Abstract
Myocardin is a serum response factor (SRF) co-activator that regulates transcription of many smooth muscle-specific genes and is essential for development of vascular smooth muscle. We used a yeast two-hybrid screen, with myocardin as bait in a search for factors that regulate myocardin transcriptional activity. From this screen, thymine DNA glycosylase (TDG) was identified as a myocardin-associated protein. TDG was originally identified as an enzyme involved in base excision repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. However, TDG has also been shown to act as a transcriptional co-activator or co-repressor. The interaction between TDG and myocardin was confirmed in vitro by glutathione S-transferase pull down and in vivo by co-immunoprecipitation assays. We found that TDG abrogates myocardin induced expression of smooth muscle-specific genes and represses the trans-activation of the promoters of myocardin of these genes. Overexpression of TDG in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous TDG in SMCs increased smooth muscle-specific myosin heavy chain (SM MHC) and Telokin gene expression. Glutathione S-transferase pull-down assays demonstrated that TDG binds to a region of myocardin that includes the SRF binding domain. Furthermore, TDG was found to compete with SRF for binding to myocardin in vitro and in vivo, suggesting that TDG can inhibit expression of smooth muscle-specific genes, at least in part, through disrupting SRF/myocardin interactions. Finally, we demonstrated that the glycosylase activity of TDG is not required for its inhibitory effects on myocardin function. This study reveals a previously unsuspected role for the repair enzyme TDG as a repressor of smooth muscle differentiation via competing with SRF for binding to myocardin.
Highlights
In the current study we show that the physical interaction between thymine DNA glycosylase (TDG) and myocardin disrupts myocardin-serum response factor (SRF) complexes and thereby attenuates smooth muscle differentation
Using GST pull-down vested from these differentiating or proliferating cells and assays we found that SRF binding to myocardin was diminished analyzed by Quantitative Real-time reverse transcription (RT)-PCR (qRT-PCR) (Fig. 9)
These results show that folin the presence of TDG and TDG binding to myo- lowing serum withdrawal, expression of TDG mRNA is cardin could be decreased by increasing amounts of SRF
Summary
Yeast Two-hybrid Screen—A fragment of the mouse myocardin cDNA encoding the N-terminal 585 amino acids was cloned into the bait vector pAS2-1 (Promega) by a standard PCR-based cloning strategy. Mammalian Expression and Reporter Gene Assays—The coding region of mouse TDG cDNA (encoding amino acids 1–397) was amplified from a yeast plasmid clone by PCR and ligated to pcDNA3.1His (Invitrogen), resulting in the expression of a fusion protein with N-terminal His and Omni epitope tags. For testing the effects of smooth muscle gene expression by depletion of endogenous TDG, control siRNA or TDG siRNA was transfected into A10 cells for 24 or 48 h and total RNA was harvested for qRT-PCR as described above. The precipitated DNA was purified and amplified by real time PCR for quantification of the target sequences using SYBR Green PCR master mix (ABgene) with SM22␣ and SM MHC gene-specific primers as described previously [18]. Sequences for human TDG primers were designed as sense: 5Ј-TCA GTG AGG TCC AGC TGA ACC ATA TG-3Ј and antisense: 5Ј-TTC CAT TAA ACA CTG CTA TTC GTG GCT G-3Ј
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