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. Author manuscript; available in PMC: 2023 Aug 9.
Published in final edited form as: Eur Respir J. 2023 Jul 7;62(1):2200474. doi: 10.1183/13993003.00474-2022

Single-Nucleus Chromatin Accessibility Identifies a Critical Role for TWIST1 in IPF Myofibroblast Activity

Eleanor Valenzi 1,*, Harinath Bahudhanapati 1,*, Jiangning Tan 1, Tracy Tabib 2, Daniel I Sullivan 1, Mehdi Nouraie 1, John Sembrat 3, Li Fan 3, Kong Chen 3, Silvia Liu 4, Mauricio Rojas 5, Audrey Lafargue 6, Dean W Felsher 7, Phuoc T Tran 6, Daniel J Kass 1,#, Robert Lafyatis 2,#
PMCID: PMC10411550  NIHMSID: NIHMS1916053  PMID: 37142338

Abstract

Background

In idiopathic pulmonary fibrosis (IPF) myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor (TF) activity by this approach is imprecise.

Methods

We performed single-nucleus ATAC sequencing (snATAC-seq) on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (n=10 IPF, 8 control) to identify differentially accessible chromatin regions and enriched TF motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells.

Results

TWIST1, and other E-box TF motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF non-myogenic (Log2FC=8.909, adj p-value=1.82E-35) and control fibroblasts (Log2FC=8.975, adj p-value=3.72E-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (Log2FC=3.136, adj p-value= 1.41E-24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts.

Conclusions

Our studies utilizing human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box TF motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a devastating fibrotic lung disease resulting in architectural distortion and impaired gas exchange, ultimately progressing to respiratory failure and death in most patients. Current therapeutics have limited effect, with no approved medications convincingly improving mortality or quality of life. While the precise pathogenesis remains unknown, current paradigms suggest repetitive microinjuries of the alveolar epithelium provoke dysregulated crosstalk with the mesenchymal compartment, leading to expansion of an activated myofibroblast population [1]. Myofibroblasts are key effectors of fibrosis by excessive deposition of extracellular matrix and by their acquired contractile capacity resulting in distorted lung architecture [2]. In IPF, myofibroblasts are also apoptosis resistant, overcoming the normal clearance mechanisms of physiological regeneration [3]. Myofibroblasts are the primary collagen-producing cell propagating fibrosis in diverse organs, with a high disease burden ranging from the dermal and lung fibrosis of systemic sclerosis, to nephrogenic fibrosis, cirrhosis, and graft-versus-host disease [4].

In recent years, the widespread adoption of single-cell RNA-sequencing (scRNA-seq) has produced multiple cell atlases of the human control and IPF lung, allowing precise characterization of cell population transcriptomes [5–8]. While gene expression provides critical information on a cell’s phenotype and active signaling pathways, defining upstream regulatory networks from the transcriptome alone is imprecise. Temporal control of gene expression is regulated by the cooperative interactions of trans-acting DNA binding proteins with cis-regulatory elements within the genome, such as promoters and enhancers [9]. These sequences dictate target gene expression in a cell-type dependent manner by recruiting sequence-specific transcription factors (TFs). The advent of single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) technology now allows the study of open chromatin regions in heterogenous cell populations directly from diseased tissues, thus connecting the input regulatory signals with the output gene expression defining each population and its effector phenotype [10]. Given the central role of myofibroblasts in pulmonary fibrosis and our current lack of any drug targeting these cells, delineating their TF regulation and chromatin accessibility may identify new targets for IPF and other fibroproliferative disorders.

Here we integrated snATAC-seq and scRNA-seq from human IPF and donor control explants to identify differentially accessible chromatin regions and TF motifs (consensus-sequence specific binding sites) within lung cell populations. We specifically focused on IPF myofibroblasts and identified enrichment of E-box TF motifs in IPF myofibroblasts compared to both IPF non-myogenic and control fibroblasts. As the TF TWIST1 is selectively expressed in IPF myofibroblasts and binds in regions of fibroblast accessible chromatin, it was particularly implicated as a positive putative regulator of IPF myofibroblast differentiation. We further investigated TWIST1 in vitro and in an animal model of pulmonary fibrosis. Our studies demonstrate a critical role for TWIST1 in regulating myofibroblast effector functions in IPF.

