Journal: Genes & Development

Article Title: ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation

doi: 10.1101/gad.345843.120

Figure Lengend Snippet: SUMOylation of SATB2 during ESC differentiation toward ectodermal progenitors. ( A ) Immunoblots detecting SATB2, NANOG, and TUBULIN in wild-type ( Satb2 wt ) and Satb2-deficient ( Satb2 Δ/Δ ) ESCs, cultured in LIF or with retinoic acid (RA) for 2 or 4 d. Blots were generated with ESCs from Satb2 wt clone 8 and Satb2 Δ/Δ clone 2. Data are representative for three independently derived Satb2 wt clones (8, 24.1, and 24.3) and Satb2 Δ/Δ clones (2, 32, and 64). Satb2 Δ/Δ ESCs were generated by treatment of Satb2 fl/fl Cre ERT/+ ESCs with 10 μM tamoxifen for 5 d. All clones were cultured on fibronectin-coated dishes before replating onto gelatin-coated dishes for differentiation. ( B ) qRT-PCR analysis to detect Satb2 and Nanog transcripts in LIF-cultured or RA-treated (d2 and d4) Satb2 wt and Satb2 Δ/Δ ESCs. Values are derived from three clones each and are calculated relative to those of Satb2 wt clone 24.1 in LIF and normalized to Pgk . ( C ) Alkaline phosphatase staining and quantification of Satb2 w t and Satb2 Δ/Δ ESCs cultured in LIF or RA for 3 d. The percentages of undifferentiated, differentiated, and mixed-type colonies are indicated in the bar plot. Images are representative of two independent experiments carried out with each clone. Values in B and C are expressed as the combined mean ± SD of all clones from either genotype. t -tests were carried out relative to Satb2 +/+ cells. Significance levels are as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( D ) Immunoblots detecting SATB2 and RanGAP in ESCs, cultured in LIF or with retinoic acid (RA) for 2, 4, or 6 d. Whole-cell lysates were purified in the presence (+) or absence (−) of the SUMO-isopeptidase inhibitor NEM (10 mM). SUMO-modified forms of SATB2 and RanGAP are marked. RanGAP was used as a positive control for SUMOylation, and Tubulin was used as a loading control. ( E ) Immunoblots of endogenous SATB2 that was immunoprecipitated (IP) with mouse anti-IgG, bead-coupled anti-SUMO1, or anti-SUMO2 antibodies and eluted with SUMO1 or SUMO2 peptides. Cell culture conditions were as in A . The asterisk indicates the IgG bands of antibodies used in the IP. ( F ) Immunoblot analysis of whole-cell extracts from wild-type ESCs randomly differentiated (−LIF) and toward ectoderm using two distinct protocols. Blots were probed with antibodies to detect indicated proteins. Ectoderm differentiation was induced by LIF withdrawal and addition of RA, or by the addition of FGF2, FGF8, and SHH. ( G ) Schematic representation of SATB2 protein, highlighting the SUMO consensus motifs at K233 and K350 in SATB2 wt and the arginine substitutions (K → R) in the double-mutant SATB2 K → R . ( H ) Immunoblots of an in vitro SUMOylation assay using recombinant purified SATB2 wt , SATB2 K → R , and increasing amounts of recombinant ZFP451protein (amino acids 2–246), comprising SUMO E3 activity . ( I ) Coimmunoprecipitation of SATB2 to detect interaction with ZFP451 and PIAS1 in Satb2 wt (wt) or Satb2 K → R ESCs cultured in LIF or in RA for 4 d. Immunoblots are representative of three or four independent experiments using wt clones DV3 and C2 ( A,B ) and Zfp451 +/+ clone 14 ( B ). In vitro SUMOylation immunoblot is representative of three independent experiments.

Article Snippet: SUMO hybridomas SUMO1 21C7 and SUMO2 8A2 were developed by M. Matunis and obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development of the National Institutes of Health and maintained at Department of Biology, The University of Iowa ( ; http://dshb.biology.uiowa.edu ).

