- Email: [email protected]
Functional analysis of a histone deacetylase-like protein of Thermus caldophilus GK24 in mammalian cell
Biochemical and Biophysical Research Communications 362 (2007) 995–1000 www.elsevier.com/locate/ybbrc
Functional analysis of a histone deacetylase-like protein of Thermus caldophilus GK24 in mammalian cell You Sun Kim 1, Young Mi Song 1, Ho Jeong Kwon
*
Chemical Genomics Laboratory, Department of Biotechnology, College of Engineering, Yonsei University, Seoul 120-749, Republic of Korea Received 15 August 2007 Available online 28 August 2007
Abstract The function of eukaryotic histone deacetylase (HDAC) has been extensively studied for its critical role in transcriptional regulation and carcinogenesis. However that of the prokaryotic counterpart remains largely unknown. Recently, we cloned HDAC-like protein in Thermus caldophilus GK24 (Tca HDAC) from a genomic library of the microorganism based on homology analysis with human HDAC1. To explore the function of Tca HDAC in mammalian cells, Tca HDAC gene expressing vector was transfected into a human fibrosarcoma cell line, HT1080. Tca HDAC was mainly localized in nuclei of the mammalian cells as a human HDAC1 was, due to an Nterminal HDAC association domain. We further generated histidine-substituted Tca HDAC mutants and investigated their role in biochemical and cellular activity of the enzyme. Tca HDAC mutants exhibited dramatic loss of enzymatic activity and conditioned media (CM) from HT1080 cells transfected with mutant Tca HDAC was unable to stimulate angiogenic phenotypes of endothelial cells in vitro whereas that of wild Tca HDAC did. Collectively, these results demonstrate that a prokaryotic histone deacetylase from T. caldophilus GK24 is functionally active in mammalian cells and its function in gene expression is conserved from prokaryotes to eukaryotes. 2007 Elsevier Inc. All rights reserved. Keywords: Histone deacetylase; Thermus caldophilus GK24; Angiogenesis; Tca HDAC; Site-directed mutagenesis
The acetylation state of histone is reversibly regulated by histone acetyltransferase (HAT) and deacetylase (HDAC). Extensive studies have shown that the change of acetylation state of histone is correlated with dramatic biological consequences in cell. Inappropriate acetylation state of histones causes abnormal outgrowth and altered pattern of cell death leading to neoplasmic transformation [1]. For instances, functional mutation in intrinsic HAT has been reported as a major cause of Rubinstein Taybi syndrome that shows high malignant frequency [2]. Furthermore, HDACs are over-expressed in several tumor cells and tissues [3–5]. Notably, a few portions of total genes are changed by abnormal HAT and HDAC as described above. In neoplasmic state of the cell, expression of tumor suppressors, such as p53, p21, and gelsolin, are repressed, whereas *
1
Corresponding author. Fax: +82 2 362 7265. E-mail address: [email protected] (H.J. Kwon). These authors contributed equally to this work.
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.101
tumor activators, such as hypoxia-induced factor-1 (HIF1), vascular endothelial growth factor (VEGF), are up-regulated [6]. One of major causes of these abnormal expressions of genes is attributable to aberrant recruitment of HDAC. Therefore, inhibition of HDAC activity as well as disruption of HDAC complex has been recognized as a potent strategy for cancer therapy. In fact, inhibition of HDAC by specific inhibitors of the enzyme shows several changes both in molecular and cellular level, including acetylation state of histone, expression of target genes, morphology, and proliferation of the cell. Hyperacetylated histones induced by HDAC inhibitor change the chromatin structure and lead to derepression of specific genes that were repressed by HDAC. It has been demonstrated that the expression of these proteins induce growth arrest, detransformation of transformed cells to normal ones, and apoptotic cell death in a variety of transformed cells as well as neuroblastoma, melanoma, and leukemia cells [7–14]. In addition, HDACs are known to be over-expressed under
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specific environmental conditions such as hypoxia, hypoglycemia, and serum deprivation [6]. Among these conditions, hypoxia is one of the key factors to trigger angiogenesis via the induction of angiogenic factors. Angiogenesis, a new blood vessel formation by endothelial cells, is controlled by oppositely regulating factors that induce or suppress endothelial cell growth. The corruption of this balance is directly related with outgrowth and spreading of cancer cells [15]. We and other group have shown that a gene-expression regulation through the acetylation of histone is highly involved in the control of angiogenesis [6,16,17]. We demonstrated that specific HDAC inhibitors, such as trichostatin A (TSA) and FK228, induce expression of p53 and von Hippel-Lindau (VHL) under hypoxia conditions, whereas they reduced the expression of HIF-1a and VEGF resulted in the inhibition of angiogenesis [6,16]. Correspondingly, over-expression of HDAC1 induced angiogenesis both in vitro and in vivo through the increased expression of VEGF as well as suppression of p53 and VHL. These results demonstrated that HDAC plays a significant role in hypoxia-induced angiogenesis that is highly related with carcinogenesis. We recently cloned a novel prokaryotic histone deacetylase of Thermus caldophilus GK24 (Tca HDAC) from the genomic library of the microorganism based on homology analysis with human histone deacetylase1 (HDAC1). Tca HDAC gene was over-expressed in Escherichia coli and purified to show a deacetylase activity [18]. Here, we investigated Tca HDAC protein in mammalian cells to elucidate the biological function of the gene. A number of experiments using wild and mutant Tca HDAC demonstrate that a prokaryotic histone deacetylase from T. caldophilus GK24 is functionally active in mammalian cells and its function in gene expression is conserved from prokaryotes to eukaryotes. Materials and methods Materials. Glutathione and reduced glutathione were purchased from Sigma (St. Louis, MO). Anti-HDAC1 and anti-tubulin antibodies were from Abcam (Cambridge, UK) and Upstate Biotechnology (Charlottesville, VA), respectively. Anti-gelsolin, anti-p21WAF1 and anti-VEGF were from Santa Cruz Biotechnology (Santa Cruz, CA). SuperFect reagent was from Qiagen (Hilden, Germany), Trizol reagent and M-MLV reverse transcriptase was from Invitrogen (Carlsbad, CA). Ultrafiltration kit was from Millipore (Bellerica, MA), transwell plate was from Corning Costar (Cambridge, MA), Matrigel was purchased from Becton Dickinson (Franklin Lakes, NJ), and Complete Mini protease inhibitor cocktail from Roche (Mannheim, Germany). Site-directed mutagenesis. The QuickChange Site-Directed Mutagenesis System (Stratagene, La Jolla, CA) was used to mutate selected codons in the clone of Tca HDAC gene. The following primers were used: to change His125 to Ala: 5 0 -GGCGGGGGTCTCCACGCAGCCCAGTAC GACCGCGC-3 0 and 5 0 -GCGCGGTCGTACTGGGCTGCGTGGAGA CCCCCGCC-3 0 . The mutated codon is underlined. PCR was performed under the following conditions: initial denaturation for 30 s at 95 C, 16 cycles of 30 s at 95 C, 1 min at 55 C, and 12 min at 68 C. After digestion of the parental DNA for 1 h at 37 C with DpnI, the amplified plasmids were transformed into E. coli XL-1 Blue competent cells. The presence of the mutation was confirmed by DNA sequencing.