Methods

This work was approved by the Institutional Review Board and the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Explanted subpleural peripheral lung tissue was digested to single-cell suspensions as previously described [11]. Single-cell suspensions were split, with a portion used for performing scRNA-seq as previously described [12], and the remaining suspension used for nuclei generation and snATAC-seq (10X Genomics). CellRanger ATAC pipeline (v1.2.0; 10X Genomics) and the R packages Signac (v1.3.0) [13], Seurat (v4.0.3), harmony (v1.0) [14], and chromVAR (v1.12.0) [15] were used for downstream analysis. Peak calling was performed by cluster using macs2 [16]. DARs were calculated by logistic regression test with number of peak region fragments as a latent variable. Wilcoxon rank sum test with Bonferroni FDR correction was used for DEG testing.

Mouse lung fibroblasts were isolated from uninjured lungs of wild type and transgenic mice as described previously [17]. Fibroblasts were lysed for immunoblotting to measure the protein expression of TWIST1, Collagen I, α-SMA, and the housekeeping protein cyclophilin A. Cells were treated with brefeldin A at one hour prior to lysis to inhibit secretion of collagen.

Primary lung fibroblasts from Twist1-Luc, ColCre+, Rosa26RTta and their littermate controls (ColCre+, Rosa26RTta) were cultured (n=3) and treated with Doxycycline and Tamoxifen. Total RNA was purified and libraries sequenced, with CLC Genomics Workbench 11 (Qiagen) used for transcript counts, quality control, alignment, DEGs, preliminary enrichment analysis, and hierarchical clustering. Additional enrichment analyses were conducted using Ingenuity Pathway Analysis (Qiagen). In vitro and in vivo data were analyzed by robust non-parametric two-way ANOVA (“WRS2” R 4.1.2 package), with statistical analysis indicated in figure legends. The raw data have been deposited in NCBI’s Gene Expression Omnibus GSE214085. Murine lung scRNA-seq data obtained from GSE141259. Detailed methods available in Supplementary File 1.

Results

Single-cell transcriptional and chromatin accessibility profiling in the IPF and control lung

We performed scRNA-seq and snATAC-seq on three IPF and two donor control lung tissue samples (Figure 1A, B) with 8,738 nuclei included for snATAC-seq analyses after filtering. Subpleural lower lobe tissue was collected at the time of lung transplant in individuals with IPF, and from organ donors without preexisting lung disease. Histologic review of adjacent tissue showed usual interstitial pneumonia for all IPF samples (Supp Fig 1). To increase the robustness of our scRNA-seq dataset, we included 13 additional samples for a total of 18 samples (n=10 IPF, 8 Control) with 65,179 cells included after filtering (Figure 1C, D). The R packages Seurat and Signac were used for dimensional reduction, clustering, differential expression/accessibility testing, and visualization at the individual sample and aggregate dataset level [13, 18]. We annotated snATAC-seq cell types by transferring predicted labels (Supp Fig 3A) of the scRNA-seq dataset based on their transcriptomes (Figure 1D), in addition to manually identifying cell types by examining gene activity matrices (a measure of chromatin accessibility within the promoter and gene). Comparison between cell-type predictions by label transfer and manual annotations indicated all major cell types were present in both datasets and consistently identified by both methods.

FIGURE 1.

