Biochimica et Biophysica Acta 1839 (2014) 579–591

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The transcriptional repression activity of STAF65γ is facilitated by promoter tethering and nuclear import of class IIa histone deacetylases Feng-Shu Hsieh a, Nai-Tzu Chen a, Ya-Li Yao b, Shi-Yun Wang a, Jeremy J.W. Chen a, Chien-Chen Lai a, Wen-Ming Yang a,c,⁎ a b c

Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan Department of Biotechnology, Asia University, Taichung 41354, Taiwan Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 40227, Taiwan

a r t i c l e

i n f o

Article history: Received 28 January 2014 Received in revised form 28 April 2014 Accepted 13 May 2014 Available online 19 May 2014 Keywords: STAF65γ Lung adenocarcinoma Transcriptional regulation YY1 Histone deacetylases

a b s t r a c t Aberrant expression levels of transcriptional regulators result in alterations in transcriptional control. STAF65γ is a structural subunit of the GCN5 transcriptional co-activator complex. Reports showed that STAF65γ is highly expressed in several human cancer cells, but the consequences of this aberrant expression pattern remain elusive. Here, we show that the STAF65γ protein is highly expressed in lung adenocarcinoma patients and high levels of STAF65γ correlate with poor prognosis. High levels of STAF65γ cause repression of the c-Myc oncogene through physical association with transcription factor YY1 and co-repressors HDACs. Physical interactions between STAF65γ and class IIa HDACs facilitate nuclear enrichment and regulate the assembly of HDAC complexes. Moreover, SUMOylation of STAF65γ is necessary for maintaining the co-repressor complex containing YY1 and class IIa HDACs at the promoter. Our findings reveal a distinct role of STAF65γ in nuclear import, transcriptional repression, and cell cycle regulation at high levels of expression, which is associated with poor clinical outcomes of lung adenocarcinoma. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Transcriptional control regulates gene expression, and anomalies in gene expression can lead to cancer [1,2]. STAF65γ (SPT3-associated factor 65γ), also named ART-1 (Adenocarcinoma Antigen Recognized by CTL-1), was first identified as a gene encoding an antigenic epitope recognized by cytotoxic T lymphocytes and up-regulated in several cancer cell lines [3]. Later, the STAF65γ protein was discovered as a component of the human SAGA (Spt–Ada–Gcn5 acetyltransferase) complex [4], which is a chromatin-modifying transcriptional activator complex conserved from yeast to mammals [5]. Similar to its yeast ortholog ySpt7 [6], STAF65γ is required for maintaining the organization of the human SAGA complex (hSAGA) for the activation of MYC-dependent [7] and p53-dependent genes [8]. Although STAF65γ as a transcriptional

Abbreviations: STAF65γ, SPT3 associated factor 65γ; HDAC, histone deacetylase; HA, hemagglutinin; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; shRNA, short hairpin RNA; FACS, fluorescence activated cell sorting; SUMO, small ubiquitin-like modifier ⁎ Corresponding author at: Institute of Molecular Biology, National Chung Hsing University, 250 Kuo Kuang Rd., Taichung 40227, Taiwan. Tel.: +886 4 2284 0486x246; fax: +886 4 2287 4879. E-mail address: [email protected] (W.-M. Yang).

http://dx.doi.org/10.1016/j.bbagrm.2014.05.007 1874-9399/© 2014 Elsevier B.V. All rights reserved.

co-activator has been well described, the effect of high levels of STAF65γ on gene regulation in cancer cells remains poorly understood. Several lines of evidence suggest that STAF65γ at high levels associate with proteins that are not components of hSAGA to perform functions other than transcriptional activation. Previously, we have shown that STAF65γ interacts with TRIP-Br1, TRIP-Br2, and TRIP-Br3 to differentially affect their transcriptional activities [9], suggesting that overexpressed STAF65γ, as opposed to the whole hSAGA co-activator complex, regulates individual TRIP-Brs on a context-specific basis. Moreover, STAF65γ participates in nuclear transport, where exogenously expressed STAF65γ restores nuclear localization of cytoplasmic TAF10 and TAF8 that lack nuclear localization signals [10,11]. Together, these data suggest that STAF65γ, which is expressed at high levels in cancer cells, has additional roles that remain to be studied. In this study, we report that in lung cancer patients the protein levels of STAF65γ were abnormally high, and high levels of STAF65γ mediated transcriptional repression of growth-regulatory genes such as c-Myc. STAF65γ acquired this co-repressor function by associating with transcription factor YY1 and regulating nuclear localization of class IIa HDACs. SUMOylation of STAF65γ also contributed to the co-repressor function by maintaining HDACs at the promoter region. Our work establishes a pathological function of STAF65γ in transcriptional repression and nuclear import of HDACs.