Expression and purification of the GST-fused Tca HDAC protein. BL21 (DE3) cell strains transformed with pGEX-4T-1 harboring mutated Tca HDAC gene were grown in LB (Luria–Bertani) medium containing ampicillin (50 lg/ml) at 37 C to an absorbance of 0.6–0.7 at 600 nm. The protein expression was induced by addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and then cells were grown for an additional 16 h. The cells were collected by centrifugation, suspended in buffer [150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4 (pH 7.3), 1% Triton X-100, 0.1% b-mercaptoethanol, 2 mM EDTA, protease inhibitor cocktail tablets, and 1 mM phenylmethylsulfonylfluoride (PMSF)], then disrupted by sonication. Sonication was performed five times on ice for 0.5 min with 1 min intervals for cooling. Following centrifugation at 15,000 rpm, 4 C for 30 min, supernatant was subjected to purification by affinity chromatography using glutathione agarose according to the manufacturer’s instructions. Bound proteins were eluted with 50 mM Tris–HCl, pH 8.0, and 10 mM reduced glutathione. The protein concentrations of these elution fractions were determined by using the BCA protein assay kit (Pierce, Rockford, IL). SDS–PAGE and Western blotting analysis. The eluted fractions were analyzed by SDS–PAGE and gels were stained with Coomassie Brilliant Blue R-250. A preparation of cell extracts and Western blot analysis were performed. Proteins resolved by SDS–PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore). The membranes were incubated with specific antibody for polyclonal antiHDAC1, p21, gelsolin, and VEGF. The Western blots were visualized with secondary antibody (Amersham Biosciences, Uppsala, Sweden) and the blots were detected with an enhanced West-pico chemiluminescence kit (ECL) as described by the manufacturer (Pierce). HDAC enzymatic assay. HDAC enzymatic assay was performed with HDAC Fluorescent Activity Assay kit (Biomol, Plymouth Meeting, PA) according to the manufacturer’s instruction, and a GENios microplate fluorometer was used with the Magellan software system (TECAN, Austria) with excitation at 360 nm and emission at 465 nm. HDAC activity was measured two times per one sample, and the mean value was collected in three independent samples. Transient transfection and conditioned medium (CM) preparation. To explore subcellular localization and angiogenic activity of Tca HDAC, conditioned medium (CM) was prepared from subconfluent HT1080 human fibrosarcoma cells in six-well plates that were transiently transfected with 1 lg mock or pEGFP-C2-Tca HDAC plasmids (Clontech, Palo Alto, CA) using SuperFect reagent according to supplier’s protocol. Briefly, on the day before transfection, HT1080 cells were inoculated in a six-well plate to 60% confluency and cultured with minimum essential medium (MEM) supplemented with 10% fetal bovine serum at 37 C and 5% CO2 in an incubator to the time of transfection. Mock or Tca HDAC plasmids, 1 lg each, was dissolved in TE (pH 7.4) with Eagle’s MEM medium without serum to a total volume of 100 ll. SuperFect transfection reagent (5 ll) was added to the DNA solution. The samples were incubated for 15 min at room temperature. HT1080 cells were washed once with phosphate-buffered saline (PBS) and 600 ll of MEM medium with 10% FBS containing the transfection mixture was added. Then, the cells were incubated with the mixture for 3 h at 37 C and washed once with PBS and cultured for 24 h in fresh serum free medium for over-expression of Tca HDAC. The CM without serum was collected, filtered through 0.45 lm porosity filter, and concentrated by filtration (10-folds) using a ultrafiltration kit (3 kDa cutoff, Millipore). Other plasmids expressing hHDAC1, mutant Tca HDAC (H125A) were transfected equally. Completion of transiently transfection was confirmed by performing RT-PCR and Western blotting. Tube formation assay. Matrigel (150 ll in a concentration of 10 mg/ml) was placed in a 48-well culture plate and polymerized for 30 min at 37 C. About 1 · 105 cells of human umbilical vein endothelial cells (HUVECs) were seeded on the surface of the Matrigel. The morphological changes in the cells were observed under a microscope and photographed at a 100· magnification using a JVC digital camera (Victor, Yokohama, Japan). Chemoinvasion assay. The invasiveness of the HUVECs was performed in vitro using a transwell chamber system with 8.0-lm pore polycarbonate filter inserts. The lower side of the filter was coated with 10 ll gelatin (1 mg/ml), whereas the upper side was coated with 10 ll of Matrigel
Y.S. Kim et al. / Biochemical and Biophysical Research Communications 362 (2007) 995–1000 (3 mg/ml). The HUVECs (1 · 105 cells) were placed in upper part of the filter and conditioned medium was applied to lower part. The chamber was then incubated at 37 C for 18 h and then fixed with methanol and stained with hematoxylin and eosin. The cell invasion was determined by counting whole cell numbers in a single filter using optical microscopy at 100· magnification. Statistical analysis. Results are expressed as means ± standard error (SE). Student’s t test was used to determine statistical significance between control and test groups. A P value of <0.05 was considered statistically significant.