FIGURE 1

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Single-cell RNA-sequencing (scRNA-seq) and single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) profiling of the human idiopathic pulmonary fibrosis (IPF) and healthy lung. a) Uniform manifold approximation and projection (UMAP) plot snATAC-seq dataset (IPF n=3, control n=2) identified by cluster number and cell type. b) Merged coverage plots demonstrating pseudo-bulk chromatin accessibility (fragment coverage by frequency of Tn5 insertion) around marker gene promoters. Y-axis cluster numbers correspond to cell clusters in figure 1a. Range of normalised accessibility for fragment coverage of each gene listed on x-axis. c) UMAP plot of scRNA-seq dataset (IPF n=10, control n=8) identified by cell type. d) Dot plot of scRNA-seq dataset showing gene expression of selected cell-type specific marker genes. The diameter of the dot corresponds to the proportion of the cells expressing the gene, and the colour density of the dot corresponds to the average expression level relative to all cell types. e) Heatmap of average number of Tn5 cut sites within the differentially accessible regions (DARs) (each row is a unique DAR) for each cell type. The colour scale represents a z-score of the number of Tn5 sites within each DAR. f ) Genomic annotation of differentially accessible region locations. Mac: macrophage; AT: alveolar type; SM: smooth muscle; NK: natural killer; pDC: plasmacytoid dendritic cell; UTR: untranslated region.

We detected all major cell types within the lung with both datasets, with 260,166 accessible chromatin regions among 8,738 nuclei. In snATAC-seq, cell types can be distinguished by whether differentially accessible regions (DARs) of the chromatin are conformationally “open” or “closed.” Epithelial and macrophage clusters had the most unique DARs (Figure 1E). Sequenced peak regions were annotated to the nearest gene and region of the genome (Figure 1F), with the majority of peaks in distal intergenic or intronic regions [19]. The distribution of peak genomic regions was similar across cell types.

Mesenchymal profiling

To more closely examine the myofibroblasts, we subclustered scRNA-seq and snATAC-seq fibroblast, smooth muscle, and pericyte populations (snATAC-seq n= 844 mesenchymal nuclei; scRNA-seq n=6,149 mesenchymal cells). By transcriptomes, we identified 3 major populations consisting of myofibroblasts, alveolar fibroblasts, and adventitial fibroblasts, and a minor population referred to as CXCL2hi fibroblasts (Figure 2A). Myofibroblasts originated primarily from IPF samples, while the non-myogenic fibroblast populations were observed in both IPF and control samples (Figure 2B). The myofibroblasts were defined by upregulation of CTHRC1, POSTN, COMP, and COL3A1, the alveolar fibroblasts by SPINT2, FGFR4, GPC3, and MACF1 (analogous to our previously described SPINT2hi fibroblasts), and the adventitial fibroblasts by upregulation of PI16, MFAP5, IGFBP6 (analogous to our previously described MFAP5hi fibroblasts) (Supp Fig 4A,5A)[11, 12, 20]. In the snATAC-seq mesenchymal subclustering, 2 clusters of myofibroblasts, 3 clusters of non-myogenic fibroblasts, and a cluster of pericytes and smooth muscle cells were present (Figure 2C). Nuclei with significant gene activity for both fibroblast and myeloid markers were likely doublet nuclei and excluded from further analyses. Control fibroblasts clustered distinctly from the IPF fibroblasts (Supp Fig 4C). We focused our analysis to the comparison of IPF myofibroblasts to IPF non-myogenic fibroblasts (including the adventitial, alveolar, and CXCL2hi fibroblast populations), as well as IPF to control fibroblasts. There were 163 DARs more accessible in IPF myofibroblasts vs 88 DARs more accessible in IPF non-myogenic fibroblasts (by Bonferroni adjusted p-value <0.05). These DARs were overall consistent across individual IPF samples (Figure 2D). These DARs were annotated to the nearest gene and utilized for pathway analysis by IPA, with the stem cell pluripotency, thioredoxin, hepatic fibrosis, and regulation of epithelial mesenchymal transition (EMT) amongst the top upregulated pathways in IPF myofibroblasts vs non-myogenic fibroblasts (Figure 2E). Of the DARs more accessible in IPF myofibroblasts, 17 were annotated to differentially expressed genes (DEGs) when comparing IPF myofibroblast to non-myogenic fibroblasts, including SPON2, PLEKHG1, SMYD3, AKAP7, LOXL2, and TSPAN2. Only 19 DARs were more accessible in IPF fibroblasts compared to 25 DARS in control fibroblasts, revealing pathways not as clearly associated with fibrosis (Figure 2F). In comparing IPF to control fibroblasts, 5 of the 19 significant DARs were annotated to genes upregulated in IPF fibroblasts including FBXL7, SPON2, ATP10D, and RUNX1. While traditionally annotated by least base pairs distance, cis-regulatory regions do not inevitably regulate the nearest gene but may instead associate with more distant genes via three-dimensional chromatin looping. DARs not located near DEGs may regulate more distant genes via such long-range interactions.

