SUMO Promotes HDAC-Mediated Transcriptional Repression Shen-Hsi Yang, Andrew D Sharrocks  Molecular Cell  Volume 13, Issue 4, Pages 611-617 (February 2004) DOI: 10.1016/S1097-2765(04)00060-7

Figure 1 SUMO-Dependent HDAC Recruitment to Elk-1 (A) Schematic of the GAL-driven TK promoter-reporter construct and GAL-Elk-1(223–428). The R motif repression (R), MAP kinase docking (D), and transcription activation (TAD) domains are indicated. The wild-type and mutant amino acid sequences in the R motif are shown. (B) Reporter gene analysis of the activities of the wild-type (WT) and mutant (K2R) GAL-Elk(223–428) fusion proteins on the GAL-driven TK promoter-reporter construct in 293 cells. The activities of each construct were tested in the presence and absence of TSA and/or DN-ubc9 as indicated. The data are presented as fold derepression of reporter activity and are calculated from the activity of the reporter under each condition in the presence of and absence of TSA. Data are normalized to the TSA-mediated derepression of the GAL4 DNA binding domain alone (taken as 1). Data showing relative luciferase activities are shown in Supplemental Figure S1 at http://www.molecule.org/cgi/content/full/13/4/611/DC1. (C) HDAC activity assays. GST-Elk(201–260) was subjected to a sumoylation reaction in vitro in the presence and absence of purified SUMO-1. The resulting two GST-Elk-1 fusions were incubated with precleared HeLa nuclear extracts. The columns were washed, and bound proteins were eluted with the indicated concentrations of KCl in a stepwise manner and assayed for histone deacetylase activity. Data are presented as the HDAC activity relative to the water control. (D) The eluted proteins in (C) were analyzed by Western blotting (IB) and probed with the indicated antibodies. (E) Reporter gene analysis of the activities of the GAL4 DBD or the indicated GAL-Elk derivatives in 293 cells in the presence and absence of cotransfected HDAC-1, HDAC-2, and HDAC-3. Data are shown as the % of inhibition of reporter activity relative to the activity of each construct in the absence of HDACs. Molecular Cell 2004 13, 611-617DOI: (10.1016/S1097-2765(04)00060-7)

Figure 2 siRNA Experiments Reveal HDAC-2 as a Critical Mediator of R Motif-Mediated Transcriptional Repression in Elk-1 (A) Schematic of the promoter-reporter construct and the GAL-Elk-1 fusion protein used in this figure (see Figure 1 for details). (B) Western blots are shown that demonstrate the specificity of the RNAi duplexes in reducing the indicated protein levels in extracts from (C). Total lysates from the presence and absence of transfected siRNAs against indicated HDACs were used and probed with the indicated antibodies. The bottom panel shows the expression of GAL-Elk(223–428) in the presence of siRNA against HDAC-2. (C and D) Reporter gene analysis of the activities of the indicated GAL fusion proteins in the presence of siRNAs against the indicated HDACs. 293 cells were transfected with GAL-Elk(223–428) (C) and GAL-p300 (D) in the presence of RNAi duplexes against either GAPDH or HDACs as indicated, and GAL-luciferase reporter gene activity was measured. Data are shown as relative to GAL-Elk(223–428) or GAL-p300 in the presence of GAPDH RNAi duplexes, taken as 1. (E) 293 cells were transfected with GAL-Elk(223–428), GAL-VP16, and GAL-C/EBPα as indicated, in the presence or absence of either GAPDH or HDAC-2-specific RNAi duplexes, and GAL-luciferase reporter gene activity was measured. Data are shown as fold induction by HDAC-2 siRNA relative to the activity of each GAL fusions in the presence of GAPDH siRNA. (F) Removal of SUMO-1 modification of Elk-1 blocks the ability of HDAC-2-specific RNAi duplexes to enhance its transactivation capacity. Reporter gene analysis of the activities of GAL-Elk(223–428) in the presence of HDAC-2-specific RNAi duplexes in cells left untreated or treated with PMA or DN-ubc9 where indicated. Data are shown as fold induction in the presence of HDAC-2 siRNA relative to the activity under each condition in the presence of GAPDH-specific RNAi (taken as 1). Data showing relative luciferase activities are shown in Supplemental Figure S2. Molecular Cell 2004 13, 611-617DOI: (10.1016/S1097-2765(04)00060-7)

Figure 3 SUMO-Dependent Acetylation Changes via Recruitment of HDAC-2 to an Elk-1-Regulated Promoter (A) ChIP analysis of transfected GAL-driven E1B promoter-reporter and the indicated GAL-Elk(223–428) derivatives in 293 cells using antisera specific for the indicated proteins or acetylated histone H4 (AcH4). Following immunoprecipitation (IP) of crosslinked lysates, real-time PCR analysis of eluted DNA was performed using oligonucleotides specific for the GAL-driven E1B promoter-reporter. All quantitative PCRs were normalized to the input control. Data are shown as binding relative to wild-type GAL-Elk(223–428) for each individual antibody used. (B and C) ChIP analysis was performed as (A) except GAL-Elk(201–260) fusion proteins were used. Data were analyzed by real-time PCR (B) or conventional PCR (C). The additional control of the Gal4 DNA binding domain alone (GAL) is included (C). (D) ChIP analysis was performed as in (A) except a GAL-driven TK promoter-reporter was used. 293 cells were serum starved for 18 hr after transfection prior to stimulation with PMA for 30 min where indicated. Coprecipitated GAL-driven TK promoter-reporter DNA, by each indicated antibody, was detected by PCR. Molecular Cell 2004 13, 611-617DOI: (10.1016/S1097-2765(04)00060-7)

Figure 4 The Role of Histone Deacetylase 2 in Regulating Basal Expression of an Elk-1 Target Gene, egr-1 (A) RT-PCR analysis of egr-1 (top) and β-actin gene (bottom) transcription in HeLa cells in the presence or absence of RNAi duplexes to either GAPDH (ctrl) or the indicated HDACs. (B and D) Reporter gene analysis of the activities of an egr-1 promoter-driven luciferase reporter construct in HeLa cells in the presence or absence of RNAi duplexes against GAPDH (ctrl) or indicated HDACs (B) and in the absence or presence of either GAPDH- (ctrl), HDAC-1-, HDAC-2-, and/or Elk-1-specific RNAi duplexes as indicated (D). Data are shown as luciferase activity relative to the activity in the presence of GAPDH RNAi duplexes (taken as 1). (C) Western blot demonstrating the depletion of endogenous Elk-1 in HeLa cells by siRNA against Elk-1 but not Pin-1 (ctrl). Duplicate samples are shown. (E) ChIP analysis of endogenous egr-1 promoter in HeLa cells in the presence or absence of transfected DN-ubc9 (lanes 1 and 2) or PMA (lanes 3 and 4). Endogenous egr-1 promoter DNA coprecipitated with the indicated antibodies was detected by PCR. Molecular Cell 2004 13, 611-617DOI: (10.1016/S1097-2765(04)00060-7)