Kuo et al. 10.1073/pnas.0504460102. |
Fig. 6. SUMO modification of mouse CBP and CBP5 fragment. COS-1 cells transfected with Flag-tagged mouse CBP (A) or Flag-CBP5 (B) along with or without EGFP-SUMO-1 (SUMO, small ubiquitin-like modifier) were precipitated with anti-Flag antibody, then followed by Western analysis with anti-SUMO-1 or anti-Flag antibody as indicated. The arrowhead depicts the SUMO-1-conjugated CBP or CBP5. The arrow and asterisk indicate CBP or CBP5 fragments and nonspecific bands, respectively. (C) Two micrograms of recombinant GST-CBP5 WT fusion proteins were subjected to in vitro sumoylation reaction as described in Materials and Methods except the sumoylation reaction was extended to 4 h. The SUMO-1 modified and unmodified GST-CBP5 fusion proteins were analyzed by immunoblotting with anti-GST or anti-SUMO-1 antibody as indicated. The arrowhead and arrow indicate the SUMO-1-modified and -unmodified GST-CBP5 fragments, respectively. The asterisk indicates the SUMO-1-conjugated bands derived from sumoylation machinery, which cannot be recognized by anti-GST antibody. Note that the migration pattern of the single, double, and triple SUMO-1-conjugated GST-CBP5 bands as indicated is similar to that of EGFP-SUMO-1-conjugated Flag-CBP5 bands in Figs. 2A and 8.
Fig. 7. The putative sumoylation sites, similar to the consensus sumoylation motif yKXE found in the CBP5 fragment, are shown in bold.
Fig. 8. Enlargement of CBP5 sumoylated bands from Fig. 2A. A scheme next to the immunoblotting is shown to illustrate the migrating position of the single and double EGFP-SUMO-1-conjugated CBP5 fragments and nonspecific bands (asterisk). Red ovals represent the relative position of EGFP-SUMO-1 conjugated to CBP5 fragment.
Fig. 9. An independent experiment in mapping CBP sumoylation sites. COS-1 cells transfected with expression construct of Flag-tagged CBP5 WT and various KR mutants along with or without EGFP-SUMO-1 as indicated were lysed in RIPA buffer with 10 mM NEM, then followed by immunoprecipitation and Western analysis with indicated antibodies. The arrowhead depicts the SUMO-1-modified CBP fragments. The arrow and asterisk indicate CBP fragments and nonspecific bands, respectively. The open arrowhead indicates the position of sumoylated CBP species missing due to mutation.
Fig. 10. Mutation of CBP SUMO modification sites attenuates the trichostatin A (TSA)-induced CBP-mediated fold increases in reporter activity. This data are identical to the normalized data shown in Fig. 3E. The relative luciferase activity is represented as means ± SD from three independent experiments, which was normalized to the activity of the Gal4 DNA binding domain (Gal-DBD) in the absence of TSA treatment (taken as 1). TSA treatment induces »1.7-fold increase of the Gal-DBD-mediated reporter activity. We consider this value as basal activity of HDACs that act independently from Gal-fusion proteins. With TSA treatment, Gal-CBP5 wild-type and 3KR mutant yield 5.7-fold and 2.3-fold induction, which corresponds to 3.4-fold and 1.4-fold presented in Fig. 3E, respectively, after normalization to the increase fold in reporter activity of the Gal-DBD induced by TSA.
Fig. 11. Mammalian two-hybrid analysis of CBP–Daxx interaction. COS-1 cells transfected with 200 ng of p4XGal-TK-Luc, 50 ng of VP16-Daxx, and 50 ng of Gal-CBP WT or 3KR mutant as indicated were subjected to reporter gene assays. The data represent mean ± SD of three independent experiments.
Fig. 12. Alignment of SUMO modification sites of mouse CBP and p300. The lysine residues for SUMO acceptor sites of CBP and p300 are indicated by red bold letters. The asterisks depict the identical amino acid residues between CBP and p300.
Table 1. Domain mapping studies of CBP associated with Daxx
Bait | Prey | |
Gal4-AD | Gal-AD-Daxx | |
LexA-SUMO-1 | - | + |
LexA-CBP1* | - | - |
LexA-CBP2* | - | - |
LexA-CBP3* | - | - |
LexA-CBP4 | - | - |
LexA-CBP5 | - | + |
LexA-CBP6 | - | - |
LexA-CBP7 | - | - |
LexA-CBP8 | - | - |
LexA-CBP9 | - | - |
LexA-CBP10 | - | - |
Various CBP fragments were fused to the LexA DNA-binding domain as baits. The LexA-SUMO-1 serves as a positive control for Daxx interaction. Daxx fused to the Gal4 activation domain (Gal4-AD) functions as prey for interaction. Yeast transformants were streaked on selection media lacking histidine and scored for growth after a 4-day incubation at 30oC. "-" and "+" indicate the absence and presence of yeast colony formation on selective medium plates, respectively.
*Due to the transactivation activity of the LexA fusion proteins of CBP1, CBP2, and CBP3, these bait transformants were streaked on selection media containing 60, 80, and 20 mM 3-aminotriazole, respectively.
Supporting Text
Plasmids and Antibodies.