FIGURE 2.

FIGURE 2

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Fibroblast subpopulations and transcription factor motif activity in idiopathic pulmonary fibrosis (IPF) myofibroblasts. a) Uniform manifold approximation and projection (UMAP) plot of single-cell RNA-sequencing (scRNA-seq) fibroblasts, smooth muscle cells and pericyte clusters by cell identity. b) UMAP plot of scRNAseq fibroblasts, smooth muscle cells and pericyte clusters from (a) with cells depicted by origination from IPF versus control samples. c) UMAP plot of single-nucleus assay for transposase-accessible chromatin sequencing fibroblast, smooth muscle cells and pericyte clusters by cell identity. d) Heatmap of average number of Tn5 cut sites within the differentially accessible regions when comparing IPF myofibroblasts to IPF nonmyogenic fibroblasts, depicted by individual sample. Each row is a unique differentially accessible region (DAR). e) Ingenuity pathway analysis pathways significantly enriched for genes annotated to the upregulated DARs in IPF myofibroblasts versus IPF nonmyogenic fibroblasts. f ) Ingenuity pathway analysis pathways significantly enriched for genes annotated to the upregulated DAR in IPF fibroblasts versus control fibroblasts. g) Transcription factors with the most significantly enriched motif activity when comparing IPF myofibroblasts to the IPF nonmyogenic fibroblasts. h) Transcription factors with the most significantly enriched motif activity when comparing all IPF fibroblasts to all control fibroblasts. EMT: epithelial–mesenchymal transition; BMP: bone morphogenetic protein; FC: fold change; adj: adjusted.

TWIST1 motif activity enrichment in IPF myofibroblasts

TF motif enrichment can be inferred for cell populations based on the enriched presence of TF-binding motifs within accessible chromatin regions, predicting critical, active TFs regulating the cell state of interest. To assess TF-motif activity we used chromVAR to determine TF-associated accessibility in our snATAC-seq dataset [21]. Specific motifs were associated with each individual cell type, with known cell-type enriched TFs validating our data and analysis, such as FOXA1 and TEAD1 in alveolar epithelial cells [22], ETS1 in natural killer cells and T lymphocytes [23], and MEF2C in smooth muscle cells [24]. Motifs highly enriched in fibroblasts compared to other cell types included ZBTB26, NFATC2, TWIST1, MYF5, HSF1, HSF2, and PBX2, amongst others (Supp Table 1, Supp Fig 6A).

On a global scale, TF expression had limited correlation with TF activity, supporting the notion that TFs may act as activators or repressors of gene expression based on post-transcriptional regulation of their activity. Of the 208 significant unique TF motif activities identified when comparing IPF to control fibroblasts, only 36 correlated to DEGs between the two populations. Comparing IPF myofibroblasts to non-myogenic fibroblasts, motifs for TWIST1, TFAP4, HAND2, ATOH7, and ZBTB18 were the most significantly enriched (Figure 2G), with TWIST1, TCF3, and NFATC3 expressed more highly by myofibroblasts. To evaluate for bias by individual sample, a leave-one-out analysis was performed for motif enrichment, with TWIST1 consistently noted amongst the top motifs in each scenario (Supplementary File 1). Motif activity for these TFs was also consistent visually across samples (Supp Fig 7A). Comparing IPF to control fibroblasts, motifs for NR3C2, ZBTB18, NR3C1, TWIST1, and TAL1::TCF3 were the most significantly enriched, with NR3C1 conversely having decreased expression in IPF fibroblasts (Figure 2H).