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2. Materials and methods 2.1. Expression plasmids The expression vector for FLAG-STAF65γ has previously been described [9]. Human STAF65γ cDNA containing amino acids 1–414 was subcloned into pcDNA3.1-HA [12], pM (Clontech), pEGFP (Clontech), and mCherry (Clontech) to express HA-STAF65γ, GAL4-STAF65γ, GFPSTAF65γ, and mCherry-STAF65γ. Deletional mutations of STAF65γ were made by PCR. STAF65γ point mutations and UBC9-DN (UBC9C93S) were obtained using the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by sequencing. The pTHE vector (doxycycline-inducible expression vector) was kindly provided by Dr. Tong-Chuan He [13]. pTHE-GFP/GFP-STAF65γ was constructed by insertion of the GFP/GFP-STAF65γ cDNA into an AgeI/XhoI-digested pTHE vector. The plasmid expressing FLAG-tagged SUMO1 was prepared by inserting RT-PCR-amplified SUMO1 cDNA into pcDNA3-FLAG [12]. HA-mHDAC7 was kindly provided by Dr. Ronald M. Evans [14]. Plasmids expressing FLAG/GFP-tagged mHDAC7 were obtained by insertion of PCR-amplified cDNA from HA-mHDAC7. HA-HDAC8 was a kind gift from Dr. James Winkler [15]. HA-tagged Ubc9 was kindly provided by Dr. Keith D. Robertson [16]. The following expression plasmids have been previously described: FLAG-HDAC1 [12]; FLAG-HDAC2 [17]; FLAG-HDAC3 [18]; FLAG-HDAC4, FLAG-HDAC5, FLAG-HDAC6 and HAHDAC10 [19]; FLAG-YY1 [20]; HA-YY1 [21]. 2.2. Reporter plasmids and RNAi DHFR-Luc and pol α-Luc were kindly provided by Dr. Peggy J. Farnham [22]. pES1.0-Luc was from Michael Centrella [23]. The following reporter plasmids have been described: pRL-TK and G5TK-Luc [9]; cMyc-Luc, p21-Luc, CAD-Luc, Rb-Luc, E2F1-Luc, c-Met-Luc and MyoDLuc [24]; YY1-Luc [21]; MITF-Luc and 6xPRS-9Luc [19]; N-CAM-Luc [25]; and Y4TK-Luc (YY1TKLuc) [26]. STAF65γ shRNA (pSUPER-STAF65γ828) was kindly provided by Dr. Ernest Martinez [7]. pGIPZ-SUMO1 (RhS4430-98481086), a small hairpin RNA (shRNA) construct, was purchased from GenDiscovery Biotechnology. YY1 shRNA (pGIPZ-YY1) has been previously described [24]. 2.3. Antibodies A STAF65γ antibody (SUPT7L, A302-803A) from Bethyl Laboratories and a MYC antibody (c-Myc IVD, BSB6868) from Bio SB were used for immunohistochemistry. Mouse monoclonal anti-HA (H9658) and anti-FLAG M2 (F1804) antibodies were from Sigma-Aldrich and phosphatase-conjugated goat anti-mouse IgG (SAB-101) antibody was from Stressgen Biotechnology. Mouse anti-GFP monoclonal antibody clone 3D8A1B8 (Gm0001-02) was obtained from Abking Biotechnologies. Mouse monoclonal anti-β-actin antibody (sc-47778) and antiYY1 antibody (sc-7341) were from Santa Cruz Biotechnology. For ChIP assays, anti-acetyl-histone H3 antibody (06-599) and anti-acetylhistone H4 antibody (06-598) from Merck Millipore and antiGAL4(DBD) antibody (sc-510) from Santa Cruz Biotechnology were used. For immunostaining, rhodamine (TRITC)-conjugated goat anti mouse IgG (115-025-003) from Jackson ImmunoResearch Laboratories was used. 2.4. Immunohistochemistry Lung adenocarcinoma, breast cancer, and colon cancer specimens were kindly provided by Taichung Veterans General Hospital. For immunohistochemistry analysis of the expression of STAF65γ and MYC in paraffin-embedded cancer tissues, an automatic immunostaining device (BenchMark XT System) and an ultraView detection kit (Ventana Medical Systems, Tucson, AZ) were used. Primary antibody against STAF65γ was used at a 1:100 dilution to stain the tissue sections

for 5 h at room temperature. Primary antibody against MYC was used at a 1:30 dilution to stain the tissue sections for 2 h at 42 °C. Counterstaining with hematoxylin was performed for 4 min. For chromogenic detection, a DAB detection kit (Ventana Medical Systems) was used according to the manufacturer's instructions. 2.5. Tumor array and survival analysis 443 lung adenocarcinomas were analyzed with microarray and patient survival [27] was recorded to evaluate the association between STAF65γ expression and clinical outcome. For tumor array analysis, probe ID 201837_s_at was used to represent STAF65γ in the Affymetrix U133A platform [27]. The median of STAF65γ expression was used as the cut-off value for assigning a high or a low expression status. For survival analysis, the Kaplan–Meier method was performed to generate survival curves representing higher and lower STAF65γ expression groups. 2.6. Cell culture, transfection, and treatment HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and penicillin–streptomycin. Cells were seeded into 60 mm-diameter tissue culture dishes the day before transfection. To inhibit HDAC4 nuclear export, transfected HEK293 cells were added to extra culture media containing Leptomycin B (Sigma) to a final concentration of 10 ng/ml and incubated for another 3 h before harvest. 2.7. RNA isolation, reverse transcription, and quantitative PCR (qPCR) Total RNA was freshly isolated from 6 × 107 transfected HEK293 cells using Total RNA Miniprep Purification Kit (Genemark, Taiwan). 3 μg of RNA were reverse-transcribed into first-strand cDNA using M-MuLV reverse transcriptase (Lucigen) with oligo dT18 primers. cDNA samples were purified and than subjected to qPCR using FastStart Universal SYBR Green Master (Rox, Roche Applied Science). TBP was used as internal control in qPCR. The relative abundance of c-Myc mRNA were determined by comparative CT method [28] using TBP as the normalizer and cells expressing FLAG vector as the calibrator. 2.8. Cell cycle analysis HEK293 cells were transfected with pTHE-GFP or pTHE-GFPSTAF65γ. After 15 h, transfected cells were serum starved. GFP or GFPSTAF65γ protein expression was induced 10 h post serum-starvation by adding serum-free culture media containing doxycycline (5 μg/ml). Treated cells were stimulated by adding culture media containing 10% (v/v) fetal bovine serum for 8 h. The distribution of cells in G1, S, and G2/M phases was quantified by fluorescence-activated cell sorting (FACS) as previously described [24]. 2.9. Luciferase assay 0.5 μg of pRL-TK, 5 μg of the reporter plasmid, and other indicated plasmid(s) were transfected into HEK293 cells. 48 h after transfection, cells were harvested and luciferase assay performed according to the dual luciferase assay protocol (Promega). 2.10. Co-immunoprecipitation and in vivo SUMOylation Co-immunoprecipitation procedure has been described [19]. For detecting SUMOylation of STAF65γ, HEK293 cells were transfected with HA-tagged STAF65γ, FLAG-SUMO1 and HA-UBC9. Transfected cells were analyzed by co-immunoprecipitation.

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2.11. Chromatin immunoprecipitation (ChIP)

2.16. Statistical analysis

Transfected HEK293 cells were cross-linked in 1.42% formaldehyde for 15 min and stopped by adding glycine. The samples were then lysed in ice-cold RIPA buffer. After sonication, 25 μg of the cleared chromatin was incubated with appropriate antibodies. The chromatinantibody complexes were washed and eluted. Samples were reversecrosslinked by incubating at 65 °C overnight. DNA was precipitated by phenol-chloroform and amplified by PCR using specific primer pairs. Exponential amplification was determined by visual comparison of reaction products from different cycles of PCR amplifying the target gene fragment (c-Myc 3′-YY1, c-Myc 5′-YY1 or G5TK-Luc) and the housekeeping gene GAPDH. Each experiment was repeated at least three times and one representative result was presented.

Student's t-test was used for pair-wise comparisons. Significance was accepted at: *P b 0.05; **P b 0.01, and ***P b 0.001.

