Reversing Mechanoinductive DSP Expression by CRISPR/dCas9-mediated Epigenome Editing

Abstract

Rationale: DSP (desmoplakin), the most abundant component of desmosomes, which maintain the mechanical integrity of epithelium, is a genome-wide association study-identified genetic risk locus in human idiopathic pulmonary fibrosis (IPF). Subjects with IPF express a significantly higher level of DSP than control subjects.

Objectives: Determine potential mechanisms by which DSP is regulated in lung fibrosis.

Methods: Matrigel-coated soft and stiff polyacrylamide gels were made to simulate the stiffness of normal and fibrotic lungs. Quantitative chromatin immunoprecipitation and electrophoretic mobility shift assay were used to evaluate transcription factor binding to the DSP promoter. Targeted DNA methylation was achieved by CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 (deactivated CRISPR-associated protein-9 nuclease)-mediated Dnmt3A (DNA methyltransferase 3A) expression under the guidance of sequence-specific single guide RNAs.

Measurements and main results: Stiff matrix promotes DSP gene expression in both human and rodent lung epithelial cells as compared with soft matrix. A conserved region in the proximal DSP promoter is hypermethylated under soft matrix conditions and becomes hypomethylated/demethylated under stiff matrix conditions. Demethylation of this conserved DSP promoter region is associated with transactivation of transcription factor EGR1 (early growth response protein 1), resulting in EGR1-dependent DSP overexpression. Targeted DNA methylation by CRISPR/dCas9/Dnmt3A-mediated epigenome editing blocks EGR1 binding to the DSP promoter and inhibits stiff matrix-induced DSP overexpression.

Conclusions: DSP is a matrix stiffness-regulated mechanosensitive gene. CRISPR/dCas9-Dnmt3A-mediated epigenome editing reverses DSP overexpression by reestablishment of the epigenetic control of DSP under the mechanically homeostatic environment. It provides a useful tool for investigations of the functional role of DSP in the pathogenesis of lung fibrosis.

Keywords: CRISPR/dCas9; desmoplakin; epigenome editing; lung fibrosis; matrix stiffness.

Figure 1.

Stiff matrix upregulates DSP (desmoplakin) expression in lung epithelial cells. Human and rodent lung epithelial cells were cultured on soft and stiff polyacrylamide gels for 48 hours. (A) Relative mRNA levels of DSP were determined by quantitative PCR (qPCR). (B) Protein levels of DSP were determined by immunoblot. GAPDH was used as reference/loading control. Results are mean ± SD of five independent experiments, each qPCR reaction performed in triplicate. *P < 0.05. A549 = human alveolar epithelial adenocarcinoma cells; mAECII = mouse type II alveolar epithelial cells; MLE = mouse lung epithelial cells; RLE-6TN = transformed rat type II alveolar epithelial cells.

Figure 2.

Stiff matrix promotes DNA demethylation in a conserved 5′ regulatory region in human and mouse DSP/Dsp (desmoplakin) genes. (A) Schematic diagram of human, mouse, and rat DSP/Dsp genes. The conserved 560-bp DNA sequences (78% homology) are indicated by ovals. Columns represent DSP/Dsp exons. (B) Analysis of CpG dinucleotides in a 30+-kb region, including the 20-kb 5′ regulatory region, exon 1, and intron 1. CpG-rich regions are indicated by areas marked with diagonals. (C) DNA methylation profiling analysis for the 560-bp conserved region in human alveolar epithelial adenocarcinoma cells and mouse primary type II alveolar epithelial cells cultured on soft and stiff matrix. Ten clones were sequenced for each condition. Solid circles indicate methylated CpG islands. Open circles indicate unmethylated CpG islands. Results are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01. AP-2 = activator protein-2; C/EBPβ = CCAAT/enhancer-binding protein β; dist = distal; EGR1 = early growth response protein 1; ETF = EGFR [epidermal growth factor receptor]-specific transcription factor; prox = proximal.

Figure 3.

