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First published online 27 January 2009
doi: 10.1242/jcs.034850

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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, King's College London BHF Centre, London SE5 9NU, UK

View larger version (49K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   Fig. 2. HDAC7 is necessary for SMC differentiation. HDAC7 siRNA (A, left panel) and shRNA lentiviral plasmid transfer (A, right panel) downregulated expression of SMC markers such as SMA, calponin and SMMHC at the protein level, and mRNA level in real-time PCR experiments (B), and SMA-Luc, SM22-Luc reporter gene expression (C) in ES cells. RLU, relative luciferase unit. The data presented are representative or means (± s.e.m.) of three independent experiments.

View larger version (58K): [in this window] [in a new window]   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 bar: 200 µm), and its statistical analysis (right panel; *P<0.05). (B) Overexpression of HDAC7 upregulated SMC marker induction whereas HDAC7 siRNA decreased SMC marker expression in SM22-LacZ embryonic cells. 20 µg and 50 µg of protein were applied to a western blot in Ad-HDAC7 and siRNA experiments, respectively. β-gal was included as an indicator for SM22 expression. The data presented are representative or means (± s.e.m.) of three independent experiments.

View larger version (56K): [in this window] [in a new window]   Fig. 4. HDAC7 undergoes splicing during ES cell differentiation into 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 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 second ATG (bold), giving rise to a short HDAC7 lacking 22 amino acids (underlined and bold), compared with the spliced one, in which the intron is removed. (B,C) RT-PCR showing HDAC7 splicing in both undifferentiated and differentiated ES cells (B) and in different passages of ES-derived SMCs (esSMC) (C). Mature SMCs were used as a positive control; β-actin was used as internal control. The ratio of spliced to unspliced HDAC7 is shown in the right panel. u, unspliced isoform; s, spliced isoform. The data presented are representative or means (± s.e.m.) of three independent experiments.

View larger version (39K): [in this window] [in a new window]   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.

View larger version (41K): [in this window] [in a new window]   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 gene expression in ES cells, *P<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 unspliced HDAC7 was introduced into ES cells by adenoviral gene transfer at MOI 20, and the spliced HDAC7 isoform was demonstrated by the presence of both Flag and HA tags. (F) HDAC7 siRNA ablated PDGF-BB-induced SM22-Luc reporter gene expression in ES cells. The data presented are representative or means (± s.e.m.) of three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 7. The short HDAC7 isoform lacking the first 22 amino acids binds and degrades MEF2C. Short HDAC7 localizes in the cytoplasm (A) and spliced HDAC7 localizes in the nucleus (B) in SMCs after adenoviral gene transfer. Scale bars: 50 µm. (C) Fractions of cytoplasm and nucleus confirmed the cellular localization of the HDAC7 isoforms. (D) MEF2C protein level was 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 ablated short HDAC7-induced MEF2C degradation. (G) Immunoprecipitation experiments demonstrate that short HDAC7 increases MEF2C ubiquitylation. The data presented are representative or averages of six independent experiments (A,B,C,D,E) or three independent experiments for F,G.

View larger version (51K): [in this window] [in a new window]   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 assay shows that the spliced HDAC7 increased the binding of myocardin to SM22 and calponin promoters. Upper panel, representative image of PCR detection; Lower panel, statistical analysis of four independent experiments (mean ± s.e.m.); *P<0.05. (C) ChIP assay reveals that the spliced HDAC7 does not suppress histone modification (AcH3 and H3K4DM; histone acetylation and dimethylation, respectively) in the SM22 and calponin gene promoters. However, in the presence of the short HDAC7 isoform, both histone modifications were dramatically decreased. Upper panel, representative image of PCR detection; lower panel, statistical analysis of four independent experiments (mean ± s.e.m.). Fold relative binding was defined as the ratio of band intensity in ChIP samples to that in Input sample, with that of Ad-tTA group set as 1.0.

View larger version (35K): [in this window] [in a new window]   Fig. 9. HDAC7 splicing induces SMC differentiation by modulating the SRF-myocardin complex. During the early stages, HDAC7 is expressed as a partially spliced form lacking the first 22 amino acids. This short HDAC7 isoform binds to MEF2C, leading to MEF2C degradation via the proteasome. The short HDAC7 also prevents spliced HDAC7 activation in the cytoplasm. The overall effect prevents smooth muscle gene expression, favouring progenitor cell differentiation towards other cell lineages. When triggered by PDGF or other stimuli, HDAC7 undergoes splicing, predominantly localises to the nucleus and possesses a higher affinity to SRF. HDAC7 modifies SRF, increasing its binding to the SM22 promoter and recruiting myocardin. Thus, the overall effect is to drive SMC marker gene expression and cell differentiation towards a SMC lineage. HDAC7u, unspliced HDAC7; HDAC7s, spliced HDAC7.

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