2.12. Glycerol gradient sedimentation HEK293 cells were transiently transfected with indicated plasmids. 48 h after transfection, cells were harvested. About 4 × 106 cells were lysed in 100 μl of PBS plus 0.1% NP-40. 100 μl of cleared lysate was applied onto a 5 ml glycerol gradient (5–45%) and spun at 46,000 rpm at 4 °C for 24 h. Fractions were collected and analyzed by Western blotting using indicated antibodies. 2.13. Immunofluorescence 2.5 × 105 HEK293 cells were seeded on chamber slides 1 day before transfection. For cells expressing fluorescent fusion proteins, the procedure was described previously [19]. For cells expressing FLAG-tagged proteins, indirect immunofluorescence was performed. Cells were incubated with anti-FLAG mAb (diluted 1:1000) followed by rhodamineconjugated secondary antibody (diluted 1:800). Images were analyzed using a fluorescence microscope. 2.14. Protein complex purification A FLAG-STAF65γ expression plasmid was transfected into HEK293 cells. 24 h after transfection, cells were harvested. Cells were subsequently lysed and briefly sonicated at 4 °C. Cell lysate was carefully applied to a gravity column (Bio-Rad Laboratories) containing equilibrated FLAG M2-Agarose (Sigma). After binding, the column was washed extensively with lysis buffer and then eluted with FLAG peptides. Fractions of eluate were collected and further analyzed by silver staining. The STAF65γ complex was compared with human SIRT6 and human Pc2 complexes, and protein bands unique to the STAF65γ complex were excised and sent for identification by LC–MS/MS. The experiments were repeated two times separately and one representative set of data is shown. 2.15. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis To identify the components in the human STAF65γ complex, eluted fractions were air-dried, concentrated and separated by 10% SDS-PAGE. Protein bands stained with Coomassie blue were cut into small pieces, incubated in 25 mM ammonium bicarbonate for 30 min, and then destained with 25 mM ammonium bicarbonate/50% acetonitrile-ACN. After reduction and alkylation, gel pieces were dehydrated and resuspended in a trypsin solution (0.02 μg/μl in 50 mM ammonium bicarbonate) for overnight digestion at 37 °C. Mass spectrometric analyses were performed on a ThermoFisher Scientific LTQ XL linear ion trap mass spectrometer (San Jose, CA). For protein database searching, the acquired MS/MS spectra were searched against the Swiss-Prot protein database (released January, 2008) using the Mascot algorithm. Homo sapiens was chosen for the taxonomic category.

3. Results 3.1. STAF65γ is highly expressed in lung adenocarcinoma and high expression of STAF65γ is associated with poor survival Elevated mRNA expression of STAF65γ has been found in several cancer cell lines and particularly in lung adenocarcinomas [3]. To examine whether lung cancer patients have increased amounts of STAF65γ, we assessed the expression of the STAF65γ protein in 10 lung adenocarcinoma specimens by immunohistochemistry. We found that STAF65γ was indeed extensively expressed in the tumor regions (Supplementary Fig. S1) among all 10 lung cancer cases (Fig. 1, A to J, and Table 1). In contrast, the non-tumor region from patient 1 showed negative or relatively low STAF65γ expression (Fig. 1L; also compare non-tumor regions in Fig. 1, A to J). Moreover, tissue sections of anthracosis (a chronic lung disease caused by deposition of coal dust or smoke) and of lymphangioleiomyomatosis (a progressive lung disease caused by a disorderly proliferation of smooth muscle cells) showed negative STAF65γ expression (Fig. 1, M and N). STAF65γ was overexpressed in breast carcinoma and in colon cancer (Fig. 1, O and P). To further determine whether STAF65γ levels are associated with prognosis, we studied the correlation between STAF65γ expression levels and clinical outcome. Within the group of patients we studied, those with higher STAF65γ levels had shorter overall survival (Fig. 1Q). 3.2. High expression of STAF65γ represses cell cycle-regulatory genes and inhibits cell cycle progression Because STAF65γ has been described as a transcriptional coactivator [7], we reasoned that the transcriptional regulatory activity of STAF65γ underlies the correlation between STAF65γ expression and cancer prognosis. We overexpressed STAF65γ as a fusion protein to the DNA binding domain of GAL4, which tethered STAF65γ to a target promoter containing five GAL4 binding sites (G5TK-Luc). We found that GAL4-STAF65γ repressed the promoter activity in a dose-dependent manner (Fig. 2A). To further elucidate whether high levels of STAF65γ inhibit transcription from specific genes, we screened a panel of native promoters using a luciferase reporter (Fig. 2B). Overexpression of STAF65γ resulted in decreased expression of several cell cycle-related genes, including c-Myc, p21, CAD, and Rb [30–33], to less than 50% of control but had mild or no effects on the other genes. Among those STAF65γ-repressed genes, we focused on c-Myc, one of the most frequently deregulated oncogenes in human tumors [34]. Because downregulation of c-Myc in lung adenocarcinoma also associates with poor prognosis [35], we suspected there was a direct correlation between up-regulation of STAF65γ and down-regulation of c-Myc. The following results further supported this idea: The repressional effect on the c-Myc promoter correlated with the expression level of STAF65γ (Fig. 2C), and the expression of c-Myc was reduced in STAF65γ-overexpressing cells as measured by RT-qPCR (Fig. 2D). The expression of MYC was low in the 10 lung adenocarcinoma specimens (Supplementary Fig. S2). In agreement with the role of MYC in G1 entry of cell cycle control, cells with elevated STAF65γ expression stayed in G0/G1 after serum stimulation, while control cells proceeded into S and G2/M phases (Fig. 2E). Together, these results indicate that STAF65γ is a co-repressor specifically involved in cell cycle regulation. 3.3. STAF65γ targets c-Myc promoter through YY1 Since STAF65γ does not contain a DNA-binding domain, it is likely that STAF65γ targets the c-Myc promoter through interaction with

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other transcription factors. We screened numerous transcription factors by co-immunoprecipitation and found that YY1 interacted with STAF65γ (Fig. 3A, lane 2). The N-terminal domain of STAF65γ was the

interaction domain for YY1 (Fig. 3A, lanes 3 and 6). Furthermore, high levels of STAF65γ and YY1 repressed c-Myc promoter activity (Fig. 3B, compare lanes 3 and 4 to lane 2, lane 7 to lane 2). Notably, the

Fig. 1. High expression of STAF65γ correlates with low survival rates of lung adenocarcinoma. (A–J) Sections of cancer tissues from 10 lung adenocarcinoma patients stained with an antibody against STAF65γ. Panel K, Negative control (NC) for patient 1 with the STAF65γ antibody omitted. Panel L, Staining of a non-tumor region (pneumocytes) from patient 1. Strong staining for tumor cells and weak staining in non-tumor regions are demarcated by black dashed lines. (M–N) STAF65γ is not overexpressed in anthracosis or lymphangioleiomyomatosis of the lung. Panel M, Section of anthracosis stained with the STAF65γ antibody. Panel N, Section of lymphangioleiomyomatosis stained with the STAF65γ antibody. (O) Section of breast adenocarcinoma stained with STAF65γ antibody. (P) Section of colorectal cancer stained with STAF65γ antibody. Inserts contain higher magnification (400×) images of areas boxed by white dashed lines. Scale bar, 200 μm. (Q) Kaplan–Meier survival analysis of two groups of lung cancer patients with different levels of STAF65γ expression. The significance of the difference between the two groups was evaluated using log rank test (p = 0.0058).

