460 Research Article Splicing of HDAC7 modulates the SRF-myocardin complex during stem-cell differentiation towards smooth muscle cells

Andriana Margariti*, Qingzhong Xiao*, Anna Zampetaki, Zhongyi Zhang, Hongling Li, Daniel Martin, Yanhua Hu, Lingfang Zeng‡ and Qingbo Xu Cardiovascular Division, Kingʼs College London BHF Centre, London SE5 9NU, UK *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 14 October 2008 Journal of Cell Science 122, 460-470 Published by The Company of Biologists 2009 doi:10.1242/jcs.034850

Summary Histone deacetylases (HDACs) have a central role in the alternative splicing during ES cell differentiation. Platelet- regulation of expression. Here we investigated whether derived growth factor enhanced ES cell differentiation into HDAC7 has an impact on embryonic stem (ES) cell SMCs through upregulation of HDAC7 splicing. Further differentiation into smooth muscle cells (SMCs). ES cells were experiments revealed that HDAC7 splicing induced SMC seeded on collagen-IV-coated flasks and cultured in the absence differentiation through modulation of the SRF-myocardin of leukemia inhibitory factor in differentiation medium to complex. These findings suggest that HDAC7 splicing is induce SMC differentiation. Western blots and double- important for SMC differentiation and vessel formation in immunofluorescence staining demonstrated that HDAC7 has a embryonic development. parallel expression pattern with SMC marker . In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, Supplementary material available online at overexpression of HDAC7 significantly increased β- http://jcs.biologists.org/cgi/content/full/122/4/460/DC1 galactosidase-positive cell numbers and activity, indicating its crucial role in SMC differentiation during Key words: Embryonic stem cells, 7, Smooth embryonic development. We found that HDAC7 undergoes muscle cell differentiation

Journal of Cell Science Introduction serum-response factor (SRF), myocardin, and myocyte enhancer Embryonic stem (ES) cells have the remarkable capability of factor 2 (MEF2). SRF has a key role during SMC differentiation differentiating into specific cell lineages, such as cardiovascular by activating the transcription of an array of muscle-specific genes cells, in response to different stimuli in vitro (Keller, 1995; Smith, (Cao et al., 2005). Myocardin is an SRF expressed 2001). Both endothelial and vascular smooth muscle lineages can specifically in smooth muscle and cardiomyocytes throughout develop from a common progenitor, the vascular progenitor cell embryonic development and adulthood (Wang et al., 2001). MEF2C (Yamashita et al., 2000). Vascular endothelial growth factor (VEGF) is mainly expressed in vessel SMCs throughout embryonic and platelet-derived growth factor BB (PDGF-BB) have distinct development (Lin et al., 1998). roles in such progenitor cell differentiation (Gerber et al., 1998; The homeostasis of histone acetylation and deacetylation is Hellstrom et al., 1999). In particular, VEGF increases endothelial known to have a central role in the regulation of , cell differentiation, whereas PDGF induces SMC differentiation through the modulation of assembly or disassembly (Gerecht-Nir et al., 2003). However, the underlying mechanism for and through co-operation with other transcription factors (Wu et vascular cell differentiation is still unclear. al., 2001; Yang and Seto, 2003). Histone deacetylases (HDACs) Vascular SMCs have a crucial function in the embryonic are part of transcriptional corepressor complexes and are key development and physiological maintenance of the cardiovascular regulators in the differentiation of stem cells towards a specific cell system (Gittenberger-de Groot et al., 1999). In adults, recent lineage (Kato et al., 2004; Sterner and Berger, 2000). Three experimental and human data suggest a potential contribution of different classes of human HDACs have been defined based on their vascular progenitors, which originate from the circulation and the homology to HDACs found in Saccharomyces cerevisiae (Taunton perivascular adventitia, to the differentiation of SMCs found within et al., 1996; Verdin et al., 2003). The class II HDACs: HDAC4, atherosclerotic lesions (Campbell and Campbell, 1994; Hu et al., HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10 contain both 2004; Mathur and Martin, 2004; Xu et al., 2003). Obviously, nuclear localisation and export signals, trafficking between the vascular progenitor cells that can differentiate into either endothelial cytoplasm and nucleus (Wang and Yang, 2001). Class II HDACs cells or SMCs could contribute to both vessel development in are reported to be important for cell differentiation in tissues such embryos and vascular disease in adults. Thus, the mechanisms that as muscles (Deng et al., 2005; Dressel et al., 2001; Karamboulas regulate SMC differentiation need to be elucidated. et al., 2006; Lu et al., 2000; McKinsey et al., 2000; McKinsey et SMC differentiation is a complicated and inadequately defined al., 2001; Miyake et al., 2003; Vega et al., 2004; Zhang et al., 2002). process. A number of factors are involved in this process, such as HDAC7 is a member of the human class II HDAC family. However, HDAC7 in ES cell differentiation 461

there is no reported direct link between HDAC7 and SMC 5-7 days in culture, the expression of HDAC7, SMA and calponin differentiation. This study aimed to investigate the role of HDAC7 was increased in a similar pattern (Fig. 1A). In addition, high levels in SMC differentiation from ES cells. We found that HDAC7 of these proteins were detected in mature SMCs (Fig. 1A). Similar expression and alternative splicing correlated with ES cell results were also obtained at the RNA level, showing a parallel differentiation towards SMCs. expression of HDAC7 and SMC markers (Myocardin, SMA, SM22a, calponin and SMMHC) (Fig. 1B). Double- Results immunofluorescence staining showed that most HDAC7-positive HDAC7 is upregulated during SMC differentiation cells were also positive for SMA and calponin. HDAC7 colocalized Our previous study demonstrated that laminar flow induced ES cell with SMA or calponin in the cytoplasm of differentiated SMCs and differentiation toward endothelial cells, and highlighted the was scarcely apparent within the nucleus (Fig. 1C). By contrast, a involvement of HDAC3 (Zeng et al., 2006). We found that there high number of mature SMCs exhibited positive HDAC7 staining, was a concomitant decrease of HDAC7 and SMC marker expression found mainly in the nucleus (Fig. 1D). Moreover, enforced by shear stress (supplementary material Fig. S1). Thus, we wondered expression of HDAC7 by adenoviral gene transfer induced SMA whether HDAC7 was important during ES cell differentiation, and calponin expression in a dose-dependent manner in especially towards SMCs. Experiments were performed to elucidate differentiated ES cells (Fig. 1E). These results suggest that HDAC7 a potential link between HDAC7 and SMC marker expression. is involved in ES cell differentiation toward SMCs. ES cells were seeded onto collagen-IV-coated flasks and cultured in the absence of leukaemia inhibitory factor (LIF) for 1 to 9 days HDAC7 is necessary for SMC differentiation to stimulate SMC differentiation, as previously described (Xiao et To test whether HDAC7 is necessary for SMC differentiation, al., 2007). Western blot analysis showed that HDAC7, smooth HDAC7 siRNA knockdown experiments were performed. As muscle actin (SMA) and calponin were expressed at very low levels expected, HDAC7 siRNA downregulated the expression of SMC in undifferentiated and early stage differentiating ES cells. Following markers such as SMA, calponin and SMMHC at the protein level Journal of Cell Science