Multiple TFs with a shared consensus binding sequence may bind to a highly similar motif, distinguished by minor differences in their position weight matrices, such as the E-box motifs of TWIST1 and HAND2. In silico motif enrichment analyses alone cannot definitively determine which TF binds to a particular motif. To confirm that TWIST1 binds to relevant regions of accessible chromatin in IPF fibroblasts, we performed ChIP-sequencing of pulmonary fibroblasts from patients with IPF (n=4) following TWIST1 antibody (Ab) immunoprecipitation and compared results to the snATAC-seq results. After quality control, pre-processing, peak calling utilizing input controls, and comparing peaks across the IPF samples, 66 statistically significant peaks occurred in multiple IPF TWIST1 Ab samples. Forty-eight percent (32/66) of overlapping peaks occurred in at least 3 of the 4 IPF samples (Supp Fig 7C), with “regulation of glutamatergic transmission” the most significantly enriched pathway amongst the genes annotated to these peaks. Despite the inevitable changes in TF-biding and chromatin structure occurring in culture, 32% of the shared IPF TWIST1 Ab peaks occurred in regions of accessible peaks in the snATAC-seq fibroblast data. In particular, TWIST1 bound in the coding sequence of KANK3 (log2FC=0.978, p-val=0.0539) and DYNCH11(log2FC=1.548, p-val=0.0166) in DARs between the IPF and control fibroblasts (Figure 3). E-box motifs were present in these regions in snATAC-seq fibroblasts. TWIST1 also bound in DARs between IPF myofibroblast and non-myogenic fibroblasts in regions annotated to CACNG8, LINC00415, RP11–34F13.3, and GPR27.

FIGURE 3.

FIGURE 3

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TWIST1 binds in areas of in vivo accessible chromatin in idiopathic pulmonary fibrosis (IPF) fibroblasts. a) Coverage plot demonstrating Tn5 insertion frequency (pseudobulk single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) tracks) in IPF and control fibroblasts in the region of KANK3 with ATAC-seq peaks depicted by red bars. TWIST1 Ab chromatin immunoprecipitation sequencing (ChIP-seq) peaks from IPF explant fibroblasts (grey tracks labelled IPF 1–4) in the same region are depicted below. Grey shading indicates the region of TWIST1 binding by ChIP-seq experiments. Scales for ATAC-seq tracks and ChIP-seq tracks are independent. b) Coverage plot demonstrating Tn5 insertion frequency (pseudobulk ATAC-seq tracks) in IPF and control fibroblasts in the region of DYNC1H1 with ATAC-seq peaks depicted by red bars. TWIST1 Ab ChIP-seq peaks from IPF explant fibroblasts (grey tracks labelled IPF 1–4) in the same region are depicted below. Light grey box indicates the region of TWIST1 binding by ChIP-seq experiments. Scales for ATAC-seq tracks and ChIP-seq tracks are independent.

As TWIST1 motif activity was enriched in IPF myofibroblasts and IPF fibroblasts and its expression is highly specific to myofibroblasts (Figure 4A–4C), we further investigated the cell populations showing increased TWIST1-associated DARs. We separated the IPF myofibroblasts into top quartile TWIST1 motif activity, and those with low TWIST1 motif activity (bottom three quartiles). The thioredoxin pathway, calcium signaling, hepatic fibrosis, and 3-phosphoinositide degradation were amongst the pathways significantly enriched by genes annotated to DARs in TWIST1hi motif activity myofibroblasts (Supp Fig 7B). In addition to TWIST1, other E-box motif TFs including HAND2, ZBTB18, NEUROG2, and NEUROD1 had the highest motif enrichment in the TWIST1hi motif activity myofibroblasts compared to those with TWIST1lo motif activity.