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Table 1 IHC analyses of STAF65γ protein in 10 lung adenocarcinoma specimens. Patient

Sex

Age (yrs)

Stage (TNM)a

Type

STAF65γ staining

1

F

51

pT3(m)N0

2 3 4 5 6 7 8 9 10

M M M M F F M M F

69 46 62 74 40 69 38 63 43

pT1bN0 pT4N2 pT2aN0 pT1aN0 pT1aN0 pT2aN0 pT1bN2 pT2aN0 pT2a(2)N0

Mixed acinar, papillary, and lepidic pattern Non-tumor region: pneumocytes Mixed acinar, solid and single cell Mixed acinar, papillary, and single cell Mixed papillary, micropapillary, solid and acinar type Mixed papillary, micropapillary, solid and acinar type Mixed acinar and papillary type Mixed acinar and papillary type Mixed acinar, papillary, and solid subtypes Mixed acinar, papillary, and solid subtypes Lepidic pattern predominant and focal acinar pattern

Nuclear staining Weak cytoplasmic/membranous staining Nuclear and focal cytoplasmic staining Nuclear and focal cytoplasmic staining Nuclear and focal cytoplasmic staining Nuclear and focal cytoplasmic staining Nuclear staining Nuclear staining Nuclear staining Nuclear and focal cytoplasmic staining Nuclear staining

a Pathologic data of cases from Taichung Veterans General Hospital were examined by the TMN staging system. Pathologic staging is based on criteria used in the American Joint Committee on Cancer 7th edition Cancer Staging Manual [29].

repressional effects were additive as knocking down the overexpressed STAF65γ relieved repression to a level comparable to YY1 alone (Fig. 3B, compare lane 5 to lanes 3 and 6). Importantly, knockdown of YY1 resulted in de-repression of the c-Myc reporter

in STAF65γ-overexpressing cells (Fig. 3B, compare lane 8 to lane 9), indicating that endogenous YY1 is required for STAF65γmediated repression of c-Myc. YY1 was also required for recruiting STAF65γ to the c-Myc promoter (Fig. 3C, compare lanes 2 and 3),

Fig. 2. High expression of STAF65γ represses cell cycle-regulatory genes and inhibits cell cycle progression. (A) GAL4-STAF65γ represses transcription in a dose-dependent manner. HEK293 cells were transiently transfected with the G5TK-Luc and pRL-TK reporter plasmids as well as increasing amounts of a GAL4 vector (0. 01, 0.1, 0.5, 3 μg) or GAL4-STAF65γ (0.01, 0.1, 0.5, 3 μg). Promoter activity was presented as relative luciferase activity of the two luciferases. Values from GAL4-STAF65γ were normalized against GAL4 alone. The results were the mean ± SD from three separate transfections. (B) STAF65γ represses promoters of genes related to cell cycle control. pRL-TK and reporter plasmids containing native promoters were transfected into HEK293 cells together with a FLAG vector (4 μg) or FLAG-STAF65γ (4 μg). Promoter activity was measured as in (A). For each native promoter, the relative luciferase activity from cells expressing FLAG-STAF65γ was normalized to the one from cells expressing the FLAG vector. (C) STAF65γ represses promoter activity of c-Myc in a dose-dependent manner. HEK293 cells were transiently transfected with pRL-TK and the reporter plasmid c-Myc Luc plus the HA vector (2 or 5 μg) or HA-STAF65γ (2 or 5 μg). Promoter activity was measured as in (A). Relative luciferase activities are presented and normalized to c-Myc Luc alone (lane 1). Statistical significance was evaluated by Student's t-test. n.s., no significance, i.e., p N 0.05; *p b 0.05; **p b 0.01; ***p b 0.001. Lower panel, expression of HA-STAF65γ. (D) STAF65γ inhibits mRNA expression of c-Myc in a dose-dependent manner. HEK293 cells were transiently transfected with FLAG vector (1 or 8 μg) or FLAG-STAF65γ (1 or 8 μg). The amount of c-Myc mRNA was measured by reverse transcription plus real-time PCR and analyzed using comparative CT analysis. (E) Elevated STAF65γ expression inhibits cell cycle progression. Left panel, schematic of the experimental design. Right panel, cell cycle profiles of GFP vector-/GFP-STAF65γ-expressing cells were analyzed in starvation (harvested at the end of starvation) or serum stimulation (harvested at the end of serum stimulation) conditions. Distribution of cells in G0/G1, S, G2/M phases were determined by FACS analysis and presented as percentages of total cells counted.

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Fig. 3. STAF65γ targets c-Myc promoter through YY1. (A) STAF65γ interacts with YY1 at the N-terminal domain. Left panel, co-immunoprecipitation analysis of YY1 and deletion mutants of STAF65γ co-expressed in HEK293 cells. Right panel, summary of YY1-interaction domain of STAF65γ. FL, full length; HFD, histone-fold domain; NLS, nuclear localization signal. (B) STAF65γ represses c-Myc promoter activity through YY1. Upper panel, c-Myc Luc and pRL-TK reporter plasmids were transfected into HEK293 cells together with other expression vectors as indicated. Luciferase activity was measured and normalized as described in Fig. 2C. Statistical significance was evaluated by Student's t-test. **p b 0.01; ***p b 0.001. The expression of YY1 and FLAG-STAF65γ proteins was confirmed by Western blot analysis (bottom). β-actin served as internal control. (C) YY1 is required for STAF65γ occupancy on the c-Myc promoter. HEK293 cells were transfected with expression plasmids as indicated. FLAG-STAF65γ-associated chromatin was immunoprecipitated using anti-FLAG antibodies. c-Myc promoter fragments were subsequently amplified by PCR using specific primers whose positions are shown in the upper panel.