Fig. 1. Expression of HDAC7 in ES cells correlates with SMC differentiation. (A) HDAC7, SMA and calponin were expressed in a similar pattern during ES cell differentiation. (B) Real-time PCR showing that the SMC gene markers are upregulated in parallel with HDAC7. (C) Double immunostaining showing colocalization of HDAC7 with SMA or calponin in the cytoplasm in differentiated ES cells. (D) HDAC7 mainly localized in the nucleus in mature SMCs. Scale bars: 50 μm. (E) Enforced expression of HDAC7 by adenoviral gene transfer increased SMA and calponin expression in differentiated ES cells. MOI, multiplicity of infection. The data presented are representative of three independent experiments. 462 Journal of Cell Science 122 (4)

(Fig. 2A) and RNA level (SMA, SM22a, calponin, SMMHC) (Fig. increased levels of HDAC7 correlated with enhanced expression 2B). HDAC7 short hairpin (sh) RNA lentiviral plasmid transfer was of SMA, and β-galactosidase (Fig. 3B, left panel). Moreover, also used to confirm the downregulation of HDAC7. As shown in HDAC7 siRNA downregulated protein levels (Fig. 3B, right panel). Fig. 2A (right panel), shRNA lentiviral plasmid suppressed HDAC7 These results suggest that HDAC7 is involved in SMC expression, concomitant with that of the SMC markers. SMA-Luc differentiation during embryonic development. and SM22-Luc reporter assays also demonstrated downregulation of promoter activity after knockdown of HDAC7 (Fig. 2C). HDAC7 undergoes splicing during ES cell differentiation To facilitate this study, HDAC7 was amplified and cloned from HDAC7 is involved in SMC differentiation during embryo predifferentiated ES cell mRNA by RT-PCR. Interestingly, most of development the clones contained an intron of 57 bases between the start codon, Next, we examined whether HDAC7 played a crucial role in SMC ATG and the second ATG codon, which contained three stop codons differentiation during embryonic development. Cells were isolated disrupting the open reading frame from the first start codon. This from SM22-LacZ mice at embryonic day (E)10-12 (expressing LacZ resulted in alternative translation from the second ATG codon, gene under the control of SM22 promoter) and cultured on collagen- giving rise to a short HDAC7 isoform lacking the first 22 N-terminal IV-coated plates or slides in differentiation medium. The effects of amino acids (Fig. 4A). HDAC7 overexpression by adenoviral gene transfer and siRNA To investigate whether HDAC7 underwent alternative splicing knockdown on SMC differentiation were determined. X-gal staining during ES cell differentiation, mRNA was extracted from showed that Ad-HDAC7 markedly increased the number of positive differentiating cells at various time points and RT-PCR was cells and the staining intensity in single cells (Fig. 3A, left panel). conducted using specific primers flanking the intron sequence (Fig. The dye was then eluted and quantified relative to protein content. 4B). Undifferentiated ES cells expressed only the HDAC7 isoform Statistical analysis showed a significant increase in X-gal staining containing the intron, whereas culture on collagen-IV-coated plates in Ad-HDAC7-infected cells compared with that in control cells in the absence of LIF for 3 days resulted in the appearance of a (Fig. 3A, right panel). Western blot analysis further confirmed that spliced isoform lacking the intron. The ratio of spliced to unspliced isoform increased as the differentiation process proceeded (Fig. 4B). Interestingly, mature SMCs mainly expressed the spliced HDAC7 isoform (Fig. 4B), suggesting that HDAC7 splicing is correlated Journal of Cell Science

Fig. 3. HDAC7 is involved in SMC differentiation during embryonic development. (A) Overexpression of HDAC7 increased SM22 expression in SM22-LacZ embryonic cells as revealed by X-gal staining (left panel; scale Fig. 2. HDAC7 is necessary for SMC differentiation. HDAC7 siRNA (A, left bar: 200 μm), and its statistical analysis (right panel; *P<0.05). panel) and shRNA lentiviral plasmid transfer (A, right panel) downregulated (B) Overexpression of HDAC7 upregulated SMC marker induction whereas expression of SMC markers such as SMA, calponin and SMMHC at the HDAC7 siRNA decreased SMC marker expression in SM22-LacZ embryonic protein level, and mRNA level in real-time PCR experiments (B), and SMA- cells. 20 μg and 50 μg of protein were applied to a western blot in Ad-HDAC7 Luc, SM22-Luc reporter gene expression (C) in ES cells. RLU, relative and siRNA experiments, respectively. β-gal was included as an indicator for luciferase unit. The data presented are representative or means (± s.e.m.) of SM22 expression. The data presented are representative or means (± s.e.m.) of three independent experiments. three independent experiments. HDAC7 in ES cell differentiation 463