Techniques: Western Blot, Cell Culture, Generated, Derivative Assay, Clone Assay, Quantitative RT-PCR, Staining, Purification, Modification, Positive Control, Immunoprecipitation, Mutagenesis, In Vitro, Recombinant, Activity Assay

Journal: Neuroscience

Article Title: NEURON-SPECIFIC SUMO KNOCKDOWN SUPPRESSES GLOBAL GENE EXPRESSION RESPONSE AND WORSENS FUNCTIONAL OUTCOME AFTER TRANSIENT FOREBRAIN ISCHEMIA IN MICE

doi: 10.1016/j.neuroscience.2016.11.036

Figure Lengend Snippet: List of primers

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-SUMO1 (21C7; DSHB Hybridoma), rabbit polyclonal anti-SUMO2/3 (Covance), rabbit polyclonal anti-GFP (Invitrogen), and mouse monoclonal anti- β -actin (Sigma).

Techniques:

Journal: Neuroscience

Article Title: NEURON-SPECIFIC SUMO KNOCKDOWN SUPPRESSES GLOBAL GENE EXPRESSION RESPONSE AND WORSENS FUNCTIONAL OUTCOME AFTER TRANSIENT FOREBRAIN ISCHEMIA IN MICE

doi: 10.1016/j.neuroscience.2016.11.036

Figure Lengend Snippet: SUMOylation after transient forebrain ischemia in SUMO knockdown (SUMO-KD) mice. Wild-type (WT) and SUMO-KD mice were subjected to sham surgery or 10 10-min forebrain ischemia and 1 h reperfusion. Ischemia-induced changes in SUMOylation, and its subcellular localization were evaluated by Western blotting (A–D) and immunohistochemistry (E, F). (A–D) Global SUMOylation in cortex (A, C) and hippocampus (B, D). Global SUMOylation induced by ischemia/reperfusion was significantly less in SUMO-KD mice. The high-molecular-weight regions, marked by brackets, were used for quantification of SUMO2/3 conjugation. Intensities of SUMO2/3 conjugates were measured by image analysis, and normalized to β-actin. Horizontal bar = median values; *p ≤ 0.05. (E, F) Immunohistochemistry analysis of SUMO2/3 (E) and SUMO1 (F) in brains of SUMO-KD mice subjected to sham or ischemia surgery. After ischemia, strong nuclear SUMO2/3 staining was observed in GFP-negative hippocampus neurons (arrows), but was absent in SUMO knockdown neurons (GFP GFP-positive; arrowheads). Nuclear rim staining by SUMO1 appeared more intense in GFP GFP-negative neurons (arrows) compared to GFP GFP-positive cells (arrowheads). Scale bar = 20 μm.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-SUMO1 (21C7; DSHB Hybridoma), rabbit polyclonal anti-SUMO2/3 (Covance), rabbit polyclonal anti-GFP (Invitrogen), and mouse monoclonal anti- β -actin (Sigma).

Techniques: Western Blot, Immunohistochemistry, Molecular Weight, Conjugation Assay, Staining

Journal: Neuroscience

Article Title: NEURON-SPECIFIC SUMO KNOCKDOWN SUPPRESSES GLOBAL GENE EXPRESSION RESPONSE AND WORSENS FUNCTIONAL OUTCOME AFTER TRANSIENT FOREBRAIN ISCHEMIA IN MICE