and overexpressing YY1 enhanced this recruitment (Fig. 3C, compare lanes 5 and 6). These results indicate that STAF65γ mediates c-Myc repression through YY1 to obtain promoter specificity. 3.4. STAF65γ mediates repression of c-Myc through histone deacetylases A decrease in acetylation of histone H3 in STAF65γ-bound chromatin suggested the involvement of histone deacetylases (HDACs) in STAF65γ-dependent transcriptional repression (Fig. 4A). STAF65γ indeed co-immunoprecipitated with HDAC1-10 (classical HDACs) and hSIRT1 (NAD+-dependent HDACs) in HEK293 cells (Fig. 4B and Supplementary Fig. S3). Among these STAF65γ-interacting HDACs, class IIa HDACs (HDAC4, HDAC5 and HDAC7) further enhanced the repressional activity of GAL4-STAF65γ (Fig. 4C). Moreover, co-expressing STAF65γ facilitated promoter recruitment of HDAC4, HDAC5 and HDAC7 with an accompanying loss of histone H3 acetylation (Fig. 4D). These data suggest that STAF65γ is sufficient to recruit class IIa HDACs to the promoter region, resulting in transcriptional repression and the increase of repressive chromatin marks.

We next asked whether STAF65γ recruits HDACs to repress its target gene, c-Myc. As expected, class IIa HDACs were highly enriched in the c-Myc promoter in the presence of STAF65γ (Fig. 4E) and enhanced STAF65γ-mediated c-Myc repression (Fig. 4F, compare lane 3 to lanes 4, 5, and 6). A slight difference within class IIa HDACs occupancy pattern on the c-Myc promoter was observed (Fig. 4E): there was an increase in the occupancy of HDAC4 and HDAC7 on c-Myc promoter 3′-YY1 binding sites (Fig. 4E, compare lane 3 to lane 4; lane 7 to lane 8) and an increase for HDAC5 on c-Myc promoter 5′-YY1 binding sites (Fig. 4E, compare lane 5 to lane 6). Taken together, these results demonstrate that high levels of STAF65γ function as a co-repressor that mediates c-Myc repression through targeting HDAC4, HDAC5, and HDAC7 to the promoter region to establish repressive chromatin marks. 3.5. The N-terminal repression domain of STAF65γ is responsible for the interaction with class IIa HDACs To map the HDAC binding domain in STAF65γ, serial deletions of STAF65γ were used in co-immunoprecipitation. HDAC4 specifically

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Fig. 4. STAF65γ recruits histone deacetylases to repress c-Myc. (A) GAL4-STAF65γ decreases acetylation of histone H3. HEK293 cells were transfected with the G5TK-Luc reporter plus GAL4 or GAL4-STAF65γ. Chromatin immunoprecipitation (ChIP) was performed with specific antibodies as indicated. PCR primers were specific for the G5TK-Luc promoter as shown in the upper panel. The experiments were repeated three times separately and one representative set of data is shown. (B) STAF65γ interacts with HDAC4, HDAC5 and HDAC7. Tagged STAF65γ and HDACs were co-expressed in HEK293 cells. Whole cell extract was immunoprecipitated with anti-FLAG antibody and bound materials were analyzed by immunoblotting with antibodies against FLAG or HA. (C) HDAC4, HDAC5 and HDAC7 further enhance the repressional activity of GAL4-STAF65γ. HEK293 cells were transiently transfected with G5TKLuc, pRL-TK, and expression plasmids indicated or corresponding empty vectors. 0.01 μg of GAL4-STAF65γ was used to observe additive effects from HDACs. Luciferase activity was measured as in Fig. 2A. Statistical significance was evaluated by Student's t-test. **p b 0.01. (D) HDAC4, HDAC5 and HDAC7 are associated with STAF65γ at the promoter region and cause hypoacetylation of STAF65γ-bound chromatin. HEK293 cells were transfected with the expression plasmids indicated. Chromatin was immunoprecipitated with anti- FLAG or anti-AcH3 antibody. PCR primers were specific for the G5TK-Luc promoter as in (A). (E) STAF65γ increases occupancy of HDAC4, HDAC5, and HDAC7 on the c-Myc promoter. HEK293 cells were transfected with FLAG-tagged HDACs and HA-STAF65γ or empty vectors. Chromatin was immunoprecipitated with anti-Flag antibodies and amplified with primers specific for the endogenous c-Myc promoter as shown in Fig. 3C. (F) HDAC4, HDAC5 and HDAC7 further enhance STAF65γ-mediated c-Myc repression. c-Myc Luc and pRL-TK reporter plasmids were transfected into HEK293 cells together with other expression vectors as indicated. Luciferase activity was measured and normalized as described in Fig. 2C. Statistical significance was evaluated by Student's t-test. **p b 0.01; ***p b 0.001. The expression of FLAG-tagged HDACs and HA-STAF65γ proteins was confirmed by Western blot analysis (bottom). β-actin served as internal control.

immunoprecipitated with the STAF65γ N-terminal domain (amino acids 1–150) (Fig. 5A, lanes 2, 3, 4, 5 and 7), but the deletion mutant without the domain did not (lane 6). Similar results were obtained for

HDAC5 (Fig. 5B) and HDAC7 (Fig. 5C). However, HDAC5 and HDAC7 might contact additional domains since HFD of STAF65γ could pull down both HDAC5 and HDAC7, but not HDAC4 (Fig. 5A, lane 9;

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Fig. 5. Class IIa HDACs associate with the N-terminal domain of STAF65γ. (A–D) HEK293 cells were co-transfected with expression vectors for deletion mutants of STAF65γ and HDACs as indicated. Cell lysate was immunoprecipitated with anti-FLAG antibody followed by immunoblotting with the indicated antibodies. IgH, immunoglobulin heavy chains. IgL, immunoglobulin light chains. All experiments were repeated three times separately and one representative set of data is shown. (E) Schematic diagram of STAF65γ deletion mutants and their interaction with HDAC4, HDAC5 and HDAC7 or JMJD2A. FL, full length. HFD, histone fold domain. NLS, nuclear localization signal. +, binding; −, no binding; Δ, weak interaction. ND, not determined.