Transient co-transfection assays revealed that only the spliced HDAC7 isoform stimulated SMA and SM22 reporter gene expression in differentiated ES cells; the unspliced isoform had little impact, whereas the short isoform exerted an inhibitory effect (Fig. 5D). In contrast to ES cells, both the unspliced and spliced HDAC7 vectors stimulated reporter gene expression in mature SMCs, the effect of which was most prominent in the spliced vector. Unspliced HDAC7 was seen to have this effect as a result of splicing in mature SMCs (Fig. 5E). These results indicate that HDAC7 splicing is necessary to induce SMC differentiation from ES cells. Further experiments were performed to assess the deacetylase activities of the short and full-length HDAC7. ES cells were infected with Ad- tTA, Ad-HDAC7-1, Ad-HDAC7-2, and the deacetylase activity was detected 48 hours later. Both isoforms maintained their deacetylase activity at similar levels (Fig. 5F), indicating that lack of the 22 amino acids in the N-terminal does not affect the deacetylase activity of the short of HDAC7, and suggesting that the 22 amino acids may exert other functions. Furthermore, we wondered whether the short HDAC7 (HDAC7- 2) had any effect on the function of the full-length HDAC7-1. ES cells were cotransfected with pShuttle2-HDAC7-1 expressing only Flag tag and different amounts of pShuttle2-HDAC7-2. Data in Fig. 5G demonstrate that HDAC7-2 suppressed HDAC7-1-induced SM22-Luc reporter gene expression in a dose-dependent manner. Additionally, overexpression of HDAC7-2 did not affect the HDAC7-1 protein level (Fig. 5G, lower panel). These results indicate that HDAC7-2 has a dominant-negative effect on spliced Fig. 4. HDAC7 undergoes splicing during ES cell differentiation into SMCs. HDAC7-mediated gene expression in SMCs. (A) Schematic illustration of HDAC7 exon 1 sequence. An additional 57 bp intron (italic and bold) exists downstream of the first ATG codon (underlined PDGF-BB-enhanced SMC differentiation is mediated through and bold). The three stop codons (italic, bold within rectangle) in the intron disrupt the open reading frame, resulting in the alternative translation from the HDAC7 second ATG (bold), giving rise to a short HDAC7 lacking 22 amino acids As PDGF was reported to be a key factor in SMC differentiation, (underlined and bold), compared with the spliced one, in which the intron is we hypothesized that there might be a link between PDGF removed. (B,C) RT-PCR showing HDAC7 splicing in both undifferentiated stimulation and HDAC7 induction. Indeed, PDGF upregulated and differentiated ES cells (B) and in different passages of ES-derived SMCs Journal of Cell Science (esSMC) (C). Mature SMCs were used as a positive control; β-actin was used HDAC7, SMA and calponin protein levels in a similar manner (Fig. as internal control. The ratio of spliced to unspliced HDAC7 is shown in the 6A). Moreover, PDGF enhanced the expression of HDAC7 reporter right panel. u, unspliced isoform; s, spliced isoform. The data presented are genes in both differentiated ES cells and SMCs (Fig. 6B), indicating representative or means (± s.e.m.) of three independent experiments. that PDGF induces HDAC7 transcription. Further experiments were performed to detect the effect of PDGF on HDAC7 splicing. PDGF significantly enhanced the effect of the with SMC differentiation. This notion was further supported by the unspliced HDAC7 plasmid on SM22 reporter gene expression in fact that increased levels of the spliced isoform of HDAC7 were ES cells to a level comparable with that of the spliced plasmid (Fig. observed in ES-derived SMCs during progressive stages of 6C), suggesting that PDGF increases HDAC7 splicing. RT-PCR differentiation (Xiao et al., 2007; Xiao et al., 2006), as indicated indicated that splicing of HDAC7 increased significantly under by RT-PCR analysis (Fig. 4C). PDGF treatment (Fig. 6D). To confirm this, enforced expression of unspliced HDAC7 was introduced to ES cells by adenoviral gene HDAC7 splicing promotes ES cell differentiation toward SMCs transfer, followed by PDGF treatment. As shown in Fig. 6E, the To further investigate the role of HDAC7 splicing in ES cell Flag-tagged HDAC7 (associated with expression of the spliced differentiation, three different HDAC7 cDNAs were cloned (Fig. isoform), was highly upregulated after PDGF treatment. No 5A) into the modified pShuttle2-Flag-HA vector, designated significant differences were observed in HDAC7 expression levels pShuttle2-HDAC7, pShuttle2-HDAC7-1, pShuttle2-HDAC7-2, in the presence of PDGF, which may be due to overexpression of respectively. The pShuttle2-HDAC7 is the unspliced isoform, exogenous HDAC7 (Fig. 6E). Finally, HDAC7 siRNA ablated containing the intron and giving rise to a short HA-tagged HDAC7 PDGF-BB-induced SM22-Luc reporter gene expression in lacking the first 22 amino acids, as pShuttle2-HDAC7-2 does. differentiated ES cells (Fig. 6F). Taken together, these results suggest However, after splicing, pShuttle2-HDAC7 will give rise to both that PDGF may promote ES cell differentiation into SMCs through Flag and HA-tagged full-length HDAC7, as pShuttle2-HDAC7-1 upregulation of HDAC7 transcription and splicing. does (Fig. 5B). When the unspliced HDAC7 vector was transferred into mature SMCs by adenoviral gene transfer, both Flag and HA HDAC7 isoform lacking the first 22 amino acids degrades tags were detected, indicating that HDAC7 underwent splicing in MEF2C via the proteasome mature SMCs (Fig. 5C). Also, the spliced HDAC7 isoform was To test whether different HDAC7 isoforms possess different cellular cloned into a pShuttle vector lacking the HA tag. localization, the HDAC7-2 and HDAC7-1 vectors were introduced 464 Journal of Cell Science 122 (4)

Fig. 5. Different HDAC7 isoforms exert different effects on SMC differentiation. (A) A schematic illustration of the location of HDAC7 primers. (B) A schematic illustration of the cloned HDAC7 splicing isoforms. (C) Western blot confirmation of the HDAC7 isoform clones. (D,E) Luciferase reporter analysis showing the different effect of HDAC7 isoforms on SMA-Luc and SM22-Luc reporters in differentiated ES cells (D) and in mature SMCs (E). *P<0.05. (F) Both HDAC7 isoforms possess similar deacetylase activity. (G) Luciferase reporter analysis shows that the short HDAC7 suppresses HDAC7-1- induced SM22 reporter gene expression in a dose-dependent manner in ES cells. ES cells were cotransfected with 0.5 μg pShuttle2- HDAC7-1, and 0, 0.5, 1.0 1.5 μg pShuttle2-HDAC7-2. Control plasmid was used to compensate the total plasmid amount. HA and Flag represent the expression of the short and spliced forms, respectively. RLU, relative luciferase unit. The data presented are representative or means (± s.e.m.) of nine independent experiments for D,E, and three for C,F,G.