Results and discussion Tca HDAC localizes in a nucleus as human HDAC1 does To explore the biological function of TcaHDAC from prokaryote and to compare it with a eukaryotic equivalent, we first analyzed the intracellular localization of Tca HDAC. Plasmids harboring human HDAC (hHDAC1) and Tca HDAC were transfected transiently into HT1080 cells and their mRNA expression level were confirmed by RT-PCR analysis. Through indirect fluorescence analysis, hHDAC1 was predominantly converged into a nucleus as like all known mammalian class HDAC. It has been well known that the localization of hHDAC1 at nuclei is a prerequisite to exhibit biological function in the cells. Likewise, wild-type Tca HDAC exhibited a superior nuclear localization implying that Tca HDAC and the hHDAC1 may show cognate activities each other (Fig. 1A). As previously reported, Tca HDAC shows a high analogy with human HDAC1 [18]. The nuclear confinement of hHDAC1 can be accounted for two different regions responsible for its nuclear import. It possesses the N-terminal motif required for homo- and hetero-oligomerization and the C-terminal lysine-rich sequence, both of which are in charge of nuclear localization of hHDAC1. The Cterminal holds a lysine-rich nuclear localization signal (NLS) sequence that is sufficient for the nuclear translocation. The core sequence resembles monopartite c-myc NLS sequence known to interact with importin a/b complex which is well studied nucleocytoplasmic transport system [19]. hHDAC1 translocation depends on this lysine-rich sequence. Besides, the N-terminal oligomerization motif also participates in hHDAC1 translocation. This motif, called HDAC association domain (HAD), is reported to associate with hHDAC1 for homo-oligomerization and hHDAC2, hHDAC3 for hetero-oligomerization comprising various histone deacetylase complexes. This association recruits other HDACs containing NLS enabling hHDAC1 to shuttle into a nucleus. Thus, the nuclear import of HDAC1 is fulfilled by two structural features [20]. Tca HDAC whose location is focused on nucleus, on the other hand, is unlikely to obey referred both mechanisms. Aligning Tca HDAC sequence with that of hHDAC1 revealed the existence of partial N-terminal HAD but lacked C-terminal NLS sequence (Fig. 1B). Since T. caldophilus GK24 is a nucleus-free prokaryotic organism, the signal toward nuclei is obviously unnecessary. Therefore,
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a nuclear local saliency of Tca HDAC can be deduced from the existence of N-terminal HAD region which may recruit the endogenous HDACs having NLS by inducing oligomerization. Histidine residues are crucial for its deacetylasing activity of Tca HDAC As histidine residues His141 and His199 of human HDAC1 are crucial for the enzyme active, we speculated that histidine residues of Tca HDAC also important for the catalytic activity and generated the site directed mutants, histidine 125, 183 to alanine [21]. The over-expression of mutant Tca HDAC H125A and H183A, which is mutated at a conserved catalytic histidine residue (corresponding to the human HDAC1 His141 and His199), displayed significantly weaker enzymatic activity compared with the wild-type protein. As shown in Fig. 2, enzyme activity was reduced dramatically. The enzyme activity of H125A exhibited weaker activity than that of H183A unlike human HDAC1 and double mutants (H125A/H183A) did not show a synergic inactivation, implying that His125 plays a more crucial role in deacetylasing activity than His183 of Tca HDAC. Tca HDAC induces angiogenesis in vitro hHDACs play a critical role in regulating gene expression and key biological processes such as tumorigenesis and angiogenesis. In addition, it has been recently demonstrated that HDAC inhibitors inhibit angiogenesis by the suppression of the production of vascular endothelial growth factor (VEGF) from tumor cells and the direct inhibition of endothelial cell migration and proliferation [6,16,22]. To explore whether Tca HDAC confers similar biological function to hHDAC1, angiogenic activity of Tca HDAC transfectants was investigated. We transiently transfected HT1080 cells with wild Tca HDAC (wt-Tca HDAC) and mutant Tca HDAC H125A (mt-Tca HDAC) vectors in serum free medium (SFM) before collecting conditioned medium (CM). The CM was prepared in SFM, concentrated by filtration, and in vitro angiogenesis assay was performed with the CMs from wild or mutant Tca HDAC transfected cells. We then directly applied CM to HUVECs to examine the effect of CM on angiogenesis in vitro. CM of mock transfectants did not induce tube-like structures. Surprisingly, however, CM collected from wtTca HDAC transfectants enhanced the formation of capillaries, which developed elongated and complex networks (Fig. 3A). The stimulation of tube formation by Tca HDAC over-expression was not detected in CM of Tca HDAC mt-transfectants, implying that HDAC activity is required for angiogenic stimulation. To validate pro-angiogenic activity of Tca HDAC, the CM was treated on HUVECs to determine chemoinvasion. Similarly, chemoinvasion of HUVECs were significantly increased by CM of wt-Tca HDAC, but not by that of Tca HDAC mt-transfectants (Fig. 3B and C).
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Fig. 1. Subcellular localization and sequence comparison with hHDAC1 of Tca HDAC. (A) The vectors encoding full-length GFP-hHDAC1 and Tca HDACs were transiently transfected into HT1080 cells. Localization of GFP-hHDAC1 and GFP-Tca HDAC was determined by fluorescence microscopy. Each image corresponds to GFP (left), GFP-hHDAC1 (middle), GFP-Tca HDAC (right), respectively. (B) Sequence of human HDAC1 (normal) and Tca HDAC (italic) was analyzed. Matching was done using NCBI bl2seq (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). Matched parts are grayshaded. Underlined are N-terminal HAD and C-terminal NLS of human HDAC1. Asterisk marks the core of NLS. The partial N-terminal HAD of Tca HDAC is indicated in bold.
Tca HDAC over-expression oppositely modulates expression of target genes
Fig. 2. Histone deacetylase enzymatic activity of mutants Tca HDAC. Mutation of His125 and His183 to Ala in Tca HDAC reduces enzymatic activity. His125 was mutated to Ala as described under ‘‘Experimental Procedures.’’ Tca HDAC wild-type or mutants fused to a GST tag were expressed in E. coli precipitated using glutathione agarose resin.