Fig. 5B, lane 9; Fig. 5C, lane 9). In contrast, JMJD2A interacted only with the C-terminal portion of STAF65γ (Fig. 5D). In summary, the Nterminal domain of STAF65γ is the interaction domain for HDACs (Fig. 5E). 3.6. STAF65γ regulates nuclear localization and complex assembly of class IIa HDACs Class IIa HDACs are dynamic and tightly regulated by developmental and physiological stimuli [36]. Because STAF65γ recruits class IIa HDACs for transcriptional repression (Fig. 4), we were interested in whether

STAF65γ affects subcellular localization and complex formation of class IIa HDACs. Class IIa HDACs normally exhibited a dynamic subcellular distribution. The majority of HDAC4, 5, and 7 were cytoplasmic or forming discrete foci (Fig. 6A to C, upper panels). Only a small percentage of cells had a pan-nuclear distribution of these HDACs. However, in the presence of high levels of STAF65γ, HDAC5 and HDAC7 showed a significant increase in pan-nuclear localization (Fig. 6A and B, middle panels). This effect was mediated by protein–protein interactions. We had created STAF65γ(322–414) that was unable to interact with HDAC5 (Fig. 5B, lane 10) and STAF65γ(151–414) that was unable to interact with

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HDAC7 (Fig. 5C, lane 6). These STAF65γ mutants could not alter the distribution of HDAC5/7 (Fig. 6A and B, lower panels). Interestingly, the subcellular localization of HDAC4 was not changed by STAF65γ. Instead, in cells with nuclear HDAC4, the nuclear localization of STAF65γ was altered from an even distribution to dots, which co-localized with or were wrapped by HDAC4 (Fig. 6C, lower panels of solvent control). To further confirm the co-localization of HDAC4 and STAF65γ in the nucleus, HEK293 cells were treated with leptomycin B (LMB) to block CRM1-dependent HDAC4 export [37,38]. LMB treatment resulted in nuclear accumulation of HDAC4 but had no effects on distribution of STAF65γ when STAF65γ was expressed alone. In LMB-treated cells, STAF65γ mostly co-existed with HDAC4 as dots (Fig. 6C, lower panels of +LMB), which demonstrate that the nuclear distribution of STAF65γ was regulated by nuclear HDAC4. Next, glycerol gradient sedimentation analysis was used to determine whether STAF65γ affects how HDACs form protein complexes. Transiently expressed HDAC4/5/7 alone formed protein assemblies of high sedimentation density (Fig. 6D, fractions 1–11). However, some of them migrated to top fractions and co-sedimented with STAF65γ when STAF65γ was also expressed (Fig. 6D, arrowheads). Meanwhile, a portion of STAF65γ co-migrated with HDACs to fractions of high sedimentation density (Fig. 6D, underlines). In contrast, the sedimentation pattern of YY1 was not altered with overexpressed STAF65γ (Fig. 6E). These results suggest that high levels of STAF65γ regulate the formation of class IIa HDAC protein complexes. To elucidate the mechanism by which STAF65γ regulates nucleocytoplasmic dynamics of class IIa HDACs, we purified a STAF65γ complex using anti-FLAG immunoaffinity chromatography from cell extract of HEK293 cells expressing FLAG-STAF65γ. Proteins from the STAF65γ complex were separated by SDS-PAGE and identified by mass spectrometry (Fig. 6F and Supplementary Fig. S4). As expected, the analysis identified several SAGA-type complex components [39], particularly TBP-associated factors (TAF4, TAF5L, TAF6L, TAF9B and TAF10). We also found importin subunits (importin α1 and β1) [40] and the cytoplasmic regulator 14-3-3 (YWHAZ and YWHAB) [41] in the STAF65γ complex (Fig. 6F and Supplementary Table S1), suggesting STAF65γ regulates subcellular distribution of class IIa HDACs by coordinating importins and 14-3-3. 3.7. SUMOylation of STAF65γ is required for maintaining HDACs on the promoter region STAF65γ can be modified by SUMO1 in vivo [42]. We identified three putative SUMO conjugation sites (K95, K276, K343) (Fig. 7A) within the repressional domains of STAF65γ (Fig. 7B). Mutations of these three lysines to arginines (STAF65γ-K3R) completely abolished SUMO1 conjugation in vivo (Fig. 7C, lane 6), while single or double mutations showed differential SUMOylation patterns (Fig. 7C, lanes 2 to 5), which demonstrated that K95, K276, K343 are SUMO1 modification sites of STAF65γ. Loss of SUMOylation negatively affected the transcriptional activity of STAF65γ. As shown in Fig. 7D, the repressional activity of GAL4-STAF65γ decreased when its SUMO modification sites were mutated (lanes 3 to 7). Similarly, knocking down SUMO1 or blocking the SUMOylation pathway by overexpressing UBC9-DN [44] compromised the repressional activity of GAL4-STAF65γ (Fig. 7E). Most important, SUMOylation is required for STAF65γ to bind the c-Myc promoter (Fig. 7F, compare lanes 2 and 4; lanes 3 and 5) and for the association of class IIa HDACs at the promoter region (Fig. 7G). These results show that SUMOylation is critical for the repressional ability of STAF65γ because it is required for targeting or maintaining HDACs on the promoter region. However, compared to wild type STAF65γ, STAF65γ-K3R was not compromised in its ability to immunoprecipitate with HDACs and YY1 or to regulate nuclear distribution of HDAC5 and HDAC7 (Supplementary Fig. S5). It appears that SUMO modifications are dispensable for physical interactions between STAF65γ and HDACs.