into SMCs by adenoviral gene transfer. As shown in Fig. 7A, the The decrease in MEF2C induced by the short HDAC7 isoform could

Journal of Cell Science short HDAC7-2 mainly localized to the cytoplasm, whereas the be ablated by the presence of proteasome inhibitor MG-132 (Fig. spliced form localized to both the cytoplasm, and more abundantly, 7F). Further experiments showed that overexpression of HDAC7- to the nucleus (Fig. 7B). To confirm this result, the cytoplasmic 2 increased the ubiquitylation of MEF2C (Fig. 7G). These results and nuclear components were fractionated (Fig. 7C). Interestingly, suggest that the short HDAC7 modulates MEF2C through overexpression of the short HDAC7 increased the accumulation of proteasome-mediated degradation and has no effect on MEF2C endogenous HDAC7 (mainly spliced) in the nucleus of SMCs (Fig. cellular compartmentalization. 7C). These data suggest that different HDAC7 isoforms exist in different cellular compartments and might have different interactions HDAC7 splicing induced SMC differentiation through with other proteins, such as transcription factors. modulation of the SRF-myocardin complex To elucidate whether different HDAC7 isoforms have different Further experiments were performed to detect the effects of HDAC7 affinities to transcription factors, we determined the effect of spliced isoforms on SRF, another key transcription factor for SMC and short forms of HDAC7 on MEF2C, a transcription factor for differentiation. Both HDAC7 isoforms did not suppress SRF at SMC differentiation. MEF2C protein level was significantly either the protein (Fig. 8A) or RNA (supplementary material Fig. decreased in the presence of the short HDAC7, but not in the S3) level. In contrast to MEF2C, spliced HDAC7 bound to SRF presence of the spliced HDAC7 isoform (Fig. 7D) (supplementary whereas short HDAC7 did not (Fig. 8A). It is reported that SRF material Fig. S2). However, the cellular localization of MEF2C activates genes involved in SMC differentiation and proliferation seemed unchanged under HDAC7-1 and HDAC7-2 overexpression by recruiting myocardin to form a complex in the SMC marker (supplementary material Fig. S2). gene promoter region (Wang et al., 2001). To test whether the To discover how HDAC7-2 modulates MEF2C levels, we then different HDAC7 isoforms affected the recruitment of SRF and performed RT-PCR in cells overexpressing HDAC7-1 and HDAC7- myocardin in the SMC marker gene promoter region, chromatin 2. The data shown in supplementary material Fig. S3 indicate that immunoprecipitation (ChIP) assays were performed. Spliced neither HDAC7-1 nor HDAC7-2 has any effect on MEF2C mRNA HDAC7 increased myocardin binding to SM22 and the calponin levels, suggesting that HDAC7-2 decreases the MEF2C protein level (CNN1) gene promoters, whereas the short form decreased both through post-translational degradation. Co-immunoprecipitation SRF and myocardin binding to these promoters (Fig. 8B). A deletion experiments in SMCs revealed that the short HDAC7 isoform bound of the CArG box in the SM22 promoter abolished HDAC7-1- to MEF2C, whereas the spliced isoform did not (Fig. 7E). Similar induced SM22-Luc reporter gene expression (supplementary results were obtained in differentiated ES cells (data not shown). material Fig. S4). These findings suggest that the spliced HDAC7 HDAC7 in ES cell differentiation 465

Fig. 6. HDAC7 is essential for PDGF-BB-promoted SMC differentiation. (A) HDAC7 expression upregulated by PDGF treatment coincided with SMA and calponin induction in ES cells. (B) PDGF treatment for 24 hours increased HDAC7 transcription in both ES and SMC cells. (C) PDGF stimulation for 24 hours enhanced the effect of unspliced HDAC7 isoform on SM22-Luc reporter P Journal of Cell Science gene expression in ES cells, * <0.05. (D) RT-PCR showing that PDGF treatment for 24 hours increased HDAC7 splicing in differentiated ES cells. The ratio of spliced to unspliced band intensity was calculated and presented in lower panel. u, unspliced isoform; s, spliced isoform. (E) Western blot analysis showing that PDGF treatment increased HDAC7 splicing. The Fig. 7. The short HDAC7 isoform lacking the first 22 amino acids binds and unspliced HDAC7 was introduced into ES cells by adenoviral gene transfer at degrades MEF2C. Short HDAC7 localizes in the cytoplasm (A) and spliced MOI 20, and the spliced HDAC7 isoform was demonstrated by the presence of HDAC7 localizes in the nucleus (B) in SMCs after adenoviral gene transfer. both Flag and HA tags. (F) HDAC7 siRNA ablated PDGF-BB-induced SM22- Scale bars: 50 μm. (C) Fractions of cytoplasm and nucleus confirmed the Luc reporter gene expression in ES cells. The data presented are representative cellular localization of the HDAC7 isoforms. (D) MEF2C protein level was or means (± s.e.m.) of three independent experiments. significantly decreased in the presence of the short HDAC7 but not in the spliced HDAC7; *P<0.05. (E) Immunoprecipitation experiments show that MEF2C binds to short HDAC7 but not to spliced HDAC7. (F) MG-132 stimulates SM22 and calponin gene transcription during SMC ablated short HDAC7-induced MEF2C degradation. (G) Immunoprecipitation differentiation by increasing the recruitment of myocardin to the experiments demonstrate that short HDAC7 increases MEF2C ubiquitylation. CArG box in the promoter region. The data presented are representative or averages of six independent As the spliced HDAC7 could associate with SRF, we wondered experiments (A,B,C,D,E) or three independent experiments for F,G. whether HDAC7 was also recruited to the promoter and affected the histone acetylation status. ChIP assays did not show HDAC7 binding to the promoters (data not shown). Unlike the short upregulation of HDAC7 transcription and splicing. Moreover, we HDAC7, overexpression of spliced HDAC7 did not suppress the provide evidence that the additional 22 amino acids in the N- acetylation of Lys9 and dimethylation of Lys4 on Histone 3 tails terminus of spliced HDAC7 affect its cellular localization and (AcH3 and H3K4DM) in the promoter regions of SM22 and the influence the affinity of HDAC7 for associated proteins. These calponin gene in ES cells (Fig. 8C). findings enhance our knowledge of SMC differentiation and provide useful insights into the mechanisms of vessel development. Discussion We observed a clear correlation between HDAC7 expression and Here, we demonstrated for the first time that HDAC7 undergoes SMC differentiation from ES cells or ex vivo cultures of embryonic alternative splicing during ES cell differentiation, and mediates SMC cells. Upregulation of HDAC7 expression increased the expression differentiation through modulation of the SRF-myocardin complex. of SMC markers, whereas downregulation significantly decreased PDGF-BB promotes ES cell differentiation into SMCs through the SMC marker expression, indicating a crucial role for HDAC7 in 466 Journal of Cell Science 122 (4)

