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

Abstract

Rationale: We previously identified conserved G/C Repressor elements in the promoters of most smooth muscle cell (SMC) marker genes and demonstrated that mutation of this element within the SM22α promoter nearly abrogated repression of this transgene after 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 (KLF)4.

Objective: The goals of the present studies were to ascertain if (1) injury-induced repression of SM22α gene after 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 (downstream activator of the mitogen-activated protein kinase pathway) and subsequent recruitment of histone de-acetylase 2 (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 wild-type or G/C Repressor mutant SM22α promoter-LacZ transgene. KLF4 and pELK-1 binding to the SM22α promoter was markedly increased after vascular injury and was G/C Repressor dependent. Sequential ChIP assays and proximity ligation analyses in cultured SMC treated with platelet-derived growth factor BB or oxidized phospholipids showed formation of a KLF4, pELK-1, and HDAC2 multiprotein complex dependent on 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.

Figure 1. The SM22 G/C Repressor mutant LacZ transgene fails to down-regulate following vascular ligation injury in vivo

A). SM22 and SM22 G/C mut LacZ mice were subjected to right common carotid ligation injury. The right and left carotids were harvested 3 days following injury, fixed, Xgal stained, and a gross morphological image was taken. B). Carotid arteries from part A were embedded in paraffin, sectioned and stained as previously described. Each picture is a representative image from N=5 animals. C). Rat aortic smooth muscle cells were plated and then transiently transfected with LT-Mirus 24 and 300 ng of alpha-actin or SM22 or SM22 with a mutant GC repressor element. 24 hrs after transfection cells were treated with PDGF-BB. 24 hrs after treatment cells were harvested and LacZ activity and protein activity were measured. * indicates significant down-regulation of the wild-type versus the G/C Repressor mutant as mentioned in Materials and methods. D). Cells were treated as mentioned in C. 24 hrs after transfection cells were treated with POVPC. Cells were harvested and assayed as mentioned in 1C.

Figure 2. KLF4 binds the SM22α promoter in vivo three days following vascular injury

A&B) C57B6 mice were subjected to right common carotid ligation injury and then ten mice each were harvested at 1, 3, 7, 14 and 21 days following vascular injury and then subjected to ChIP analysis for KLF4 (A) or to an intronic region of the SM22 gene (B). C&D) SM22 and SM22 G/C mut LacZ mice were subjected to right common carotid injury and harvested 3 days after vascular ligation injury, tissues from ten mice were pooled, and then subjected to ChIP assay for KLF4 (C) or Sp3 (D) . In each ChIP IP, qPCR analysis was conducted on both the Endogenous and LacZ transgene as indicated in the Figure. * indicates significant binding as mentioned in Materials and methods.

Figure 3. KLF4 binds to the SM22α LacZ transgene in vitro and binding is attenuated by mutation of the G/C Repressor element after PDGF-BB or POVPC treatment in rat aortic smooth muscle cells

A) Rat aortic smooth muscle cells were plated at 1×10^4. 24 hr later cells were transiently transfected using LT-Mirus with 300 ng of SM22α WT or G/C Repressor MT and increasing concentrations of pcDNA-KLF4. 24 hours following transfection, media was removed and replaced and 24 hours later cells were harvested and subjected to Bgal and protein assays. Results are the average of three independent experiments performed in triplicate. B&C) Rat aortic smooth muscle cells stably transfected with either SM22α or G/C Repressor mutant were plated at 1×10^4 and allowed to grow to confluency and then switched to serum free media for three days. Following serum starvation, cells were treated with 20 ng/ml PDGF-BB (B) or 5 ug/ml of POVPC (C) for 12 hours and then subject to ChIP analysis. * indicates significant binding as mentioned in materials and methods. D) Cells were plated and 24 hours following plating cells were treated with either si-control or si-KLF4. 24 hours following siRNA transfection, cells were treated with either vehicle or 20 ng/ml PDGF-BB for 12 hours. Cells were harvested and ChIP analysis was performed as described in part A. * indicates significant binding as mentioned in Fig 2D.