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4. Discussion Our results here highlight two activities of STAF65γ that have not been reported before: First, STAF65γ cooperates with class IIa HDACs at the YY1-binding sites on the c-Myc promoter to repress c-Myc expression. Second, STAF65γ selectively enhances nuclear localization of HDAC5 and HDAC7. These activities underlie the mechanism of turning STAF65γ from a transcriptional co-activator into a repressor and indicate potential roles of STAF65γ in lung adenocarcinoma, which has high levels of STAF65γ expression. 4.1. STAF65γ acts as a co-repressor at high levels of expression STAF65γ represses transcription in a transcription factor (YY1)dependent manner (Fig. 3). Moreover, STAF65γ mediates changes in chromatin modifications through enhancing promoter occupancy of HDAC4, HDAC5 and HDAC7 (Fig. 4). As a transcriptional regulator recruited to the promoter by a DNA-binding transcription factor and itself recruiting histone modifiers, STAF65γ fits the description of a corepressor [45]. Moreover, the mode of recruitment by STAF65γ suggests that STAF65γ is a molecular scaffold that bridges YY1 and specific HDACs on the c-Myc promoter. The scaffold function of STAF65γ has been reported in the context of the hSAGA coactivator complex [7]. The functional switch from a co-activator to a co-repressor scaffold most likely depends on high expression levels as STAF65γ becomes an avid partner of HDACs (Figs. 4 and 5; Supplementary Fig. S3) and forms distinct protein networks other than hSAGA in this condition of expression. Two well-known co-activators, p300 and CREB binding protein (CBP), were reported to possess repressive abilities [46–48]. p300 functions as a co-repressor in a cell cycle-dependent manner while dCBP represses transcription within chromatin [49,50]. These findings echo our results that STAF65γ acts as a co-repressor in a dose-dependent manner. The hSAGA complex has been found to partner with MYC to activate transcription and to regulate cell proliferation [7]. This result shows that STAF65γ, which is a member of the hSAGA complex, is a co-activator of MYC at physiological levels of expression. In this context, STAF65γ participates in the activation of MYC-dependent gene expression and the promotion of cell proliferation [7]. Our findings here show that when highly expressed, instead of being a partner of MYC, STAF65γ becomes an upstream regulator of c-Myc by controlling its expression. Therefore, in addition to turning into a transcriptional repressor, this “promotion” of STAF65γ from a partner to an upstream regulator of MYC might be of particular importance when the normal expression level of STAF65γ is perturbed, which results in rapid de-regulation of MYC-dependent gene expression. Whether this change in the chain of command can apply to other target genes of STAF65γ remains to be investigated. In conclusion, our results highlight a novel co-activator/co-repressor switch mechanism mediated by differential expression levels. They also reveal that high expression levels favor SUMOylation of STAF65γ (see below), which might mediate the switch from co-activator to corepressor. Further investigation will reveal mechanistic details. 4.2. SUMOylation regulates the co-repressor function of STAF65γ We found that three lysines are SUMOylated in STAF65γ and mutation of these lysines abolishes the repressional activity of STAF65γ (Fig. 7). These results demonstrate that STAF65γ is a SUMOylated protein and SUMOylation is required for the repressional activity of STAF65γ. SUMOylation of STAF65γ affects the recruitment of HDACs to chromatin but does not affect nuclear import or protein–protein interactions with HDACs and YY1 in soluble fractions, suggesting that SUMOylation of STAF65γ regulates protein–protein interactions only in the context of chromatin. These results also suggest that SUMOylation of STAF65γ is transiently stimulated when STAF65γ is recruited to the chromatin,

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which enhances recruitment of HDACs. Whether DNA or core histones are required for this stimulation remains to be investigated. A recent report shows that the SUMO E2 enzyme UBC9 is overexpressed in lung cancer [51], which correlates with SUMOylation of STAF65γ found in this report. Furthermore, the co-repressor CoREST1 directly binds to SUMO2 to assemble an LSD1/CoREST1/HDAC complex to repress neuronal-specific gene expression in non-neuronal cells, providing a direct, mechanical link between SUMOylation and transcriptional repression [52]. Our results, together with these reports, suggest that STAF65γ is more accessible to SUMOylation in lung cancer and functions as a co-repressor to recruit HDACs. In addition to SUMOylation, STAF65γ is subjected to various posttranslational modifications. Our mass spectrometric analysis of the STAF65γ complex also revealed several possible PTMs on STAF65γ, including ubiquitination and acetylation (unpublished results). It has also been reported that STAF65γ is a phosphorylated protein [11]. It would be rewarding to investigate the role of other PTMs and their interplay in the regulation of STAF65γ. 4.3. STAF65γ differentially regulates nucleocytoplasmic dynamics of class IIa HDACs We identified STAF65γ as a novel regulator of the subcellular distribution and complex composition of class IIa HDACs (Fig. 6). The STAF65γ protein complex contains importin subunit α and β, suggesting that STAF65γ regulates nucleocytoplasmic dynamics of class IIa HDACs through the importin pathway. As a scaffold protein, STAF65γ might force HDAC5 and HDAC7 into a complex with importin. It has been demonstrated that overexpression of STAF65γ drives cytoplasmic TAF10 or an NLS-deficient mutant TAF8 into the nucleus [10,11], strongly supporting this hypothesis. Moreover, we also found that the master cytoplasmic regulator 14-3-3 in the STAF65γ complex. 14-3-3 is known to bind phosphorylated class IIa HDACs to mask their NLS [41], which might suggest that STAF65γ interacts with 14-3-3 and then releases HDACs from 14-3-3. We suspect that STAF65γ acts as a negative regulator of 14-3-3, thereby indirectly retaining class IIa HDACs in the nucleus. Further investigation is required to examine these proposed mechanisms. Unexpectedly, STAF65γ did not affect the subcellular localization of HDAC4. Instead, HDAC4 partially altered the diffuse nucleoplasmic localization of STAF65γ into nuclear speckles. From a previous finding in which HDAC4 specifically targets MEF2C to punctate nuclear bodies and represses MEF2C-dependent transcription [53], we postulate that STAF65γ as a scaffold protein could be important in the organization of a repressive complex to regulate the differentiation of muscle cells. Interestingly, like STAF65γ, class IIa HDACs were reported to be highly expressed in different cancer tissues [54]. Whether high levels of STAF65γ and HDACs coordinate to affect the progression of lung cancer remains to be investigated.