SMC differentiation. A recent study showed that HDAC7-knockout mice had fewer SMA-positive cells in the vessel wall of embryos, and the staining of SMA in the dorsal aorta was less prominent. Moreover, fewer than normal SMCs were observed surrounding dorsal aortae in the knockout embryos at E11, indicating that HDAC7 has an essential role in SMCs. However, in the same study, the histological sections of mouse embryos harbouring the HDAC7 mutation expressing LacZ did not show β-galactosidase staining in SMCs of the ascending aorta (Chang et al., 2006). This discrepancy might arise from the deletion of intron 2. It is possible that there are a number of important regulatory enhancer- like regions existing in intron 2 (19,729 bp), which are necessary for HDAC7 expression in SMCs. An important finding of this study is that HDAC7 undergoes alternative splicing during ES cell differentiation into SMCs. HDAC7 mRNA from undifferentiated ES cells contains a 57 bp intron, in which three stop codons disrupt the open reading frame from the initiation ATG codon, resulting in alternative translation from a second ATG codon and giving rise to a short HDAC7 isoform lacking 22 N-terminal amino acids. During differentiation towards the SMC lineage, the intron is excised, giving rise to the full-length HDAC7 protein. Mature SMCs predominantly expressed the spliced HDAC7 isoform. The removal of 22 amino acid residues is likely to alter the conformation of HDAC7 and therefore alter its interactions with other proteins. HDAC7 is a shuttle protein, trafficking between the cytoplasm

Journal of Cell Science and the nucleus during cell differentiation (Kao et al., 2001; Karvonen et al., 2006). The full- length HDAC7 (spliced isoform) exists in both Fig. 8. HDAC7 splicing induces SMC differentiation through modulation of the SRF-myocardin complex. (A) SRF associated with the spliced HDAC7, but not with the short isoform. (B) ChIP the cytoplasm and nucleus in mature SMCs. assay shows that the spliced HDAC7 increased the binding of myocardin to SM22 and calponin However, the short HDAC7 protein, which is promoters. Upper panel, representative image of PCR detection; Lower panel, statistical analysis of translated from unspliced HDAC7 mRNA, four independent experiments (mean ± s.e.m.); *P<0.05. (C) ChIP assay reveals that the spliced predominantly exists in the cytoplasm. One HDAC7 does not suppress histone modification (AcH3 and H3K4DM; histone acetylation and SM22 possible explanation is that the short HDAC7 has dimethylation, respectively) in the and calponin gene promoters. However, in the presence of the short HDAC7 isoform, both histone modifications were dramatically decreased. Upper panel, a higher affinity for the anchorage proteins in the representative image of PCR detection; lower panel, statistical analysis of four independent cytoplasm, but a lower affinity for trafficking- experiments (mean ± s.e.m.). Fold relative binding was defined as the ratio of band intensity in associated motor molecules than the spliced ChIP samples to that in Input sample, with that of Ad-tTA group set as 1.0. isoform. Thus, the overexpression of the short HDAC7 isoform occupies all the binding sites of the anchorage proteins, driving the accumulation of endogenous SMCs with PDGF-BB dramatically reduced SMC α-actin synthesis spliced HDAC7 in the nucleus. However, the association of HDAC7 in the prolonged absence of serum (Holycross et al., 1992), with anchorage proteins might be necessary for HDAC7 activation. suppressing SMC differentiation (Wang et al., 2004). Our Hence, during overexpression of short HDAC7, endogenous experiments showed that SMC differentiation from ES cells was HDAC7 accumulates in the nucleus, but is inactive. Importantly, enhanced through short stimulations with PDGF-BB (within 24 both isoforms maintain their deacetylase activity, indicating that hours) in the absence of serum, indicating that the duration of serum the N-terminus is responsible for the different localizations and deprivation and the proliferation stage of cells are important functions. parameters in SMC differentiation mediated by PDGF-BB. This Previous reports identified PDGF-BB as a key regulator for SMC highlights the fact that PDGF-BB has different functions in mature differentiation (Nishishita and Lin, 2004). Recent reports indicate SMCs and ES cells. In our previous study, proteomic analysis that vascular progenitor cells are able to differentiate into indicated that ES-derived SMCs are different from mature SMCs, contractile-type SMCs in the absence of VEGF or into synthetic- and that PDGF-BB increased SMC marker gene expression (Yin type SMCs in the presence of PDGF-BB, respectively (Miyata et et al., 2006). Therefore, it seems that PDGF-BB can initiate and al., 2005). However, reports have also shown that treatment of enhance stem or progenitor cell differentiation towards SMCs HDAC7 in ES cell differentiation 467