Figure 4. pElk-1 binds to the SM22α promoter three days following carotid ligation in vivo

A) C57B6 mice were subjected to right common carotid injury as mentioned in Fig 2A and then subjected to ChIP analysis for pElk-1. * indicates significant binding compared to non-injured controls. Results are the average of three independent experiments. B) SM22 and SM22 G/C mut LacZ mice were subjected to right common carotid injury and then harvested 3 days after vascular injury, tissues from ten mice were pooled, and then subjected to ChIP assay for pELK-1. In each ChIP IP, qPCR analysis was conducted on both the Endogenous and LacZ transgene as indicated in the Figure. * indicates significant binding as mentioned in Fig 2D. C&D) Stably transfected rat aortic smooth muscle cells were plated at 1×10^4, allowed to grow to confluency and then switched to serum free media for three days. Following serum starvation, cells were treated with 20 ng/ml PDGF-BB (C) or 5 ug/ml of POVPC (D) for 12 hours and then subject to ChIP analysis. * indicates significant binding as mentioned in Fig 2D.

Figure 5. KLF4 and pELK-1 interact following PDGF-BB and POVPC using Proximity Ligation Assay (PLA)

A). Human coronary SMCs were plated and 24 h later switched to serum free medium. B and C). After deprivation with Serum Free Medium for 24h, cells were treated with PDGF-BB (10ng/ml), POVPC (10μg/ml) for 24h. Simultaneously, cells were treated with Erk inhibitors PD98059 (10μM) and U0126 (10μM). PLA amplification corresponding with the interaction of KLF4 and pELK-1 is visualized as red spots localized mainly into the nucleus. Interaction between KLF4 and pELK-1 is induced by PDGF-BB and POVPC treatment. This induction is abolished by the addition of Erk inhibitors. Scale bars, 100μg. D). Quantitation of number of spots per nucleus. * indicates specific binding compared to non-treated serum starved smooth muscle cells with a p-value <0.05.

Figure 6. H3 acetylation of the SM22α promoter was reduced and was G/C Repressor dependent

A&B). SM22 and SM22 G/C mut LacZ mice were subjected to right common carotid injury and then harvested 3 days after vascular injury, tissues from ten mice were pooled, and then subjected to ChIP assay for AcH3 (A) or HDAC2 (B). In each ChIP IP, qPCR analysis was conducted on both the Endogenous and LacZ transgene as indicated in the Figure. * indicates significant binding as mentioned in Fig 2D. C&D) Rat aortic smooth muscle cells were plated at 1×10^4, allowed to grow to confluency and then switched to serum free media for three days. Following serum starvation, cells were treated with 20 ng/ml PDGF-BB (C) or 5 ug/ml of POVPC (D) for 12 hours and then subject to ChIP analysis. * indicates significant binding as mentioned in Fig 2D.

Figure 7. Triple Sequential ChIP assays demonstrate that KLF4, pELK-1 and HDAC2 occupy the same piece of chromatin and their binding is attenuated with the G/C Repressor mutation

A) Rat aortic smooth muscle cells were prepared as mentioned previously. Following serum starvation, cells were treated with 20 ng/ml PDGF-BB for 12 hours and then subject to ChIP analysis. Immunoprecipitations were performed in the sequence mentioned in the y-axis. B) IgG was used as a negative control within the sequence of triple immunoprecipitations. Reciprocal immunoprecipitation were performed but are not pictured. * indicates significant binding as mentioned in Fig 2D. C) Triple sequential ChIP analyses were performed as mentioned in part A with a modification to the sequence of the pull-down as indicated on the y axis. D) IgG was used as a negative control during the sequence of the pull-down as mentioned previously in part B.

Figure 8. KLF4, pELK-1 and HDAC2 bind α–actin, SM-MHC but not c-Fos in vivo following caortid ligation injury

A) mice were ligated as mentioned in Fig 2. following ligation, right and left carotid were harvested and subject to ChIP analysis for (A) SRF, (B) KLF4, (C) pELK-1, (D) HDAC2, and (E) H4ac binding at the α-actin, SM-MHC, and c-Fos promoters. * denotes significant decreases in binding over vehicle treated controls via students t-test with p-value <0.05.