doi: 10.1016/j.neuroscience.2016.11.036

Figure Lengend Snippet: Overview of microarray data. Data analysis was performed on the global gene expression profiles of 12 samples from four groups: wild-type (WT) sham (WS), and ischemia (WI); and SUMO-KD (TG) sham (TS), and ischemia (TI). (A) The sampling regions. The region of hippocampal CA1 subfield that was dissected out and used for microarray analysis and qPCR is marked with white dot lines in a representative brain slice of SUMO-KD mice with GFP fluorescence. (B) Principal component analysis (PCA). The individual samples were plotted in a 3-dimensional space based on three principal components. Four groups of samples are clustered according to the genotype and surgery. (C) Venn diagram. The numbers of differentially regulated genes that were identified by pairwise comparisons of groups, with a cut-off of ≥ 2-twofold increase (↑) or decrease (↓) in gene expression are shown. (D) Verification of SUMO1-3 knockdown in SUMO-KD mice. The RNA samples from the sham group that were used for the microarray study, were analyzed to determine the levels of SUMO1-3 mRNA levels in WT and SUMO-KD mice. All individual data were normalized to β-actin. To calculate fold change, the mean values of WT mouse samples were set to 1.0. Horizontal bar = median values; *p ≤ 0.05.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-SUMO1 (21C7; DSHB Hybridoma), rabbit polyclonal anti-SUMO2/3 (Covance), rabbit polyclonal anti-GFP (Invitrogen), and mouse monoclonal anti- β -actin (Sigma).

Techniques: Microarray, Expressing, Sampling, Slice Preparation, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Homeostatic Synaptic Scaling Is Regulated by Protein SUMOylation

doi: 10.1074/jbc.M112.356337

Figure Lengend Snippet: Synaptic scaling decreases SENP1 and increases SUMO-1 conjugation. A , a 24-h treatment with 1 μ m TTX increased surface expression of GluA1 and GluA2 in cortical neurons. Surface GluA1/2 was normalized to total GluA1/2. Data are the means ± S.E. of four experiments. B , quantification of SUMO conjugation after a 24-h treatment with 1 μ m TTX and representative SUMO-1 and SUMO-2/3 blots. The total levels of SUMO-1 or SUMO-2/3 (the entire lane) were normalized to the levels of β-tubulin ( n = 3). C , quantification and representative blots of SENP1 and SENP3 normalized to β-tubulin levels with and without TTX treatment ( n = 6 for SENP1, and n = 3 for SENP3). D , overexpression of SENP1(active) prevented TTX-induced synaptic scaling. GluA1 after TTX treatment was normalized to total GluA1 levels and is expressed as a percentage of the control value, represented by the dashed line ( n = 4). E , SENP1(active) or SENP1(C603S) overexpression did not alter surface expression of GluA1, but SUMO-1/Ubc9 overexpression did ( n = 4). *, p < 0.05; **, p < 0.01 (Student's t test); ns , not significant.

Article Snippet: Antibodies were from the indicated manufacturers: monoclonal anti-GluA1 and anti-GluA2 (Millipore), monoclonal anti-Arc/Arg3.1 and anti-SUMO-1 (D11, Santa Cruz Biotechnology; or clone 21C7, Developmental Studies Hybridoma Bank, University of Iowa), polyclonal anti-SUMO-2 and anti-SENP3 (Cell Signaling), polyclonal anti-SENP1 (Imgenex), and monoclonal β-tubulin (Sigma-Aldrich).

Techniques: Conjugation Assay, Expressing, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Homeostatic Synaptic Scaling Is Regulated by Protein SUMOylation

doi: 10.1074/jbc.M112.356337

Figure Lengend Snippet: TTX increases surface expression of GluA1 and expression of SUMO-1. A , control and 24-h TTX (1 μ m )-treated cultured hippocampal neurons showing regions of interest used for quantification of expression levels. Enlarged regions of interest are indicated by boxes. Scale bars = 20 μm. B , quantification of surface expression of GluA1 and total SUMO-1, expressed as a percentage of control non-TTX-treated cells ( n = 3, i.e. three separate experiments, each assaying three to five regions of interest in 10 different cells). C and D , SENP1(C603S)- and SENP1(active)-overexpressing cells, respectively. E , effects of SENP1(active) overexpression on SUMO-1 and surface GluA1 in neurons without TTX treatment. Surface GluA1 is expressed as a percentage of control cells expressing SENP1(C603S) ( n = 3, as described above). F , surface GluA1 in cells treated with TTX expressing either SENP1(C603S) or SENP1(active) is expressed as a percentage of surface GluA1 in cells without TTX treatment ( n = 3, as described above). G , SUMO-1 levels in cells treated with TTX and infected with either SENP1(C603S) or SENP1(active) virus are expressed as a percentage of SUMO-1 levels in cells without TTX treatment ( n = 3, as described above). *, p < 0.05; ns , not significant. **, p < 0.01.