through regulation of SMC-specific transcription factors. In this study, we showed that PDGF-BB induced SMC differentiation through upregulation of HDAC7 transcription and its alternative splicing. Similarly to other HDACs, HDAC7 can also associate with transcription factors and other HDACs, modulating the acetylation status of transcription factors. Different isoforms might have different affinities for these proteins, eliciting different roles during ES cell differentiation. MEF2C is a key transcription factor for SMC differentiation. MEF2C has a specific temporal and spatial expression in the embryo and participates in vascular development; mice lacking MEF2C have no differentiated SMCs in the vasculature (Lin et al., 1998). HDAC7 represses MEF2C-dependent transcription via a physical interaction with the MADS domain of MEF2C. This was mapped to residues 72-172 of HDAC7, a region conserved among three classes of HDACs: HDAC4, HDAC5 and HDAC7 (Dressel et al., 2001; Kao et al., 2001). In our study, spliced HDAC7 does not bind to MEF2C and has no effect on the MEF2C protein level. However, the short HDAC7 binds to MEF2C and induces MEF2C degradation via the proteasome. The short HDAC7 protein might deacetylate MEF2C and expose lysine residues for ubiquitylation, because the ubiquitylation of MEF2C was increased in cells overexpressing HDAC7-2. Enhanced ubiquitylation was also observed in the input samples after HDAC7 treatment. Ubiquitylation and acetylation can occur at the same lysine residues Fig. 9. HDAC7 splicing induces SMC differentiation by modulating the SRF- in target proteins, thus acetylation will block the lysine residue, myocardin complex. During the early stages, HDAC7 is expressed as a preventing ubiquitylation. Overexpression of HDAC7 might partially spliced form lacking the first 22 amino acids. This short HDAC7 isoform binds to MEF2C, leading to MEF2C degradation via the proteasome. deacetylate these acetylated lysine residues, exposing them for The short HDAC7 also prevents spliced HDAC7 activation in the cytoplasm. ubiquitylation. Therefore, ubiquitylated proteins will be increased The overall effect prevents smooth muscle gene expression, favouring in the presence of HDAC7. progenitor cell differentiation towards other cell lineages. When triggered by SRF is a key transcription factor in SMC differentiation, and PDGF or other stimuli, HDAC7 undergoes splicing, predominantly localises to the nucleus and possesses a higher affinity to SRF. HDAC7 modifies SRF, it binds to CArG box DNA to recruit downstream accessory factors increasing its binding to the SM22 promoter and recruiting myocardin. Thus, to regulate SMC transcription (McDonald et al., 2006). The the overall effect is to drive SMC marker gene expression and cell coactivator myocardin interacts with SRF through a basic and differentiation towards a SMC lineage. HDAC7u, unspliced HDAC7; HDAC7s, spliced HDAC7. Journal of Cell Science glutamine-rich domain near the N-terminus (Wang et al., 2004) and increases association of SRF with methylated histone and CArG box chromatin during activation of SMC gene expression (McDonald et al., 2006). Moreover, myocardin might also utilize short HDAC7 increase indirectly the deacetylation and a specific epigenetic element (H3K4dMe) to control SRF dimethylation of histone H3 in SM22 and calponin promoter, association with CArG box chromatin (McDonald et al., 2006). because this isoform does not localize to the nucleus? Spliced HDAC7 binds to SRF and increases the recruitment of In summary, HDAC7 mRNA undergoes alternative splicing myocardin to SRF. ChIP assays clearly show the increase of during ES cell differentiation towards a SMC lineage. At an early myocardin binding to SM22 and calponin gene promoters in cells stage, HDAC7 is expressed as a partially spliced isoform, which overexpressing HDAC7-1. The deletion of the CarG boxes contains a 57 bp intron altering the open reading frame from the abolished HDAC7-1-induced SM22-luc reporter gene expression, primary ATG codon. This gives rise to a short HDAC7 isoform, suggesting the involvement of SRF in HDAC7-mediated SMC transcribed from the second ATG codon and resulting in an HDAC7 differentiation. These findings support the idea that splicing of isoform lacking the first 22 amino acids. When triggered by PDGF HDAC7 induces SMC marker expression through modulation of or other stimuli, HDAC7 mRNA undergoes splicing to remove the the SRF-myocardin complex. The short HDAC7 isoform intron, giving rise to full-length HDAC7. The spliced HDAC7 will significantly suppressed the binding of SRF and myocardin to the be activated in the cytoplasm and translocated into the nucleus, SM22 and calponin gene promoters, although endogenous spliced where it associates with and modulates SRF, increasing SRF HDAC7 accumulated in the nucleus in the presence of exogenous binding to myocardin and the recruitment of SRF-myocardin short HDAC7. This provides further evidence that short HDAC7 complex to the SM22 promoter. Thus, the overall effect is to drive prevents HDAC7 activation in the cytoplasm, but how HDAC7 SMC marker gene expression and cell differentiation towards an is anchored and activated in cytoplasm needs more detailed SMC lineage. By contrast, the short HDAC7 isoform binds to investigation. MEF2C, leading to MEF2C degradation via the proteasome. The Moreover, future studies are necessary to shed light on the binding short HDAC7 is predominantly localized in the cytoplasm, which of SRF to spliced HDAC7, and to provide answers to a number of may prevent activation of spliced HDAC7 in the cytoplasm. The questions such as: (1) does spliced HDAC7 deacetylate the lysine overall effect prevents SMC gene expression, favouring progenitor residues of SRF, altering the conformation of SRF and increasing cell differentiation towards other cell lineages. This hypothesis is the affinity of SRF to myocardin? (2) How does the exogenous illustrated in Fig. 9. 468 Journal of Cell Science 122 (4)