Inhibition of DNA methyltransferase promotes DSP (desmoplakin)/Dsp expression on soft matrix. (A) Human (A549) and (B) mouse (primary mouse type II alveolar epithelial cells) lung epithelial cells were cultured on soft and stiff matrix in the presence or absence of 5 mM 5-aza-2′-deoxycytidine (5-aza-CdR) for 48 hours. Levels of DSP/Dsp mRNA and protein were determined by quantitative PCR (qPCR) and immunoblot analysis. GAPDH/Gapdh were used as reference/loading controls. DMSO is a solvent of 5-aza-CdR. (C and D) DNA methylation profiles in the conserved 560-bp regulatory region were determined by sodium bisulfite sequencing. Ten randomly picked DNA clones were sequenced for each condition. Solid circles indicate methylated CpG islands. Open circles indicate unmethylated CpG islands. Results are mean ± SD of three or four independent experiments, each qPCR reaction performed in triplicate. *P < 0.05. AP-2 = activator protein-2; C/EBPβ = CCAAT/enhancer-binding protein β; dist = distal; EGR1 = early growth response protein 1; ETF = EGFR [epidermal growth factor receptor]-specific transcription factor; prox = proximal.

Figure 4.

EGR1 (early growth response protein 1) transactivation is associated with stiff matrix–induced DSP (desmoplakin) overexpression. (A) Schematic depicts the wild-type (open shapes) and mutated (solid shapes) 560-bp regulatory sequences. Promoter activity was determined by a luciferase-based assay. (B) The bindings of EGR1 to the distal and proximal EGR1 binding sites (EBS) under soft versus stiff matrix conditions were measured by quantitative chromatin immunoprecipitation. (C) The bindings of nuclear EGR1 from human alveolar epithelial adenocarcinoma cells with unmethylated or methylated DSP promoter probes were determined by electrophoretic mobility shift assay. (D) Levels of EGR1 in the whole-cell lysates, cytoplasmic fraction, and nuclear fraction were determined by immunoblot. GAPDH and lamin B were used as loading controls for the cytoplasmic and nuclear proteins, respectively. (E) Knockdown of EGR1 by siRNA was confirmed by immunoblot. Effects of EGR1 knockdown on stiff matrix–induced DSP expression were determined by immunoblot. Results are mean ± SD of at least three or five independent experiments. *P < 0.05. Ab = antibody; C/EBPβ = CCAAT/enhancer-binding protein β; Ctrl = control; dist = distal; ETF = EGFR [epidermal growth factor receptor]-specific transcription factor; Met = methylated; prox = proximal.

Figure 5.

Targeted DNA methylation by CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 (deactivated CRISPR-associated protein-9 nuclease)–Dnmt3A (DNA methyltransferase 3A)–mediated epigenome editing reverses mechanoinductive DSP (desmoplakin) expression. (A) Schematic depiction of targeted DNA methylation. (B) Lentivirus-mediated expression of BFP (blue fluorescent protein), dCas9-BFP-Dnmt3A, and dCas9-BFP-∆Dnmt3A fusion proteins were determined by immunoblot. (C) Confocal fluorescent microscopy showed expression of guide RNA (gRNA)-mCherry (red) and dCas9-BFP-Dnmt3A (blue) in infected human alveolar epithelial adenocarcinoma (A549) cells. Scale bar, 20 μm. (D) Purification of A549 cells with dual expression of gRNA-mCherry (red) and dCas9-BFP-Dnmt3A (blue) by fluorescence-activated cell sorting. The boxed area indicates 10.8% cells with dual expression at relatively higher levels (top 45% red and top 15% blue). (E) DNA methylation profiling analysis for cells with dual expression of gRNA and dCas9-BFP-Dnmt3A/∆Dnmt3A. Ten clones were sequenced for each condition. Solid circles indicate methylated CpG islands. Open circles indicate unmethylated CpG islands. (F) Relative mRNA levels of DSP were determined by quantitative PCR. (G) Protein levels of DSP were determined by immunoblot. GAPDH was used as reference/loading control. Results are mean ± SD of three to five independent experiments, each quantitative PCR reaction performed in triplicate. *P < 0.05; **P < 0.01. Ctrl = control; dist = distal; EGR1 = early growth response protein 1; PAM = protospacer adjacent motif; prox = proximal; sgRNA = single guide RNA.

Figure 6.

Human DSP (desmoplakin) intron 5 contains DNA enhancer activity. (A) Schematic depictions of human DSP gene and wild-type full-length and deletion mutations of intron 5. The SNP at rs2076295, the core enhancer region, and the presumed enhancer suppressor and additional regulatory elements are indicated. DNA enhancer activity assays were performed as described in Methods. (B) DNA enhancer activity assays for DSP intron 5 with the major (955T) and minor (955G) variant allele. (C) Effects of matrix stiffness on the enhancer activity of DSP intron 5 with 955T, intron 5 with 955G, and the core enhancer region (R50); PGL4.24, empty vector. Results are mean ± SD of three or five independent experiments, each performed in triplicate. *P < 0.05.