To evaluate whether Tca HDAC modulates human HDAC target genes and angiogenesis factor, we transfected HT1080 cells with vector expressing wt-Tca HDAC or mt-Tca HDAC. In contrast to the suppressed levels of p21 and gelsolin, expression of VEGF was significantly up-regulated by wt-Tca HDAC. Compared to wt-Tca HDAC, however, mt-Tca HDAC did not induce a significant change in expression of p21, gelsolin and VEGF. mt-Tca HDAC inactivated both the deacetylation and transcriptional repression activities of Tca HDAC (Fig. 4). In this study, the biological role of prokaryotic HDAC was evaluated. Basic properties of Tca HDAC was very close to that of human HDAC1 in deacetylasing activity, nuclear localization and target gene regulation involved in progressing angiogenesis. These similarities can be
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Fig. 3. Angiogenic stimulation of Tca HDAC over-expression. HT1080 cells were transiently transfected with empty, wt-Tca HDAC or mt-Tca HDAC vectors and the conditioned medium (CM) was collected for 24 h. (A) HUVECs was incubated for 8 h in the CM on 48-well plates coated Matrigel, and cell morphology was photographed under a microscope. CM collected from HDAC transfectants stimulates tube formation in vitro. (B) Invasiveness of HUVECs was performed in vitro using transwell chambers system of 8.0-lm pores polycarbonate filter inserts for 16 h. The cells that invaded through the Matrigel-coated inserts were stained, counted and photographed under a light microscope at 100· magnification. chemoinvasion of HUVECs were significantly increased by CM of wt-Tca HDAC, but not by CM of mt–Tca HDAC transfectants. (C) The percentage of invasion was quantified as described in Experimental Design. Values are means ± SD from five different experiments (P < 0.001).
Fig. 4. Regulation of HDAC target genes by Tca HDAC over-expression. (A) HT1080 cells were transiently transfected with mock, hHDAC1, wt-Tca HDAC or mt-Tca HDAC (H125A) expression vectors, total cell lysate was isolated and immunoblot analysis was performed with each specific antibody. (B) Bar graphs display a relative induction of each band (j, gelsolin; Q, p21WAF1; h, VEGF).
deduced by a sequence homology analysis. Tca HDAC gene codes a 375 amino acid polypeptide. It has a partial N-terminal HDAC association domain (HAD) at a fore-
part of catalytic domain. As described, this domain is responsible for oligomerization of several HDACs as well as Rb-binding protein 48 (RbAp48) and Sin3A/Sin3B con-
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stituting diverse HDAC complexes [20]. The biological resemblance might result from the interaction between Tca HDAC and other HDAC complex-composing subunits via N-terminal HAD. And these complexes would facilitate the access of Tca HDAC to target proteins to trigger its catalytic activity. This conclusion enables us to infer an inborn characteristics of Tca HDAC as a bacterial gene-expression regulator in T. caldophilus GK24, the thermophilic bacteria. In addition, the structural simplicity of primitive system may satisfy the partial HAD sequence for an interaction with others, which has been supplemented in an evolutionary process. Although there is no proven HDAC activity in prokaryotes, various bacterial histone-like proteins and their regulation of gene expression were revealed [23]. Post-translational modifications of bacterial proteins such as acetylation were also reported for their biological processes [24]. Eventually, our findings suggest that function of the gene keeps intact not only among eukaryotes but also to prokaryotes and would contribute to a research ascending against an immense evolutional track. Acknowledgments This study was supported by grants from Forest Science & Technology Projects provided by Korea Forest Service and from the Brain Korea 21 Project, Republic of Korea. References [1] P.A. Marks, R.A. Rifkind, Erythroleukemic differentiation, Annu. Rev. Biochem. 47 (1978) 419–448. [2] F. Petrij, R.H. Giles, H.G. Dauwerse, J.J. Saris, R.C. Hennekam, M. Masuno, N. Tommerup, G.J. van Ommen, R.H. Goodman, D.J. Peters, Rubinstein Taybi syndrome caused by mutations in the transcriptional co-activator CBP, Nature 376 (1995) 348–351. [3] P.P. Pandolfi, Transcription therapy for cancer, Oncogene 20 (2001) 3116–3127. [4] P. Dhordain, R.J. Lin, S. Quief, D. Lantoine, J.P. Kerckaert, R.M. Evans, O. Albagli, The LAZ3 (BCL06) oncoprotein recruits a SMRT/ Msin3A/histone deacetylase containing complex to mediate transcriptional repression, Nucleic Acids Res. 26 (1998) 4645–4651. [5] J.H. Choi, H.J. Kwon, B.I. Yoon, J.H. Kim, S.U. Han, H.J. Joo, D.Y. Kim, Expression profile of histone deacetylase1 in gastric cancer tissues, Jpn. J. Cancer Res. 92 (2001) 1300–1304. [6] M.S. Kim, H.J. Kwon, Y.M. Lee, J.H. Baec, J.E. Jang, S.W. Lee, E.J. Moon, H.S. Kim, S.K. Lee, H.Y. Chung, C.W. Kim, K.W. Kim, Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes, Nat. Med. 7 (2001) 437–443. [7] N. Tsuji, M. Kobaysshi, K. Nagashima, Y. Wakisaka, K. Koizumi, A new antifungal antibiotics, trichostatin, J. Antibiot. (Tokyo) 29 (1976) 1–6.