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patients with higher STAF65γ levels had shorter overall survival. These findings argue that high expression of STAF65γ plays a significant role in lung cancer. Overexpression of c-Myc has been observed from different cancer cell lines and tissues [55]. It is accepted that MYC is required for cellcycle entry and overexpression of c-Myc results in cell proliferation. However, under certain circumstances, overexpression of c-Myc is disadvantageous for cancer progression. It has been reported that glucose or glutamine withdrawal triggers apoptosis of MYC-overexpressing cells [56,57]. In cancer with such a high metabolic demand, STAF65γ might be selected to slow down the frequency of cell cycle entry. Our results that STAF65γ represses other cell cycle-related genes support this notion. Moreover, mice with overexpression of c-Myc also showed suppressed tumorigenicity of lung cancer cells [58], and low or negative c-Myc expression was reported in lung adenocarcinoma with a poor clinical outcome [35]. These lines of evidence support that STAF65γmediated repression of c-Myc is an important factor underlying cancer progression. Further studies on which stages of cancer progression are controlled by STAF65γ will uncover the detailed mechanism. 5. Conclusion In summary, we show that high levels of STAF65γ have two novel functions: mediating gene repression and facilitating nuclear import of proteins. As STAF65γ selectively enhances nuclear localization of HDAC5 and HDAC7 to the YY1 binding sites of the c-Myc promoter, it represses the expression of c-Myc. We propose that high levels of STAF65γ might have additional targets, altering gene expression levels and changing the localization and complex assembly of proteins, which are important during the course of cancer and perhaps during tissue differentiation as well. Acknowledgments This work was supported by grants from the National Science Council (NSC 99-2311-B-005-005-MY3 and NSC 102-2311-B-005-005 to W.-M. Y.; NSC 98-2311-B-468-001-MY3 to Y.-L. Y.) and Asia University (101-asia-39 to Y.-L. Y.). We greatly appreciate the plasmid and expression constructs from Keith D. Robertson, Stuart L. Schreiber, Ronald M. Evans, James Winkler, and Tong-Chuan He, Mark Groudine, Xiao-Fan Wang, Peggy J. Farnham, Michael Centrella, Joseph R. Nevins, and Ernest Martinez. We thank I-Lu Lai, Mei-Ju Hsieh, Chia-Chin Lee, and Ya-Fang Shi for making the expression plasmids. We thank Kazusa DNA Research Institute for providing JMJD2A cDNA. We especially thank Shyh-Chang Chen of Department of Pathology and Medical Laboratory and Mei-Chun Liu of Instrument Technology Research Center, Taichung Veterans General Hospital, as well as Chia-Chun Yang, Yi-Hua Lai, and Chih-Wei Liu for technical assistance. We also thank Hsin-Chi Lan and Ya-Chen Liang for helpful discussions and critical reading of this manuscript.

4.4. STAF65γ is associated with cancer progression Appendix A. Supplementary data The extensive expression of STAF65γ in tumor sections but not in normal regions of lung adenocarcinoma specimens (Fig. 1) is the first report to correlate STAF65γ with cancer. Furthermore, lung cancer

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2014.05.007.

Fig. 6. STAF65γ regulates nuclear localization and complex assembly of HDACs. (A) STAF65γ alters nuclear distribution of HDAC5 through protein–protein interaction. HEK293 cells were transfected with FLAG-HDAC5 and GFP-tagged STAF65γ constructs as indicated. Subcellular localization of the HDAC5 and STAF65γ proteins was determined by fluorescence microscopy. N (big spots), proteins aggregated into big spots in the nucleus. CNN, more proteins in the cytoplasm than in the nucleus. (B) STAF65γ enhances nuclear localization of HDAC7 through protein–protein interaction. HEK293 cells were transfected with GFP-HDAC7 alone or in the presence of mCherry-tagged STAF65γ constructs as indicated. Dots (C, N), protein dots in both the cytoplasm and the nucleus. (C) HDAC4 recruits STAF65γ to punctate nuclear bodies. HEK293 cells were transfected with FLAG-HDAC4 in the presence of GFP-STAF65γ. Subcellular localization of HDAC4 was detected by indirect immunofluorescence using antibodies against the epitope tags and rhodamine-conjugated secondary antibodies. Upper panel, solvent control (95% ethanol). Lower panel, with LMB treatment (Leptomycin B, 10 ng/ml). (D) Glycerol gradient analysis of HDAC4, HDAC5 and HDAC7 in STAF65γ-overexpressing cells. HEK293 cells were co-transfected with expression plasmids as indicated. Cell lysate was sedimented through a 5–45% glycerol gradient and analyzed by Western blotting with indicated antibodies. Thick lines indicate overlaps of protein distribution. Arrowheads highlight the overlap of HDACs and STAF65γ in fractions of low sedimentation density. (E) Glycerol gradient analysis of YY1 (as control) in STAF65γ-overexpressing cells. (F) Composition of the FLAG-STAF65γ complex in HEK293 cells by silver-staining analysis. Proteins identified by LC–MS/MS were shown at the corresponding positions.

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Fig. 7. SUMOylation of STAF65γ is required for maintaining STAF65γ and HDACs on the c-Myc promoter. (A) Putative SUMOylation sites in the STAF65γ protein are highly conserved in vertebrates. Comparison of the three putative SUMOylation sites of human, mouse, zebrafish and chicken STAF65γ using SUMOsp 2.0 [43]. (B) The repressional domains of STAF65γ contain amino acids 1–150 and 231–414. Left panel, schematic diagram of GAL4-STAF65γ fusion proteins used in the reporter assay. FL, full length. HFD, histone fold domain. NLS, nuclear localization signal. The three putative SUMO conjugation sites (K95, K276, and K343) within the repressional domains were indicated by short arrows. Right panel, reporter assay from HEK293 cells transiently transfected with the G5TK-Luc and pRL-TK reporter plasmids as well as a GAL4 vector (3 μg) or GAL4-STAF65γ deletion mutants (3 μg). A high amount of DNA was used to distinguish the repressional activities between GAL4-STAF65γ deletion mutants. Luciferase activity was measured and normalized as in Fig. 2A. (C) K95, K276, and K343 are the SUMOylation sites in STAF65γ. HEK293 cells were transfected with expression plasmids as indicated. Western blotting was performed using anti-HA antibodies to detect the SUMOylation status of STAF65γ. STAF65γ-K3R was a SUMOylation-deficient mutant in which all three acceptor lysines were mutated to arginines. (D) STAF65γ SUMOylation mutants show decreased repressional activity. Reporter plasmids were co-transfected into HEK293 cells with either GAL4-STAF65γ (0.1 μg) or GAL4 tagged STAF65γ SUMOylation mutants (0.1 μg) as indicated. Luciferase activity was analyzed as in (B). *p b 0.05; **p b 0.01. (E) Suppression of SUMOylation decreases the repressional activity of GAL4-STAF65γ. HEK293 cells were transiently transfected with reporter plasmids. Combinations of GAL4, GAL4-STAF65γ, GAL4-STAF65γ-K3R, SUMO1 shRNA and UBC9-DN (dominant negative mutant of UBC9) were also expressed as indicated. Luciferase activity was analyzed as in (B). ***p b 0.001. (F) SUMOylation-deficient STAF65γ does not associate with the c-Myc promoter. HEK293 cells were transfected with plasmids as indicated. Chromatin was immunoprecipitated with anti-FLAG antibodies and amplified with primers specific for the endogenous c-Myc promoter as illustrated in Fig. 3C. (G) SUMOylation-deficient STAF65γ does not associate with HDAC4, HDAC5 or HDAC7 at the promoter region. HEK293 cells were transfected with the expression plasmids indicated. Chromatin immunoprecipitation was performed as described in Fig. 4D.

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