Figure 2. Collagen 1 is directly repressed by KLF11 in mesenchymal cells coupled to the HP1-HMT pathway.

(A) To illustrate that KLF11 was a direct regulator of the collagen I promoter, chromatin immunoprecipitation was performed on LX2 cells transduced with empty vector or KLF11. Immunoprecipitation of KLF11 displayed amplification of the collagen 1a2 promoter by PCR, thus verifying the direct binding of KLF11 to the promoter of collagen1a2. Input controls for empty vector and KLF11 are included to ensure equal volume of precipitated DNA as well as the results of amplification from immunoprecipitates using a non-specific IgG antibody. (B) Further evidence that collagen is a direct target of the sequence specific transcription factor KLF11 was observed in luciferase assays. Sections of the collagen 1a2 promoter containing GC consensus sequences were cloned upstream of a luciferase reporter. The reporter gene was significantly repressed upon overexpression of KLF11 in LX2 cells (84 ± 1.94% compared to empty vector, p<0.05). Examination of the effect of overexpression of KLF11-EAPP mutant reveals only a slight change in the repression of the collagen I promoter (41 ± 4.5% repression compared to empty vector, p<0.05). However, overexpression of the KLF11ΔHP1 mutant lead to completion de-repression of the collagen I promoter (206 ± 17% compared to empty vector, p<0.05), indicating that the repression of the gene occurs through the HP1-HMT chromatin remodeling pathway. (C) PCR of collagen 1a2 expression in LX2 cells transduced with empty vector, KLF11, or KLF11ΔHP1 demonstrate that expression of the gene is repressed in the presence of KLF11 (0.59 ± 0.11 fold compared to empty vector) but depressed in the presence of the inactivating KLF11ΔHP1 mutant (0.92 ± 0.14 fold compared to empty vector).