FIGURE 4.

FIGURE 4

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TWIST1 expression and motif activity in idiopathic pulmonary fibrosis (IPF) and control fibroblasts. a) Uniform manifold approximation and projection (UMAP) plot of TWIST1 motif activity in the mesenchymal single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) clustering, depicted by scaled expression with red representing the highest motif activity. b) Violin plot of TWIST1 motif activity comparing IPF versus control fibroblasts only. c) UMAP plot of TWIST1 gene expression in the mesenchymal single-cell RNA-sequencing (scRNA-seq) clustering demonstrating high TWIST1 expression in the myofibroblasts only. d) Coverage plot demonstrating Tn5 insertion frequency by snATAC-seq fibroblast cluster in the TWIST1 gene region. Red boxes represent regions of statistically significant differential accessibility in IPF myofibroblasts versus nonmyogenic fibroblasts. e) Ingenuity pathway analysis pathways significantly enriched for genes annotated to the upregulated differentially accessible regions (DAR) in TWIST1-open chromatin myofibroblasts versus TWIST1-closed chromatin myofibroblasts. f ) Dot plot of E-box transcription factor gene expression in IPF and control mesenchymal populations by scRNA-seq with deeper colour indicating higher level of gene expression and circle size indicating the percentage of cells in the population expressing the gene. NFAT: nuclear factor of activated T-cells; EMT: epithelial–mesenchymal transition; FGF: fibroblast growth factor.

TWIST1 expression was upregulated in IPF myofibroblasts (Log2FC=3.136, adjusted p-value= 1.41E-24) vs non-myogenic fibroblasts. Compared to other E-box motif TFs, TWIST1 was the most specific to myofibroblasts and showed the greatest upregulation in IPF (Figure 4F), supporting our hypothesis that TWIST1 dysregulation perpetuates IPF myofibroblast activity. In comparing TWIST1hi IPF fibroblasts (expression level >0.5, top 6.5% expression) to TWIST1lo IPF fibroblasts (expression level<0.5), we identified 338 DEGs including upregulation of POSTN, ASPN, LOXL2, MMP14, and COL8A1, amongst others, in the TWIST1hi population. Enriched pathways in TWIST1hi IPF fibroblasts included pulmonary fibrosis idiopathic signaling, hepatic fibrosis, axonal guidance signaling, and S100 family signaling.

Two regions of TWIST1, in the promoter and the second intron, had significant differential accessibility in comparing myofibroblast to non-myogenic fibroblasts (Figure 4D). In addition, we also examined myofibroblasts with accessible chromatin in TWIST1 (51.2% of myofibroblasts) versus those with closed chromatin in TWIST1 (48.8% of myofibroblasts). Pathways enriched amongst genes annotated to DARs in TWIST1-open myofibroblasts included FGF signaling, regulation of EMT, Gαq signaling, and ribonucleotide reductase signaling, amongst others (Figure 4E). Regulation of EMT was enriched based on enhanced accessibility for CD70, FGF13, FGFR1, MAPK3, PARD6B, SNAI2, STAT3, and TWIST1, supporting the previously identified central role of TWIST1 in EMT [25], as well as supporting the validity of our data.

To investigate whether TWIST1 motifs were enriched in non-mesenchymal cells known to transition to mesenchymal and myofibroblast phenotypes in fibrosis- via EMT or endothelial mesenchymal transition- we evaluated enriched motifs in IPF vs control epithelial and endothelial populations. TWIST1 motifs were significantly enriched in IPF endothelial nuclei vs controls, however the NFAT-related factors (NFATC4, NFATC3, and NFATC2), several AP-1 family TFs, and NR3C1 were the top enriched motifs for this comparison (Supp Table 6). TWIST1 motifs were not significantly enriched in the aggregate IPF epithelial cells versus controls, or in the comparison for alveolar type 1, alveolar type 2, basal, or ciliated cells, however the more limited number of nuclei for these populations limited the number of significant motifs identified. These data support the primary action of TWIST1 within the myofibroblast, rather than the epithelial populations, consistent with its increased gene expression in the myofibroblast population.

