1. p300 Transcriptional Repression Is Mediated by SUMO Modification
David Girdwood, Donna Bumpass, Owen A Vaughan, Alison Thain, Lisa A Anderson, Andrew W Snowden, Elisa Garcia-Wilson, Neil D Perkins, Ronald T Hay 
Molecular Cell 
Volume 11, Issue 4, Pages (April 2003)
DOI: /S (03)

2. Figure 1
p300 Repression Activity Maps to Amino Acids 1017–1029 of p300
(A) Schematic diagram of p300 domains and the p300 and Gal4-p300 expression constructs. Cysteine- and histidine-rich domains (CH1, CH2, and CH3), histone acetyl transferase (HAT) domain, Bromo domain, and Cell Cycle Regulatory domain 1 (CRD1) are indicated. Sequences fused to the C-terminal end of the Gal4-p300 N construct are displayed.
(B) U-2 OS cells were transfected with 5 μg of Bax CAT reporter plasmid alone or with 0.5 μg CMV-p53 together with 0.5 μg expression plasmid for p300 or p300Δ1004–1045 as indicated. Cells were harvested 24 hr after transfection and CAT assays performed.
(C) Samples from (B) were analyzed for p300 expression by Western blotting with a p300 antibody.
(D) Expression plasmids encoding Gal4 or Gal4-p300 fusion proteins (20 ng) were cotransfected into U-2 OS cells as indicated with a Gal4 E1B CAT reporter plasmid (5 μg) and either an RSV control or RSV p21 expression plasmid (4 μg). Results presented in (B) and (D) are the means of three separate experiments, and standard deviations are shown.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
The CRD1 Motif Corresponds to Two Potential SUMO Modification Sites
(A) The indicated sequences were fused to the Gal4-p300 N construct. Mutations were within the context of the minimal CRD1 (mCRD1) motif. The PML/E1B sequence was created from unrelated SUMO modification sites present in PML (PRKVIKME) and adenovirus type 2 E1B 55 kDa protein.
(B) Alanine scan of the minimal CRD1 motif. Expression plasmids encoding Gal4-p300 fusion proteins containing the indicated mutations of CRD1 (20 ng) were cotransfected into U-2 OS cells with a Gal4 E1B CAT reporter plasmid (5 μg).
(C) Mutation of both putative acceptor lysines for SUMO modification abolishes CRD1-mediated repression. Expression plasmids (20 ng) encoding Gal4-p300 fusion proteins containing either the wild-type mCRD1 motif or the indicated mutations of mCRD1 (K1020A, K1024A, K1020A/K1024A and K1020R/K1024R) were cotransfected into U-2 OS cells with a Gal4 E1B CAT reporter plasmid (5 μg) and either an RSV control or an RSV p21 expression plasmid (4 μg).
(D) Two heterologous SUMO modification sites can substitute for CRD1. Expression plasmids (20 ng) encoding Gal4-p300 fusion proteins containing either the wild-type mCRD1 motif or the PML/E1B SUMO modification sites, were cotransfected into U-2 OS cells with a Gal4 E1B CAT reporter plasmid (5 μg) and either an RSV control or an RSV p21 expression plasmid (4 μg). Results shown in (B,) (C), and (D) are the means of three separate experiments, and standard deviations are shown.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 SUMO Modification of p300 In Vivo
(A) Endogenous p300 is modified by endogenous SUMO in vivo. HeLa S3 spinner cells were lysed and immunoprecipitated with crosslinked p300 or 336 control antibodies as described (Desterro et al., 1998). Bound proteins were analyzed by Western blotting with a rabbit antibody which recognizes SUMO-1.
(B) Endogenous p300 is modified by SUMO in vivo. A HeLa cell line containing an integrated 6His SUMO-1 gene was lysed under denaturing conditions and 6His SUMO-1 linked proteins were isolated on Ni-NTA agarose. Bound proteins were analyzed by Western blotting with an antibody which recognizes p300. HeLa cells lacking the 6His SUMO-1 gene were used as a control.
(C) The repression domain of p300 is required for modification with SUMO-1, SUMO-2, and SUMO-3. COS7 cells were transfected with expression plasmids for either 6His-p300 or 6His-p300 lacking the CRD1 region (Δ1004–1045) in the presence of either HA-SUMO-1, HA-SUMO-2, or HA-SUMO-3. Cells were lysed under denaturing conditions and 6His proteins isolated on Ni-NTA agarose. Bound proteins were analyzed by Western blotting with an antibody to the HA tag. A proportion of the unfractionated extract was analyzed by Western blotting with anti-HA and p300 antibodies to monitor expression levels.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 SUMO Modification of p300 In Vitro
p300 (1–1301) was labeled with 35S Met by in vitro translation in a wheat germ extract. In vitro SUMO modification reactions using labeled p300 as substrate were carried out in the presence of purified components (+, SUMO, SAE, Ubc9). Where indicated, either SUMO (-SUMO), SAE (-SAE), or Ubc9 (-Ubc9) were omitted from the reaction or GST-SUMO was substituted for SUMO (GST-SUMO). A control reaction lacking these components was also analyzed (-). Reactions were carried out with either SUMO-1 (A), SUMO-2 (B), or SUMO-3 (C) as indicated and the products analyzed by SDS-PAGE and phosphorimaging.
