad.350914.123

Figure Lengend Snippet: NFATC2IP binds the SMC5/6 complex through its SUMO-like domains. ( A ) Schematic of pairwise matrix screens by AlphaFold-Multimer to predict protein–protein interactions among NFATC2IP and the SMC5/6 complex subunits. Predicted interactions where at least three out of five models met the cutoff scores of pDockQ < 0.23 and interface predicted aligned error (PAE) < 15 Å are shown. Large proteins (SMC5, SMC6, SLF1, and SLF2) were divided into fragments according to either experimentally determined or AlphaFold-Multimer-predicted structural domain boundaries. Fragments of the same protein are indicated with dashed lines connecting the nodes. For SMC5, these are SMC5_1 (“head” region, residues 1–204 and 951–1101), SMC5_2 (“hinge” region, resides 461–647), and SMC5_3 (“coiled-coil” region, resides 205–460 and 648–950). For SMC6, these are SMC6_1 (“head” region, resides 1–201 and 952–1091), SMC6_2 (“hinge” region, residues 476–662), and SMC6_3 (“coiled-coil” region, residues 202–475 and 663–951 [derived from the human SMC5/6 complex structure]) . For SLF1, these are SLF1_1 (residues 1–364) and SLF1_2 (residues 365–1058). For SLF2, these are SLF2_1 (residues 1–600) and SLF2_2 (residues 601–1173). ( B ) PAE plots of the interaction between the coiled-coil region of SMC5 (SMC5_3 fragment of the pairwise modeling) and NFATC2IP, ranked by predicted template model (pTM) scores. ( C ) Subcellular fractions of 293T cells transiently expressing the indicated 3xFLAG-tagged NFATC2IP constructs were subjected to immunoprecipitation with anti-FLAG antibodies. Input samples and immunoprecipitation products were immunoblotted with the indicated antibodies. α-Tubulin, lamin B1, and histone H3 were included as controls for cytoplasmic, nucleoplasmic, and chromatin subcellular fractionations, respectively. (FL) Full-length. ( D , top panel) Representative model of the AlphaFold-Multimer predictions of protein–protein interactions among the NFATC2IP SLDs (yellow), NSMCE2 (sea green), UBC9 (purple), and a portion of the SMC5cc region (cyan). ( Bottom panel) The PAE plots of the prediction models associated with the top panel. ( E ) Overview of the representative AlphaFold-Multimer prediction model (colors are as in D ) overlaid with the structure of the Smc5/Nse2 complex with the Ubc9∼SUMO mimetic (PDB: 7P47; translucent). Proteins in the crystal structure are indicated alongside the AlphaFold-Multimer model at corresponding positions. ( F ) Multiple sequence alignment of amino acid sequences of the human NSMCE2 protein ( Homo sapiens ) and its orthologs in S. cerevisiae , zebrafish ( Danio rerio ), and mice ( Mus musculus ) with the associated UniProt entry codes and amino acid positions using the Clustal Omega sequence alignment tool. Amino acid residues of the SUMO-interacting motif (SIM2) on the C-terminal end of Nse2 for positioning backside SUMO in yeast are highlighted with a blue box and red text. ( G ) Functional model of the NFATC2IP–SMC5/6–NSMCE2–UBC9 complex. The arrow indicates the potential positive regulation of NSMCE2-dependent SUMOylation by NFATC2IP.

Article Snippet: SUMO-conjugated polypeptides in subcellular fractionation samples were purified with biotin SUMO capture reagent (Biotin S-Cap, LifeSensors, Inc. SM-101) following the manufacturer's protocol.

Techniques: Protein-Protein interactions, Derivative Assay, Expressing, Construct, Immunoprecipitation, Sequencing, Functional Assay

Journal: Genes & Development

Article Title: NFATC2IP is a mediator of SUMO-dependent genome integrity

doi: 10.1101/gad.350914.123

Figure Lengend Snippet: NFATC2IP promotes SUMOylation in chromatin. ( A , B ) Immunoblot analysis of whole-cell extracts of parental RPE1-hTERT Cas9 TP53 −/− (WT) or isogenic NFATC2IP -KO cells that were transiently transfected with plasmids for overexpression of His 6 -SUMO1 ( A ) or His 6 -SUMO2 ( B ), followed by purification of His-tagged peptides using Ni-NTA agarose beads under denaturing conditions. The immunoblots were probed with antibodies to the indicated proteins. RanGAP1 was used as a SUMOylation control. GAPDH was used as a loading control. ( C ) Immunoblot analysis of chromatin subfractions of extracts derived from parental RPE1-hTERT Cas9 TP53 −/− (WT) or isogenic NFATC2IP -KO cells. SUMO-conjugated proteins were isolated by binding to the biotinylated S-Cap peptide, followed by affinity pull-down with streptavidin-conjugated magnetic beads. (Input) Input control fraction, (FT) unbound flow-through after binding of biotin S-Cap to streptavidin beads, (PD) proteins from S-Cap pull-down eluted from streptavidin beads, (WCE) whole-cell extract. Immunoblots were probed using antibodies to the indicated proteins. ɑ-Tubulin, lamin B1, and histone H3 were included as controls for cytoplasmic, nucleoplasmic, and chromatin subcellular fractions, respectively. ( D ) Immunoblot analyses of chromatin subfractions of extracts derived from parental RPE1-hTERT Cas9 TP53 −/− (WT) or isogenic NFATC2IP -KO cells that either were left untransduced or were transduced with a lentivirus encoding the indicated protein. The subfractionated chromatin extracts were subjected to isolation of SUMO-conjugated proteins via binding to the biotinylated S-Cap peptide, followed by affinity pull-down with streptavidin-conjugated magnetic beads. (Input) Input control fraction before binding to streptavidin-conjugated magnetic beads, (PD) proteins from S-Cap pull-down eluted from streptavidin beads, (WCE) whole-cell extract. Immunoblots were probed using antibodies to the indicated proteins. ɑ-Tubulin, lamin B1, and histone H3 were included as controls for cytoplasmic, nucleoplasmic, and chromatin subcellular fractions, respectively.

Article Snippet: SUMO-conjugated polypeptides in subcellular fractionation samples were purified with biotin SUMO capture reagent (Biotin S-Cap, LifeSensors, Inc. SM-101) following the manufacturer's protocol.

Techniques: Western Blot, Transfection, Over Expression, Purification, Control, Derivative Assay, Isolation, Binding Assay, Magnetic Beads, Transduction