Article Snippet: Antibodies were from the indicated manufacturers: monoclonal anti-GluA1 and anti-GluA2 (Millipore), monoclonal anti-Arc/Arg3.1 and anti-SUMO-1 (D11, Santa Cruz Biotechnology; or clone 21C7, Developmental Studies Hybridoma Bank, University of Iowa), polyclonal anti-SUMO-2 and anti-SENP3 (Cell Signaling), polyclonal anti-SENP1 (Imgenex), and monoclonal β-tubulin (Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Over Expression, Infection

Journal: The Journal of Biological Chemistry

Article Title: Homeostatic Synaptic Scaling Is Regulated by Protein SUMOylation

doi: 10.1074/jbc.M112.356337

Figure Lengend Snippet: Arc SUMOylation is required for synaptic scaling. A , Arc was SUMOylated in HEK293T cells at Lys-110 and Lys-268. Shown is a Western blot for Arc in HEK293T lysates from cells transfected with either Arc or Arc-ΔKK (K110A/K268A mutant) with or without Ubc9. B , immunoprecipitation of SUMO-Arc from rat brain lysate in the absence and presence of N -ethylmaleimide. Immunoprecipitation ( IP ) was performed with anti-Arc antibody (clone H-6), and Western blotting ( WB ) was performed with anti-SUMO-1 antibody (clone 21C7). C , bacterial SUMOylation assay of Arc. GST-Arc from bacteria expressing SUMOylation machinery (E1, E2, and SUMO-1) was purified on glutathione-Sepharose, and half was treated with 20 n m recombinant SENP1 for 1 h at room temperature. Eluates from glutathione-Sepharose were blotted for Arc and SUMO-1. D , overexpressed Arc-ΔKK displayed the same response to TTX as Arc-WT in neurons treated for 24 h with 1 μ m TTX. The levels of Arc were normalized to tubulin levels ( n = 6). E , representative immunostaining of non-TTX- and TTX-treated hippocampal neurons infected with the GFP control, GFP-Arc-WT, or GFP-Arc-ΔKK Sindbis virus. Images are shown of GFP and surface GluA1 ( red ) fluorescence for each neuron. F , quantification of GluA1 surface expression in cells without TTX treatment, expressed as a percentage of surface expression in GFP-infected cells ( n = 4, each repeat assaying 10 neurons). G , quantification of GluA1 surface expression after a 24-h treatment with 1 μ m TTX in cells infected with viruses, expressed as a percentage of surface expression in GFP-infected cells without TTX ( n = 4, as described above). *, p < 0.05; **, p < 0.01 (Student's t test).

Article Snippet: Antibodies were from the indicated manufacturers: monoclonal anti-GluA1 and anti-GluA2 (Millipore), monoclonal anti-Arc/Arg3.1 and anti-SUMO-1 (D11, Santa Cruz Biotechnology; or clone 21C7, Developmental Studies Hybridoma Bank, University of Iowa), polyclonal anti-SUMO-2 and anti-SENP3 (Cell Signaling), polyclonal anti-SENP1 (Imgenex), and monoclonal β-tubulin (Sigma-Aldrich).