Ten CREB-binding protein (CBP) fragments as illustrated in Fig. 1D were individually PCR amplified and further subcloned into the pRcRSV-NLS-Flag vector and the pBTM116 vector for mammalian and yeast cell expression, respectively. The mammalian constructs expressing Flag-tagged or Gal4 fusion of individual or combined sumoylation site mutants in the context of the CBP5 fragment or CBP full-length and the yeast LexA-CBP5-3KR construct (3KR, K999/1034/1057R triple lysine mutant) were created by using a QuikChange site-directed mutagenesis kit (Stratagene). pEGFP-SUMO-1, pCMV-HA-SENP2, pVP16-Daxx, and pGST-CBP5 were constructed by subcloning cDNAs of SUMO-1, SENP2, Daxx, and CBP5 into the pEGFP-C1 (Clontech), pCMV-HA, pVP16 (Clontech), and pGEX-4T1 (Amersham Pharmacia) vectors, respectively. The Stat1-mediated reporter construct p3xLy6E-Luc was described in ref. 1. All constructs were verified by DNA sequencing. pSUPER-Daxx was described in ref. 2. Anti-CBP (sc-369), anti-GFP (sc-8334), anti-HDAC3 (sc-11417) (HDAC, histone deacetylase), and control rabbit IgG (sc-2027) antibodies were purchased from Santa Cruz Biotechnology, anti-SUMO-1 antibody from Zymed, anti-Flag antibody (M2) and anti-Daxx (D7810) from Sigma, anti-HA antibody from Covance, anti-actin antibody from Chemicon, and anti-HDAC1 (ab7028) and anti-HDAC2 (ab7029) from Abcam.RT-PCR and Semiquantitative and Real-Time PCR Analysis of IRF1.
One microgram of RNA of each sample was then reverse transcribed by using ThermoScript RT-PCR system (Invitrogen) in 20 ml of reaction mix. A 2-ml aliquot of the RT reaction product was used for semiquantitative PCR analysis with specific IRF1 primers (forward primer: 5'-ATGAGACCCTGGCTAGAG-3' and reverse primer: 5'-AAGCATCCGGTACACTCG-3') for an initial denaturation step at 95°C for 1 min; 25 cycles of 30 s at 95°C, 35 s at 57°C, and 30 s at 72°C; and a final elongation step at 68°C for 10 min. As an internal control, an aliquot of each sample was analyzed for the level of glyceraldehydes 3'-phosphate dehydrogenase (GAPDH) RNA by semiquantitative PCR with the forward primer (5'-CCACCCATGGCAAATTCC-3') and reverse primer (5'-TCTAGACGGCAGGTCAGG-3'). The PCR products were then subjected to electrophoresis on 1.5% agarose gel containing ethidium bromide. The real-time quantitative PCR were performed on the Applied Biosystem PRISM 7700 sequence detector with SYBR Green dye (Applied Biosystems) for detection as described in the manufacturer’s guidelines. For each sample, the average threshold (Ct) value was resulted from quadruplicate assays, and the DCt value was determined by subtracting the average GAPDH Ct value from the average IFR1 Ct value. Three independent experiments were performed for measuring IRF1 levels of Flag-CBP WT- and 3KR-transfected 293 cells.HeLa Nuclear Extract Preparation.
HeLa cells (5 × 107) were harvested, washed, and resuspended in 500 ml of buffer A [10 mM Hepes, pH 7.9/1.5 mM MgCl2/0 mM KCl/0.5 mM DTT/1´ protease inhibitor mixture (Complete, Roche Molecular Biochemicals)] and incubated on ice for 15 min. Cells were disrupted by passage through a syringe five times, then nuclei were purified by centrifugation. The isolated nuclei were resuspended in 150 ml of buffer C [20 mM Hepes, pH 7.9/420 mM NaCl/1.5 mM MgCl2/0.2 mM EDTA/0.5 mM DTT/25% glycerol/1´ protease inhibitor mixture (Complete, Roche Molecular Biochemicals)] with or without 10 mM N-ethylmaleimide (NEM) and incubated on ice with a vigorous agitation for 30 min. Nuclear extract (supernatants) were recovered after centrifugation then diluted with 9´ Nonidet P-40 buffer for immunoprecipitation.Chromatin Immunoprecipitation (ChIP) Procedures.
We basically followed the ChIP protocol described by Winmann and Farnham (3). Transfected 293 cells were subjected to fixation with 1% formaldehyde for 20 min at room temperature. Glycine was added to a final concentration of 125 mM to stop cross-linking. After chromatin extraction, shearing, and preclearing steps, the samples were split for immunoprecipitation with 5 mg of anti-Daxx, anti-Gal4, anti-HDAC1, anti-HDAC2, anti-HDAC3, or no antibody (as input chromatin control). Bound DNA–protein complexes were washed and then eluted as described in ref. 3, and the cross-links were removed by incubating in 250 mM NaCl at 65°C for 4 h. The resulting samples were precipitated and resuspended for proteinase K digestion and followed by DNA purification with the QIAquick PCR purification kit (Qiagen) and eluted with 50 ml of 10 mM Tris/1 mM EDTA, pH 8. Five percent of the ChIP product or 0.5% of the input chromatin control was used in each PCR reaction. PCR was performed by using the following parameters: 1 min at 95°C; 25 cycles of 30 sec at 95°C, 1 min at 57°C, and 30 sec at 72°C; and 5 min at 72°C. Primers set used for ChIP of p5XGal-E1B-Luc were 5'-ACTCATCAATGTATCTTATC-3' and 5'-AATGCCAAGCTGGAATTCGA-3'. PCR products were run on a 1.5% agarose gel and analyzed by ethidium bromide staining.1. Wen, X., Lin, H. H., Shih, H. M., Kung, H. J. & Ann, D. K. (1999) J. Biol. Chem. 274, 38204–38210.
2. Lin, D. Y., Fang, H. I., Ma, A. H., Huang, Y. S., Pu, Y. S., Jenster, G., Kung, H. J. & Shih, H. M. (2004) Mol. Cell. Biol. 24, 10529–10541.
3. Weinmann, A. S. & Farnham, P. J. (2002) Methods 26, 37–47.