[8] M. Yoshida, M. Kijima, M. Akita, T. Beppu, Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A, J. Biol. Chem. 265 (1990) 17174–17179. [9] M. Kijima, M. Yoshida, K. Sugita, S. Horinouchi, T. Beppu, Trapoxin, an antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase, J. Biol. Chem. 268 (1993) 22429– 22435. [10] H.L. Newmark, J.R. Lupton, C.W. Young, Butyrate as a differentiating agent; pharmacokinetics, analogues and current status, Cancer Lett. 78 (1994) 1–5. [11] H.J. Kwon, T. Owa, C.A. Hassig, J. Shimada, S.L. Schreiber, Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase, Proc. Natl. Acad. Sci. 95 (1998) 3356–3361. [12] R.D. Glick, S.L. Swendeman, D.C. Coffey, R.A. Ridkind, P.A. Marks, V.M. Richon, Hybrid polar histone deacetylase inhibitor induces apoptosis and CD95/CD95 ligand expression in human neuroblastoma, Cancer Res. 59 (1999) 4392–4399. [13] L. Qui, M.J. Kelso, C. Hansen, M.L. West, D.P. Fairlie, P.G. Parsons, Anti-tumor activity in vitro and in vivo of selective differentiating agents containing hydroxamate, Br. J. Cancer 80 (1999) 1252–1258. [14] Y. Lim, I. Han, H.J. Kwon, E.S. Oh, Trichostatin A-induced detransformation correlates with decreased focal adhesion kinase phosphorylation at tyrosine 861 in ras-transformed fibroblasts, J. Biol. Chem. 277 (2002) 12735–12740. [15] J. Folkman, Fighting cancer by attacking its blood supply, Sci. Am. 275 (1996) 150–154. [16] H.J. Kwon, M.S. Kim, M.J. Kim, H. Nakajiam, K.W. Kim, Histone deacetylase inhibitor FK228 inhibits tumor angiogenesis, Int. J. Cancer 97 (2002) 290–296. [17] C.F. Deroanne, K. Bonjean, S. Servotte, L. Devy, A. Colige, N. Clausse, S. Blacher, E. Verdin, J.M. Foidart, B.V. Nusgens, V. Castronovo, Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling, Oncogene 21 (2002) 427–436. [18] Y.M. Song, Y.S. Kim, D.I. Kim, D.S. Lee, H.J. Kwon, Cloning, expression, and biochemical characterization of a new histone deacetylase-like protein from Thermus caldophilus GK24, Biochem. Biophys. Res. Commun. 361 (2007) 55–61. [19] E. Conti, M. Uy, L. Leighton, G. Blobel, J. Kuriyan, Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha, Cell 94 (1998) 193–204. [20] J. Taplick, V. Kurtev, K. Kroboth, M. Posch, T. Lechner, C. Seiser, Homo-oligomerisation and nuclear localisation of mouse histone deacetylase 1, J. Mol. Biol. 308 (2001) 27–38. [21] C.A. Hassig, J.K. Tong, T.C. Fleischer, T. Owa, P.G. Grable, D.E. Ayer, S.L. Schreiber, A role for histone deacetylase activity in HDAC1-mediated transcriptional repression, Proc. Natl. Acad. Sci. 95 (1998) 3519–3524. [22] M. Goh, F. Chen, M.T. Paulsen, A.M. Yeager, E.S. Dyer, M. Ljungman, Phenylbutyrate attenuates the expression of Bcl-X-L, DNA-PK, caveolin-1, and VEGF in prostate cancer cells, Neoplasia 3 (2001) 331–338. [23] C.J. Dorman, P. Deighan, Regulation of gene expression by histonelike proteins in bacteria, Curr. Opin. Genet. Dev. 13 (2003) 179–184. [24] R. Barak, M. Eisenbach, Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis, Mol. Microbiol. 40 (2001) 731–743.
Functional analysis of a histone deacetylase-like protein of Thermus caldophilus GK24 in mammalian cell You Sun Kim 1, Young Mi Song 1, Ho Jeong Kwon
*
Chemical Genomics Laboratory, Department of Biotechnology, College of Engineering, Yonsei University, Seoul 120-749, Republic of Korea Received 15 August 2007 Available online 28 August 2007
Abstract The function of eukaryotic histone deacetylase (HDAC) has been extensively studied for its critical role in transcriptional regulation and carcinogenesis. However that of the prokaryotic counterpart remains largely unknown. Recently, we cloned HDAC-like protein in Thermus caldophilus GK24 (Tca HDAC) from a genomic library of the microorganism based on homology analysis with human HDAC1. To explore the function of Tca HDAC in mammalian cells, Tca HDAC gene expressing vector was transfected into a human fibrosarcoma cell line, HT1080. Tca HDAC was mainly localized in nuclei of the mammalian cells as a human HDAC1 was, due to an Nterminal HDAC association domain. We further generated histidine-substituted Tca HDAC mutants and investigated their role in biochemical and cellular activity of the enzyme. Tca HDAC mutants exhibited dramatic loss of enzymatic activity and conditioned media (CM) from HT1080 cells transfected with mutant Tca HDAC was unable to stimulate angiogenic phenotypes of endothelial cells in vitro whereas that of wild Tca HDAC did. Collectively, these results demonstrate that a prokaryotic histone deacetylase from T. caldophilus GK24 is functionally active in mammalian cells and its function in gene expression is conserved from prokaryotes to eukaryotes. 2007 Elsevier Inc. All rights reserved. Keywords: Histone deacetylase; Thermus caldophilus GK24; Angiogenesis; Tca HDAC; Site-directed mutagenesis
The acetylation state of histone is reversibly regulated by histone acetyltransferase (HAT) and deacetylase (HDAC). Extensive studies have shown that the change of acetylation state of histone is correlated with dramatic biological consequences in cell. Inappropriate acetylation state of histones causes abnormal outgrowth and altered pattern of cell death leading to neoplasmic transformation [1]. For instances, functional mutation in intrinsic HAT has been reported as a major cause of Rubinstein Taybi syndrome that shows high malignant frequency [2]. Furthermore, HDACs are over-expressed in several tumor cells and tissues [3–5]. Notably, a few portions of total genes are changed by abnormal HAT and HDAC as described above. In neoplasmic state of the cell, expression of tumor suppressors, such as p53, p21, and gelsolin, are repressed, whereas *
1
Corresponding author. Fax: +82 2 362 7265. E-mail address: [email protected] (H.J. Kwon). These authors contributed equally to this work.