Techniques: Western Blot, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Purification, Recombinant, Immunostaining, Infection, Fluorescence

Journal: bioRxiv

Article Title: Ubiquitin and SUMO conjugation as biomarkers of Acute Myeloid Leukemias response to chemotherapies

doi: 10.1101/825182

Figure Lengend Snippet: (A) Flowchart followed for UbL signature characterization . Extracts from parental and chemoresistant HL-60 or U937 cells were first supplemented with recombinant UbLs (to avoid rate-limiting amounts of the modifiers) and UbL-vinyl-sulfones (to inhibit UbL deconjugating activities). They were then incubated with protein Protoarrays. After extensive washes, the arrays were incubated with, first, a primary mouse anti-SUMO-1 antibody and a rabbit anti-Flag antiserum recognizing the Flag-tag present on the recombinant Ubiquitin added to the reaction and, then, appropriate fluorescent secondary antibodies. Fluorescence signals were processed using the PAA R package. The statistical analysis was performed to identify a UbL signature of chemoresistance, as described in Methods. Three independent experiments were performed for each cell line. ( B) IC 50 of chemosensitive and chemoresistant AML cell lines . IC 50 of chemosensitive parental HL-60 and U937 (wt) cells and of their resistant counterparts (see Materials and Methods) (ARA-R and DNR-R) were assayed after 24 hrs of exposure to drugs. n=3, Mean +/-SEM with * corresponding to p<0.05. (C) Identification of ubiquitylated- and SUMOylated proteins . Normalized Ub and SUMO-1 fluorescence data obtained on all arrays incubated with extracts were compared to averaged signals on control arrays (extracts supplemented with NEM to inhibit UbL conjugation activities) to identify robustly UbL-conjugated proteins. Proteins showing significant differences between the 2 groups when using both the Welch- and the Wilcoxon-Mann-Whitney statistical tests and having mean fluorescence intensities values higher than 800 (arbitrary threshold) on Protoarrays were selected for further analysis. The Venn diagram shows the number or proteins identified as modified by SUMO-1 and/or ubiquitin.

Article Snippet: Next, they were incubated in the washing buffer under agitation (50 rpm) at 4°C for 1 hr with 1 μg/mL of rabbit anti-Flag- (SIGMA, F7425) or mouse anti-SUMO-1 (21C7 from the Developmental Studies Hybridoma Bank) antibodies.

Techniques: Recombinant, Incubation, FLAG-tag, Fluorescence, Conjugation Assay, MANN-WHITNEY, Modification

Journal: bioRxiv

Article Title: Ubiquitin and SUMO conjugation as biomarkers of Acute Myeloid Leukemias response to chemotherapies

doi: 10.1101/825182

Figure Lengend Snippet: (A) Identification of UbL-modified biomarkers of AML chemoresistance. Modification levels of the proteins modified by Ubiquitin (left panel) or SUMO-1 (central panel) selected in were compared between all parental (U937 and HL-60) and drug-resistant (ARA-R or DNR-R) sublines. Differentially modified proteins with significant p-values in both Wilcoxon signed-rank- and one sample t-test and with a drug-resistant vs parental cell ratio higher than 1.25 or lower than 0.8 are indicated in red for ubiquitylated proteins and in blue for SUMOylated ones. The Venn diagram shows the overlap between differentially ubiquitylated- and SUMOylated proteins. (B) Identification of UbL-conjugated biomarkers specific for HL-60 and U937 cell resistance to Ara-C or DNR . Statistical analyses between drug-resistant and parental cells were performed separately for U937 and HL-60 cell lines and for each drug resistance. The number of proteins showing a significant p-value in one sample t -test and a ratio between drug-resistant and parental cells higher than 1.5, or lower than 0.66, are shown. (C) Ontology analysis of the UbL signature. An ontology analysis of the 122 proteins of the UbL signature was performed using the Panther software.

Article Snippet: Next, they were incubated in the washing buffer under agitation (50 rpm) at 4°C for 1 hr with 1 μg/mL of rabbit anti-Flag- (SIGMA, F7425) or mouse anti-SUMO-1 (21C7 from the Developmental Studies Hybridoma Bank) antibodies.

Techniques: Modification, Software