Cooperative Binding of KLF4, pELK-1, and HDAC2 to a G/C Repressor Element in the SM22α Promoter Mediates Transcriptional Silencing During SMC Phenotypic Switching In Vivo
Rationale: We previously identified conserved G/C Repressor elements in the promoters of most SMC marker genes, and demonstrated that mutation of this element within the SM22α promoter nearly abrogated repression of this transgene following vascular wire injury or within lesions of ApoE-/- mice. However, the mechanisms regulating the activity of the G/C Repressor are unknown, although we have previously shown that phenotypic switching of cultured SMC is dependent on Krupple-Like Factor 4.
Objective: The goals of the present studies were to: 1) injury-induced repression of SM22αgene following vascular injury is mediated through KLF4 binding to the G/C Repressor element; and 2) the transcriptional repressor activity of KLF4 on SMC marker genes is dependent on cooperative binding with pELK-1 and subsequent recruitment of HDAC2 which mediates epigenetic gene silencing.
Methods and Results: Chromatin immunoprecipitation (ChIP) assays were performed on chromatin derived from carotid arteries of mice having either a wildtype or G/C Repressor mutant SM22α promoter-LacZ transgene. KLF4 and pELK-1 binding to the SM22α promoter was markedly increased following vascular injury and was G/C Repressor dependent. Sequential ChIP assays and proximity ligation analyses in cultured SMC treated with PDGF BB or oxidized phospholipids showed formation of a KLF4, pELK-1, and HDAC2 multi-protein complex dependent upon the SM22α G/C Repressor element.
Conclusions: Silencing of SMC marker genes during phenotypic switching is partially mediated by sequential binding of pELK-1, and KLF4 to G/C Repressor elements. The pELK-1-KLF4 complex in turn recruits HDAC2 leading to reduced histone acetylation and epigenetic silencing.
- Gene transcription
- Smooth muscle
- Smooth muscle cells
- Transcription factors
- Transgenic mice
- Vascular disease
- Received March 19, 2012.
- Accepted July 17, 2012.
- Copyright © 2012, American Heart Association