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.101
tumor activators, such as hypoxia-induced factor-1 (HIF1), vascular endothelial growth factor (VEGF), are up-regulated [6]. One of major causes of these abnormal expressions of genes is attributable to aberrant recruitment of HDAC. Therefore, inhibition of HDAC activity as well as disruption of HDAC complex has been recognized as a potent strategy for cancer therapy. In fact, inhibition of HDAC by specific inhibitors of the enzyme shows several changes both in molecular and cellular level, including acetylation state of histone, expression of target genes, morphology, and proliferation of the cell. Hyperacetylated histones induced by HDAC inhibitor change the chromatin structure and lead to derepression of specific genes that were repressed by HDAC. It has been demonstrated that the expression of these proteins induce growth arrest, detransformation of transformed cells to normal ones, and apoptotic cell death in a variety of transformed cells as well as neuroblastoma, melanoma, and leukemia cells [7–14]. In addition, HDACs are known to be over-expressed under
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specific environmental conditions such as hypoxia, hypoglycemia, and serum deprivation [6]. Among these conditions, hypoxia is one of the key factors to trigger angiogenesis via the induction of angiogenic factors. Angiogenesis, a new blood vessel formation by endothelial cells, is controlled by oppositely regulating factors that induce or suppress endothelial cell growth. The corruption of this balance is directly related with outgrowth and spreading of cancer cells [15]. We and other group have shown that a gene-expression regulation through the acetylation of histone is highly involved in the control of angiogenesis [6,16,17]. We demonstrated that specific HDAC inhibitors, such as trichostatin A (TSA) and FK228, induce expression of p53 and von Hippel-Lindau (VHL) under hypoxia conditions, whereas they reduced the expression of HIF-1a and VEGF resulted in the inhibition of angiogenesis [6,16]. Correspondingly, over-expression of HDAC1 induced angiogenesis both in vitro and in vivo through the increased expression of VEGF as well as suppression of p53 and VHL. These results demonstrated that HDAC plays a significant role in hypoxia-induced angiogenesis that is highly related with carcinogenesis. We recently cloned a novel prokaryotic histone deacetylase of Thermus caldophilus GK24 (Tca HDAC) from the genomic library of the microorganism based on homology analysis with human histone deacetylase1 (HDAC1). Tca HDAC gene was over-expressed in Escherichia coli and purified to show a deacetylase activity [18]. Here, we investigated Tca HDAC protein in mammalian cells to elucidate the biological function of the gene. A number of experiments using wild and mutant Tca HDAC demonstrate that a prokaryotic histone deacetylase from T. caldophilus GK24 is functionally active in mammalian cells and its function in gene expression is conserved from prokaryotes to eukaryotes. Materials and methods Materials. Glutathione and reduced glutathione were purchased from Sigma (St. Louis, MO). Anti-HDAC1 and anti-tubulin antibodies were from Abcam (Cambridge, UK) and Upstate Biotechnology (Charlottesville, VA), respectively. Anti-gelsolin, anti-p21WAF1 and anti-VEGF were from Santa Cruz Biotechnology (Santa Cruz, CA). SuperFect reagent was from Qiagen (Hilden, Germany), Trizol reagent and M-MLV reverse transcriptase was from Invitrogen (Carlsbad, CA). Ultrafiltration kit was from Millipore (Bellerica, MA), transwell plate was from Corning Costar (Cambridge, MA), Matrigel was purchased from Becton Dickinson (Franklin Lakes, NJ), and Complete Mini protease inhibitor cocktail from Roche (Mannheim, Germany). Site-directed mutagenesis. The QuickChange Site-Directed Mutagenesis System (Stratagene, La Jolla, CA) was used to mutate selected codons in the clone of Tca HDAC gene. The following primers were used: to change His125 to Ala: 5 0 -GGCGGGGGTCTCCACGCAGCCCAGTAC GACCGCGC-3 0 and 5 0 -GCGCGGTCGTACTGGGCTGCGTGGAGA CCCCCGCC-3 0 . The mutated codon is underlined. PCR was performed under the following conditions: initial denaturation for 30 s at 95 C, 16 cycles of 30 s at 95 C, 1 min at 55 C, and 12 min at 68 C. After digestion of the parental DNA for 1 h at 37 C with DpnI, the amplified plasmids were transformed into E. coli XL-1 Blue competent cells. The presence of the mutation was confirmed by DNA sequencing.
Expression and purification of the GST-fused Tca HDAC protein. BL21 (DE3) cell strains transformed with pGEX-4T-1 harboring mutated Tca HDAC gene were grown in LB (Luria–Bertani) medium containing ampicillin (50 lg/ml) at 37 C to an absorbance of 0.6–0.7 at 600 nm. The protein expression was induced by addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and then cells were grown for an additional 16 h. The cells were collected by centrifugation, suspended in buffer [150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4 (pH 7.3), 1% Triton X-100, 0.1% b-mercaptoethanol, 2 mM EDTA, protease inhibitor cocktail tablets, and 1 mM phenylmethylsulfonylfluoride (PMSF)], then disrupted by sonication. Sonication was performed five times on ice for 0.5 min with 1 min intervals for cooling. Following centrifugation at 15,000 rpm, 4 C for 30 min, supernatant was subjected to purification by affinity chromatography using glutathione agarose according to the manufacturer’s instructions. Bound proteins were eluted with 50 mM Tris–HCl, pH 8.0, and 10 mM reduced glutathione. The protein concentrations of these elution fractions were determined by using the BCA protein assay kit (Pierce, Rockford, IL). SDS–PAGE and Western blotting analysis. The eluted fractions were analyzed by SDS–PAGE and gels were stained with Coomassie Brilliant Blue R-250. A preparation of cell extracts and Western blot analysis were performed. Proteins resolved by SDS–PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore). The membranes were incubated with specific antibody for polyclonal antiHDAC1, p21, gelsolin, and VEGF. The Western blots were visualized with secondary antibody (Amersham Biosciences, Uppsala, Sweden) and the blots were detected with an enhanced West-pico chemiluminescence kit (ECL) as described by the manufacturer (Pierce). HDAC enzymatic assay. HDAC enzymatic assay was performed with HDAC Fluorescent Activity Assay kit (Biomol, Plymouth Meeting, PA) according to the manufacturer’s instruction, and a GENios microplate fluorometer was used with the Magellan software system (TECAN, Austria) with excitation at 360 nm and emission at 465 nm. HDAC activity was measured two times per one sample, and the mean value was collected in three independent samples. Transient transfection and conditioned medium (CM) preparation. To explore subcellular localization and angiogenic activity of Tca HDAC, conditioned medium (CM) was prepared from subconfluent HT1080 human fibrosarcoma cells in six-well plates that were transiently transfected with 1 lg mock or pEGFP-C2-Tca HDAC plasmids (Clontech, Palo Alto, CA) using SuperFect reagent according to supplier’s protocol. Briefly, on the day before transfection, HT1080 cells were inoculated in a six-well plate to 60% confluency and cultured with minimum essential medium (MEM) supplemented with 10% fetal bovine serum at 37 C and 5% CO2 in an incubator to the time of transfection. Mock or Tca HDAC plasmids, 1 lg each, was dissolved in TE (pH 7.4) with Eagle’s MEM medium without serum to a total volume of 100 ll. SuperFect transfection reagent (5 ll) was added to the DNA solution. The samples were incubated for 15 min at room temperature. HT1080 cells were washed once with phosphate-buffered saline (PBS) and 600 ll of MEM medium with 10% FBS containing the transfection mixture was added. Then, the cells were incubated with the mixture for 3 h at 37 C and washed once with PBS and cultured for 24 h in fresh serum free medium for over-expression of Tca HDAC. The CM without serum was collected, filtered through 0.45 lm porosity filter, and concentrated by filtration (10-folds) using a ultrafiltration kit (3 kDa cutoff, Millipore). Other plasmids expressing hHDAC1, mutant Tca HDAC (H125A) were transfected equally. Completion of transiently transfection was confirmed by performing RT-PCR and Western blotting. Tube formation assay. Matrigel (150 ll in a concentration of 10 mg/ml) was placed in a 48-well culture plate and polymerized for 30 min at 37 C. About 1 · 105 cells of human umbilical vein endothelial cells (HUVECs) were seeded on the surface of the Matrigel. The morphological changes in the cells were observed under a microscope and photographed at a 100· magnification using a JVC digital camera (Victor, Yokohama, Japan). Chemoinvasion assay. The invasiveness of the HUVECs was performed in vitro using a transwell chamber system with 8.0-lm pore polycarbonate filter inserts. The lower side of the filter was coated with 10 ll gelatin (1 mg/ml), whereas the upper side was coated with 10 ll of Matrigel
Y.S. Kim et al. / Biochemical and Biophysical Research Communications 362 (2007) 995–1000 (3 mg/ml). The HUVECs (1 · 105 cells) were placed in upper part of the filter and conditioned medium was applied to lower part. The chamber was then incubated at 37 C for 18 h and then fixed with methanol and stained with hematoxylin and eosin. The cell invasion was determined by counting whole cell numbers in a single filter using optical microscopy at 100· magnification. Statistical analysis. Results are expressed as means ± standard error (SE). Student’s t test was used to determine statistical significance between control and test groups. A P value of <0.05 was considered statistically significant.