(D) SUMO modification of the CRD motif in vitro. The indicated quantities (μg) of a GST fusion protein containing the mCRD1 motif (GST-TELKTEIKEEEDQ) was incubated under modification conditions with 125I-SUMO-1, SAE, and Ubc9. Reaction products were analyzed by SDS-PAGE and phosphorimaging. The positions of free SUMO (SUMO-1), one SUMO addition (+1 SUMO-1), and two SUMO additions (+2 SUMO-1) are indicated. A previously described GST-PML fusion protein was analyzed as a positive control (Tatham et al., 2001).
(E) Mutational analysis of the CRD domain. A series of GST-fusion proteins in which the indicated residues were altered were constructed and the proteins expressed and purified from bacteria.
(F) SUMO modification of the CRD mutants. GST fusion proteins containing the indicated mutated CRD motif were incubated under modification conditions with 125I-SUMO-1, SAE, and Ubc9. Reaction products were analyzed by SDS-PAGE and phosphorimaging. The position of free SUMO (SUMO-1), one SUMO addition (+1 SUMO-1), and two SUMO additions (+2 SUMO-1) are indicated. A previously described GST-PML fusion protein was analyzed as a positive control (Tatham et al., 2001).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Transient Localization of p300 and SUMO-1
HeLa cells in 35 mm, thin glass-bottom dishes were transfected with pEYFP-SUMO1 and NLS-CFP-P300 (full length) with FuGENE 6. After 24 hr, cells at 37°C images were acquired using a DeltaVision microscope system. 10 × 1 μm sections for YFP and CFP were taken at 15 min intervals over a 2 hr period. Images were deconvoluted using SoftWorx (Applied Precision). Inserts show higher magnification images of a nuclear domain where p300 and SUMO transiently colocalize, although many other regions where they colocalize are evident.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
SUMO Modification of the CRD Domain Is Required for Repression
(A) SUMO-specific protease 3 (SSP3) relieves p300-mediated repression. Expression plasmids encoding the indicated Gal4 or Gal4-p300 fusion proteins (5 ng) were cotransfected into U-2 OS cells with a Gal4 AdMLP luciferase reporter plasmid (2 μg) and either pcDNA3 or pcDNA3 expression constructs for SSP3 or C548A SSP3 (50 ng).
(B) Dose response of SSP3 action. An expression plasmid encoding Gal4-p300 N fusion protein (5 ng) was cotransfected into U-2 OS cells with a Gal4 AdMLP luciferase reporter plasmid (2 μg) and increasing amounts of pcDNA3 expression constructs for either SSP3 or C548A SSP3. Results are displayed as fold activation compared to transfection with the pcDNA3 empty.
(C) A dominant-negative version of Ubc9 relieves repression in vivo. Expression plasmids encoding Gal4 or Gal4-p300 fusion proteins (20 ng) were cotransfected into U-2 OS cells as indicated with a Gal4 E1B CAT reporter plasmid (5 μg) and either pcDNA3 or pcDNA3 C93S Ubc9 (2 μg). Results shown in (A), (B), and (C) are the means of three separate experiments, and standard deviations are shown.
(D) Expression of SSP3 and C93S Ubc9 blocks SUMO modification of p300 in vivo. COS7 cells were cotransfected with expression plasmids for 6His-p300 and HA-SUMO-1 in the presence of either SSP3 or C93S Ubc9 (dnUbc9). Cells were lysed and either processed directly for Western blotting with anti-p300, anti-SSP3, and anti-Ubc9 antibodies or 6His-proteins isolated on Ni-NTA agarose. Bound proteins were analyzed by Western blotting with an antibody to the HA tag.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
SUMO-Mediated p300 Repression Is Mediated by Histone Deacetylase
(A) Trichostatin A alleviates CRD1-dependent p300 repression. HEK 293 cells were transfected with 5 μg Gal4 E1B CAT reporter together with 10 ng of Gal4, 10 ng of Gal4-p300 (192–1044), or 0.2 ng of Gal4-p300 (192–1004) expression plasmids. Trichostatin A was added to cells after 4 hr and processed for CAT assay 20 hr later. Results shown are the means of three separate experiments, and standard deviations are shown. Note that 50-fold less of the Gal4-p300 (192–1004) was used compared to the Gal4-p300 (192–1044) plasmid so that both gave similar levels of reporter activity in the absence of Trichostatin A.
(B) SUMO- modified CRD1 domain binds HDAC6 in vitro. GST-p300-CRD1 or GST-p were modified by SUMO-1 in vitro and the modified species recovered on glutathione agarose. 35S-labeled in vitro-translated HDAC4 and HDAC6 were incubated with glutathione agarose bound GST, unmodified GST-p300 proteins, or the SUMO-modified GST-p300 proteins as indicated. After extensive washing, bound proteins were analyzed by SDS PAGE and phosphorimaging. 10% of the 35S-labeled HDAC4 and HDAC6 input was also analyzed.
(C) Gal4 or Gal4-p300 N+mCRD1 expression plasmids (15 ng) were cotransfected into U-2 OS cells together with a Gal4-dependent luciferase reporter (1.5 μg) and either an RSV control or RSV p21 expression plasmid (1.5 μg) as indicated. The HDAC6 expression plasmid or pCDNA3 control was included as indicated. Results presented are the means of three separate experiments, and standard deviations are shown.
(D) Gal4-p300 N– and Gal4-p300 N+mCRD1 expression plasmids (0.5 ng) were cotransfected into U-2 OS cells together with a Gal4-dependent luciferase reporter (1.5 μg) and 2 μg of either the scrambled siRNA plasmid or a pool of the three HDAC6 siRNA expression plasmids as indicated. Results presented are the means of three separate experiments, and standard deviations are shown.
Molecular Cell  , DOI: ( /S (03) )