Vascular SMCs have a crucial role in both physiological RNA extraction and reverse transcriptase-polymerase chain maintenance of the cardiovascular system during embryonic reaction (RT-PCR) Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the development, and in the pathophysiology of vascular diseases, such manufacturer’s protocol. 2 μg RNA were reverse transcribed into cDNA with random as atherosclerosis, in adults (Gittenberger-de Groot et al., 1999; Hu primer by MMLV reverse transcriptase (RT) (Promega). 50 ng cDNA (relative to et al., 2002; Liu et al., 2004). In this study, we found that alternative RNA amount) was amplified by standard PCR with Taq DNA polymerase (Invitrogen) splicing of HDAC7 has an important role in determining ES cell and primers. HDAC7 differentiation towards a SMC lineage. Thus, targeting Plasmid construction splicing will provide a new therapeutic strategy for intervention in Full-length mouse HDAC7 cDNA fragment was obtained by RT-PCR amplification vascular diseases. from 3 day pre-differentiated ES cell RNA with the primer set HDAC7c-1 and HDAC7c-2 (Fig. 6A; supplementary material Table S1), and subcloned into KpnI/XbaI Materials and Methods sites of the modified pShuttle2-Flag-HA vector, designated pshuttle2-HDAC7. The spliced HDAC7, designated as pshuttle2-HDAC7-1 and the short HDAC7, designated Materials as pshuttle2-HDAC7-2 isoforms were created by PCR-based mutagenesis with specific Goat anti-HDAC7 (C-18, sc-11491), rabbit anti-HDAC7 (sc-1142), goat anti-MEF2C primer sets (Fig. 6A; supplementary material Table S1). HDAC7, SM22 and SMA gene (sc-13268), rabbit ant-Ub (FL-76, sc-9133), goat myocardin (sc-34238), rabbit anti- promoter sequences were amplified from human genomic DNA as follows: 458 bp SRF (sc-335) and rabbit anti-histone H4 (H-97, Sc-10810)] antibodies were purchased HDAC7, 5-AAGCCAGCAAGATCCTCATTG-3 and 5-ACAGATGGCCGT- from Santa Cruz Biotech (Santa Cruz, CA). Mouse anti-α-tubulin (Clone B-5-1-2, GAGGTCATG-3 (Acc. No. AK036586); 415 bp SMA 5-ACGGCCGCCTC- T 5168), rabbit-anti-HDAC7 (KG-17, H 2662), mouse anti-HA (Clone HA-7, CTCTTCCTC-3 and 5-GCCCAGCTTCGTCGTATTCC-3 (BC064800); 507 bp H9658) and monoclonal anti-HA-agarose-conjugated antibody (A2095) were from SM22, 5-GCAGTCCAAAATTGAGAAGA-3 and 5-CTGTTGCTGCC CAT - Sigma (St Louis, MO). Two sets of antibodies against calponin (Sigma, C2687 and TTGAAG-3 (BC003795); 384 bp MHC, 5-AGGCAGACCTCATGCAGCTC-3 and Abcam, ab46794) and smooth muscle myosin heavy chain (SMMHC) (Sigma, M7786 5-GAGCTTGGCTTTGACAGCAC-3 (BC026142); 343 bp-actin, 5-CA- and AbD Serotec, AHP1117) were used in this study. Antibodies against monoclonal anti-α smooth muscle actin (SMA) (Sigma, Clone 1A4, A5228), and Flag (F9291) CAACTGGGACGACATGGAG-3 and 5 -TTCATGAGGTAGT CAGTCTGG-3 were from Sigma. Antibody against β-galactosidase (beta-Gal) (rabbit) was from Delta (M12481); 842 bp Calponin, 5 -TAACCGA GG TCCTGCCTACG-3 and 5 - Biolabs. Antibodies against acetyl histone H3 and dimethylated Lys4 histone H3 were TGTGGGTGGGCTCACTCAGC-3 (Z19542); 1055 bp HDAC7-P, 5 -CTAGA- from Upstate. All antibodies were raised in the mouse except those indicated. All CAAGCTTACAGAGAGAGGGAGCAGG-3 and 5 -CACTCCCTCGAGGACA - HDAC7 SM22 secondary antibodies were from Dakocytomation, (Glostrup, Denmark). The GTCTGTGGCTG-3 (AC004466). , and SMA gene promoter deacetylase activity was detected with an HDAC activity assay kit (colorimetric) sequences were amplified from mouse genomic DNA as follows: 1113 bp SMA-P, (ab1432) from Abcam. 5-TGCATGAGCCGTGGGAG-3 (16-32 bp); 5-ACTTA CCCTGACAGCGAC-3 (1128-1111 bp) (M57409); 1350 bp SM22-P, 5-TTCAGGACGTAATCAGTG-3 (4- Cell culture 21 bp); 5-AGCTTCGGTGTCTGGGCTG-3 (1371-1353 bp) (AH003214). Mouse ES cells (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA) were cultured Promoter sequences were cloned into pGL3-luc basic vector (Promega), designated in gelatin-coated flasks in Dulbecco’s modified essential medium (DMEM) (ATCC) pGL3-HDAC7-Luc, pGL3-SM22-Luc and pGL3-SMA-Luc, respectively. SM22 supplemented with 10% fetal bovine serum (FBS) (ATCC), 10 ng/ml LIF (Chemicon), reporter construct with deleted CArG-boxes was generated (Primer set: forward, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin in a 5-ACCGGAAAGACACCAAGTTGG-3; reverse, 5-ACCAGCCTGTGTGGAG- humidified incubator supplemented with 5% CO2, split at a 1:6 ratio every other day. TGAG-3 ). All the constructs were verified by DNA sequencing. Cell passages 3-20 were used in this study. Mature SMCs were isolated from mouse aorta as described previously (Leitges et al., 2001), and maintained in DMEM Transient transfection supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, split at For transient transfection, the ES cells were cultured on collagen-IV-coated 12-well a 1:3 ratio every 3 days. HEK293 cell line was purchased from ATCC and cultured plate for 3 days, then transfected with reporter gene (0.33 μg/well) alone or together according to the company’s recommendation. with expression plasmid (0.16 μg/well), using Fugene-6-Reagent (Roche Molecular Biochemicals), according to the manufacturer’s instructions. Renilla luciferase (0.1 Journal of Cell Science ES cell differentiation μg/well) was included in all transfection assays as internal control. The modified For differentiation, ES cells were seeded on mouse collagen IV (5 μg/ml)-coated pShuttle2 vector was used as mock control. Luciferase and Renilla activities were flasks or plates in differentiation medium [DM, MEM alpha medium (Gibco) detected 48 hours after transfection using a standard protocol. Relative luciferase unit supplemented with 10% FBS (Gibco, lot 3095073K), 0.05 mM 2-mercaptoethanol, (RLU) was defined as the ratio of Firefly versus Renilla with that of the control (set 100 U/ml penicillin, and 100 μg/ml streptomycin] for 1 to 9 days before further as 1.0). For verification of HDAC7 expression vectors, HEK293 cells were transfected treatment. The medium was refreshed every other day. Shear experiments were with 1 μg/well of HDAC7 isoforms in six-well plates with Fugene 6 reagent, followed performed as described previously (Zeng et al., 2006). For PDGF treatment, the by western blot analysis. pretreated ES cells were cultured in serum-free MEM alpha medium supplemented with 1% bovine serum albumin (BSA), 10 ng/ml (Sigma), 0.05 mM 2- Nucleofection mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin for 1 hour, followed For transient co-transfection experiments, different amount of pShuttle-HDAC7-2 by the addition of 25 ng/ml PDGF-BB (Sigma) and further incubation for 3, 6 or 24 expression plasmid (0, 0.5, 1.0 and 1.5 μg per 1106 cells) with pShuttle-HDAC7- hours. 1 (0.5 μg per 1106 cells) were introduced into ES cells by nucleofector II (Amaxa, Germany) with mouse ES cell nucleofection kit (Amaxa, VPH-1001) and using Immunoblotting program A-30 according to the manufacturer’s instructions. The appropriate amount Cells were harvested and lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM of empty vector pShuttle was included as plasmid amount compensation and internal NaCl, 1 mM EDTA pH 8.0) supplemented with protease inhibitors and 0.5% Triton control. Nucleofected cells were plated in dishes coated with 5 μg/ml collagen IV X-100 by sonication for whole cell lysate, or with hypotonic buffer (10 mM HEPES- and cultured for 2 days in DM. Total protein were harvested and subjected to western KOH pH 7.2, 1.5 mM MgCl , 10 mM KCl) and high-salt buffer (20 mM HEPES- 2 blot analysis. KOH pH 7.2, 25% glycerol, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA) supplemented with protease inhibitors and 0.5% NP-40 for nuclear and cytoplasmic fractions. siRNA experiments The HDAC7 siRNA (sc-35547) and control CTL3 siRNA (sc-62166) were purchased Indirect immunofluorescent assay from Santa Cruz Biotech, or control siRNA (Cat. No: 4611) was purchased from ES cells were seeded on collagen-IV-coated slides and cultured in DM. SMCs were Ambion Ltd (Huntingdon, UK). ES cells were cultured on collagen IV-coated six- seeded on gelatin-coated slides and cultured up to 70% confluence. An indirect well plates for 5 days; 80 nM HDAC7 siRNA or CTL3 siRNA oligonucleotides were immunofluorescent assay was performed. Goat anti-HDAC7 and FITC-conjugated introduced into each well with siIMPORTER transfection reagent (Millipore) alone donkey anti-goat IgG antibodies were used for HDAC7 staining, whereas Cy3- or together with reporter plasmids pGL3-SM22-Luc, and pGL3-SMA-Luc (0.17 μg conjugated mouse anti-SMA antibody and mouse anti-calponin or TRITC-conjugated each reporter per well) according to the protocol provided. All siRNA experiments swine anti-mouse IgG antibodies were used for staining SMA and calponin, were carried out in triplicate. Control siRNA-transfected cells were included as respectively. The cells were mounted with fluorescent mounting medium and controls, and 0.05 μg of Renilla luciferase was used as internal control. All observed under a fluorescence microscope, and SP5 confocal microscope. Images transfections were carried out in triplicate. The cells were harvested 72 hours after were assessed by Zeiss Axioplan 2 Imaging microscope with Plan-NEOPLUAR transfection and the luciferase activity assay or western blot analysis was performed. 40/0.75 objective lenses, AxioCam camera and Axiovision software at room HDAC7 was also suppressed by HDAC7 shRNA lentiviral plasmids transfer temperature, and were processed using Adobe Photoshop software. (NM_019572 Sigma), according to the protocol provided. HDAC7 in ES cell differentiation 469