Results and discussion Tca HDAC localizes in a nucleus as human HDAC1 does To explore the biological function of TcaHDAC from prokaryote and to compare it with a eukaryotic equivalent, we first analyzed the intracellular localization of Tca HDAC. Plasmids harboring human HDAC (hHDAC1) and Tca HDAC were transfected transiently into HT1080 cells and their mRNA expression level were confirmed by RT-PCR analysis. Through indirect fluorescence analysis, hHDAC1 was predominantly converged into a nucleus as like all known mammalian class HDAC. It has been well known that the localization of hHDAC1 at nuclei is a prerequisite to exhibit biological function in the cells. Likewise, wild-type Tca HDAC exhibited a superior nuclear localization implying that Tca HDAC and the hHDAC1 may show cognate activities each other (Fig. 1A). As previously reported, Tca HDAC shows a high analogy with human HDAC1 [18]. The nuclear confinement of hHDAC1 can be accounted for two different regions responsible for its nuclear import. It possesses the N-terminal motif required for homo- and hetero-oligomerization and the C-terminal lysine-rich sequence, both of which are in charge of nuclear localization of hHDAC1. The Cterminal holds a lysine-rich nuclear localization signal (NLS) sequence that is sufficient for the nuclear translocation. The core sequence resembles monopartite c-myc NLS sequence known to interact with importin a/b complex which is well studied nucleocytoplasmic transport system [19]. hHDAC1 translocation depends on this lysine-rich sequence. Besides, the N-terminal oligomerization motif also participates in hHDAC1 translocation. This motif, called HDAC association domain (HAD), is reported to associate with hHDAC1 for homo-oligomerization and hHDAC2, hHDAC3 for hetero-oligomerization comprising various histone deacetylase complexes. This association recruits other HDACs containing NLS enabling hHDAC1 to shuttle into a nucleus. Thus, the nuclear import of HDAC1 is fulfilled by two structural features [20]. Tca HDAC whose location is focused on nucleus, on the other hand, is unlikely to obey referred both mechanisms. Aligning Tca HDAC sequence with that of hHDAC1 revealed the existence of partial N-terminal HAD but lacked C-terminal NLS sequence (Fig. 1B). Since T. caldophilus GK24 is a nucleus-free prokaryotic organism, the signal toward nuclei is obviously unnecessary. Therefore,
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a nuclear local saliency of Tca HDAC can be deduced from the existence of N-terminal HAD region which may recruit the endogenous HDACs having NLS by inducing oligomerization. Histidine residues are crucial for its deacetylasing activity of Tca HDAC As histidine residues His141 and His199 of human HDAC1 are crucial for the enzyme active, we speculated that histidine residues of Tca HDAC also important for the catalytic activity and generated the site directed mutants, histidine 125, 183 to alanine [21]. The over-expression of mutant Tca HDAC H125A and H183A, which is mutated at a conserved catalytic histidine residue (corresponding to the human HDAC1 His141 and His199), displayed significantly weaker enzymatic activity compared with the wild-type protein. As shown in Fig. 2, enzyme activity was reduced dramatically. The enzyme activity of H125A exhibited weaker activity than that of H183A unlike human HDAC1 and double mutants (H125A/H183A) did not show a synergic inactivation, implying that His125 plays a more crucial role in deacetylasing activity than His183 of Tca HDAC. Tca HDAC induces angiogenesis in vitro hHDACs play a critical role in regulating gene expression and key biological processes such as tumorigenesis and angiogenesis. In addition, it has been recently demonstrated that HDAC inhibitors inhibit angiogenesis by the suppression of the production of vascular endothelial growth factor (VEGF) from tumor cells and the direct inhibition of endothelial cell migration and proliferation [6,16,22]. To explore whether Tca HDAC confers similar biological function to hHDAC1, angiogenic activity of Tca HDAC transfectants was investigated. We transiently transfected HT1080 cells with wild Tca HDAC (wt-Tca HDAC) and mutant Tca HDAC H125A (mt-Tca HDAC) vectors in serum free medium (SFM) before collecting conditioned medium (CM). The CM was prepared in SFM, concentrated by filtration, and in vitro angiogenesis assay was performed with the CMs from wild or mutant Tca HDAC transfected cells. We then directly applied CM to HUVECs to examine the effect of CM on angiogenesis in vitro. CM of mock transfectants did not induce tube-like structures. Surprisingly, however, CM collected from wtTca HDAC transfectants enhanced the formation of capillaries, which developed elongated and complex networks (Fig. 3A). The stimulation of tube formation by Tca HDAC over-expression was not detected in CM of Tca HDAC mt-transfectants, implying that HDAC activity is required for angiogenic stimulation. To validate pro-angiogenic activity of Tca HDAC, the CM was treated on HUVECs to determine chemoinvasion. Similarly, chemoinvasion of HUVECs were significantly increased by CM of wt-Tca HDAC, but not by that of Tca HDAC mt-transfectants (Fig. 3B and C).
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Fig. 1. Subcellular localization and sequence comparison with hHDAC1 of Tca HDAC. (A) The vectors encoding full-length GFP-hHDAC1 and Tca HDACs were transiently transfected into HT1080 cells. Localization of GFP-hHDAC1 and GFP-Tca HDAC was determined by fluorescence microscopy. Each image corresponds to GFP (left), GFP-hHDAC1 (middle), GFP-Tca HDAC (right), respectively. (B) Sequence of human HDAC1 (normal) and Tca HDAC (italic) was analyzed. Matching was done using NCBI bl2seq (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). Matched parts are grayshaded. Underlined are N-terminal HAD and C-terminal NLS of human HDAC1. Asterisk marks the core of NLS. The partial N-terminal HAD of Tca HDAC is indicated in bold.
Tca HDAC over-expression oppositely modulates expression of target genes
Fig. 2. Histone deacetylase enzymatic activity of mutants Tca HDAC. Mutation of His125 and His183 to Ala in Tca HDAC reduces enzymatic activity. His125 was mutated to Ala as described under ‘‘Experimental Procedures.’’ Tca HDAC wild-type or mutants fused to a GST tag were expressed in E. coli precipitated using glutathione agarose resin.
To evaluate whether Tca HDAC modulates human HDAC target genes and angiogenesis factor, we transfected HT1080 cells with vector expressing wt-Tca HDAC or mt-Tca HDAC. In contrast to the suppressed levels of p21 and gelsolin, expression of VEGF was significantly up-regulated by wt-Tca HDAC. Compared to wt-Tca HDAC, however, mt-Tca HDAC did not induce a significant change in expression of p21, gelsolin and VEGF. mt-Tca HDAC inactivated both the deacetylation and transcriptional repression activities of Tca HDAC (Fig. 4). In this study, the biological role of prokaryotic HDAC was evaluated. Basic properties of Tca HDAC was very close to that of human HDAC1 in deacetylasing activity, nuclear localization and target gene regulation involved in progressing angiogenesis. These similarities can be
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Fig. 3. Angiogenic stimulation of Tca HDAC over-expression. HT1080 cells were transiently transfected with empty, wt-Tca HDAC or mt-Tca HDAC vectors and the conditioned medium (CM) was collected for 24 h. (A) HUVECs was incubated for 8 h in the CM on 48-well plates coated Matrigel, and cell morphology was photographed under a microscope. CM collected from HDAC transfectants stimulates tube formation in vitro. (B) Invasiveness of HUVECs was performed in vitro using transwell chambers system of 8.0-lm pores polycarbonate filter inserts for 16 h. The cells that invaded through the Matrigel-coated inserts were stained, counted and photographed under a light microscope at 100· magnification. chemoinvasion of HUVECs were significantly increased by CM of wt-Tca HDAC, but not by CM of mt–Tca HDAC transfectants. (C) The percentage of invasion was quantified as described in Experimental Design. Values are means ± SD from five different experiments (P < 0.001).
Fig. 4. Regulation of HDAC target genes by Tca HDAC over-expression. (A) HT1080 cells were transiently transfected with mock, hHDAC1, wt-Tca HDAC or mt-Tca HDAC (H125A) expression vectors, total cell lysate was isolated and immunoblot analysis was performed with each specific antibody. (B) Bar graphs display a relative induction of each band (j, gelsolin; Q, p21WAF1; h, VEGF).
deduced by a sequence homology analysis. Tca HDAC gene codes a 375 amino acid polypeptide. It has a partial N-terminal HDAC association domain (HAD) at a fore-
part of catalytic domain. As described, this domain is responsible for oligomerization of several HDACs as well as Rb-binding protein 48 (RbAp48) and Sin3A/Sin3B con-
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stituting diverse HDAC complexes [20]. The biological resemblance might result from the interaction between Tca HDAC and other HDAC complex-composing subunits via N-terminal HAD. And these complexes would facilitate the access of Tca HDAC to target proteins to trigger its catalytic activity. This conclusion enables us to infer an inborn characteristics of Tca HDAC as a bacterial gene-expression regulator in T. caldophilus GK24, the thermophilic bacteria. In addition, the structural simplicity of primitive system may satisfy the partial HAD sequence for an interaction with others, which has been supplemented in an evolutionary process. Although there is no proven HDAC activity in prokaryotes, various bacterial histone-like proteins and their regulation of gene expression were revealed [23]. Post-translational modifications of bacterial proteins such as acetylation were also reported for their biological processes [24]. Eventually, our findings suggest that function of the gene keeps intact not only among eukaryotes but also to prokaryotes and would contribute to a research ascending against an immense evolutional track. Acknowledgments This study was supported by grants from Forest Science & Technology Projects provided by Korea Forest Service and from the Brain Korea 21 Project, Republic of Korea. References [1] P.A. Marks, R.A. Rifkind, Erythroleukemic differentiation, Annu. Rev. Biochem. 47 (1978) 419–448. [2] F. Petrij, R.H. Giles, H.G. Dauwerse, J.J. Saris, R.C. Hennekam, M. Masuno, N. Tommerup, G.J. van Ommen, R.H. Goodman, D.J. Peters, Rubinstein Taybi syndrome caused by mutations in the transcriptional co-activator CBP, Nature 376 (1995) 348–351. [3] P.P. Pandolfi, Transcription therapy for cancer, Oncogene 20 (2001) 3116–3127. [4] P. Dhordain, R.J. Lin, S. Quief, D. Lantoine, J.P. Kerckaert, R.M. Evans, O. Albagli, The LAZ3 (BCL06) oncoprotein recruits a SMRT/ Msin3A/histone deacetylase containing complex to mediate transcriptional repression, Nucleic Acids Res. 26 (1998) 4645–4651. [5] J.H. Choi, H.J. Kwon, B.I. Yoon, J.H. Kim, S.U. Han, H.J. Joo, D.Y. Kim, Expression profile of histone deacetylase1 in gastric cancer tissues, Jpn. J. Cancer Res. 92 (2001) 1300–1304. [6] M.S. Kim, H.J. Kwon, Y.M. Lee, J.H. Baec, J.E. Jang, S.W. Lee, E.J. Moon, H.S. Kim, S.K. Lee, H.Y. Chung, C.W. Kim, K.W. Kim, Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes, Nat. Med. 7 (2001) 437–443. [7] N. Tsuji, M. Kobaysshi, K. Nagashima, Y. Wakisaka, K. Koizumi, A new antifungal antibiotics, trichostatin, J. Antibiot. (Tokyo) 29 (1976) 1–6.
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