Adenoviral DNA transfer fluorescence was monitored in an ABI Prism 7000 Sequence Detector system (Applied Ad-HDAC7 viral DNA was constructed from the corresponding plasmids into BD Biosystems). Relative mRNA expression level was defined as the ratio of target gene Adeno-X Viral DNA system (Clontech), the resulting viral particles were created and expression level to 18S rRNA expression level with that of the control sample set as amplified in HEK293 cells with protocol provided. For adenoviral DNA transfer 1.0. experiments, ES cells were seeded on collagen IV-coated dishes and cultured in DM for 3 days, then infected with Ad-HDAC7 at specific MOI (multiplicity of infection) Statistical analysis as indicated in figures and further incubated in DM for 2 days, followed by western Data are expressed as the mean ± s.e.m. and were analyzed with a two-tailed Student’s blot analysis. The empty virus Ad-tTA was used as a control virus and to compensate t-test for two-groups or pair-wise comparisons. A value of P<0.05 was considered the MOI. to be significant. Ex vivo embryonic cell culture E10-E12 embryos were harvested from SM22-LacZ mice (Moessler et al., 1996) and We thank John Paul Kirton for critical reading of the manuscript. cells were dispensed through 1 ml syringe and No.19G needle. The cells were then This work was supported by Grants from British Heart Foundation seeded in collagen-IV-coated dishes in DM and incubated for 24 hours. After treatment and Oak Foundation. with trypsin, 5105 cells/2 ml were then seeded in collagen-IV-coated six-well plates in DM for another 24 hours. The cells were infected with Ad-HDAC7 virus at 10 MOI or transfected with HDAC7 siRNA, and incubated for 72 hours before X-gal References Campbell, J. H. and Campbell, G. R. (1994). The role of smooth muscle cells in staining and western blot analysis were performed. Ad-tTA virus and control siRNA atherosclerosis. Curr. Opin. Lipidol. 5, 323-330. were used as controls. Images were assessed and processed as described above except Cao, D., Wang, Z., Zhang, C. L., Oh, J., Xing, W., Li, S., Richardson, J. A., Wang, 20/0.5 objective lenses were used instead. After the pictures were taken, the dye D. Z. and Olson, E. N. (2005). Modulation of smooth muscle gene expression by was extracted from the stained samples with DMSO, followed by detection of association of histone acetyltransferases and deacetylases with myocardin. Mol. Cell. absorbance at 590 nm and protein level quantification. The readout of A590nm was Biol. 25, 364-376. μ normalized per g protein with that of Ad-tTA set as 1.0. All animal experiments Chang, S., Young, B. D., Li, S., Qi, X., Richardson, J. A. and Olson, E. N. (2006). were performed according to protocols approved by the Institutional Committee for Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase Use and Care of Laboratory Animals. 10. Cell 126, 321-334. Deng, X., Ewton, D. Z., Mercer, S. E. and Friedman, E. (2005). Mirk/dyrk1B decreases Co-immunoprecipitation the nuclear accumulation of class II histone deacetylases during skeletal muscle Cells were infected with Ad-HDAC7-1, HDAC7-2 or Ad-tTA. 48 hours after infection, differentiation. J. Biol. Chem. 280, 4894-4905. the cells were lysed by rotation for 1 hour at 4°C. 1 mg whole lysate was subjected Dressel, U., Bailey, P. J., Wang, S. C., Downes, M., Evans, R. M. and Muscat, G. E. to a standard co-immunoprecipitation procedure. Lysates were pre-cleared with normal (2001). A dynamic role for HDAC7 in MEF2-mediated muscle differentiation. J. Biol. 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