Increased expression of Twist1 in collagen-producing cells is associated with increased collagen synthetic activity

We have previously observed that IPF patients with the highest expression of TWIST1 by whole lung microarray analysis exhibit the most impaired gas exchange [26]. Combining these data with our ATAC-seq observations led us to consider how Twist1 overexpression in fibroblasts may impact an animal model of pulmonary fibrosis. We examined the effect of induced expression of Twist1 in lung Col1a2+ expressing fibroblasts (Twist1-LUC, Figure 5A). Lung fibroblasts were isolated and cultured in the presence of tamoxifen and doxycycline, to induce Twist1 expression, with and without TGF-β. In unstimulated Twist1-LUC fibroblasts, we observed increased expression of collagen I and the myofibroblast marker α-smooth muscle actin (α-SMA/ACTA2) compared to wild type fibroblasts (Twist1-WT) (Figure 5B–D). TGF-β augmented both SMA/ACTA2 and collagen 1 in the presence of Twist1 overexpression, suggesting that TWIST1 mediates a TGF-β independent pathway. We confirmed increased expression of Twist1 in Twist1-Luc fibroblasts (Figure 5B, E). Although Twist1 expression may promote a “pro-survival” phenotype in certain tumors, we found that Twist1 overexpression did not promote proliferation or resistance to apoptosis by pulmonary fibroblasts in vitro (Supp Fig 8A–C).

FIGURE 5.

FIGURE 5

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Overexpression of Twist1 in Col1a2+ cells leads to increased collagen I levels in vitro and in vivo. a) A triple transgenic animal was bred where Cre recombinase is under control of the col1a2 enhancer element (col1a2-Cre-ER(T)). In the presence of tamoxifen (TAM), the STOP signal is excised leading to expression of the reverse tetracycline transactivator (rtTA). In the presence of doxycycline (DOX) and the rtTA, the tetO7 operator is activated leading to expression of Twist1 and luciferase. b) Lung fibroblasts from Twist1-WT (wild-type) and Twist1-Luc (Twist1 overexpressors) were incubated in the presence of TAM and DOX with and without transforming growth factor (TGF)-β (2 ng·mL−1 ). Cells were lysed and subjected to immunoblotting for collagen I, α-smooth muscle actin (SMA), Twist1 and the loading control, cyclophilin A. In the presence of TAM/DOX, increased c) collagen, d) α-SMA and e) Twist1 in Twist1-LUC fibroblasts. This was amplified in the presence of TGF-β (n=3). Data were analysed by robust nonparametric two-way ANOVA. p-values for the effects of Twist1 and TGF-β and the interaction are presented in the panels. f ) Twist1-WT and Twist1-Luc mice were injured with bleomycin. Animals were sacrificed at 14 days. Lungs were excised, and the ratio of the lung mass before and after freeze-drying was determined. g) Determination of acid-soluble collagen content showed a significant increase in bleomycin-induced collagen in Twist1-Luc mice compared to Twist1-WT mice (by robust nonparametric two-way ANOVA; n=6–8). h) A comparable degree of histological injury was observed in Twist1-WT and Twist1-Luc mice. Haematoxylin and eosin (H&E) and trichrome images are presented. Incidentally noted multinucleated giant cells are identified and magnified by the black arrows. Scale bar=200 μm, inset ×100 magnification. NS: nonsignificant.

Next, we explored the effect of increased Twist1 in fibroblasts in vivo following bleomycin injury (Figure 5F–G, Supp Fig 9A). Uninjured Twist1-Luc mice showed no pathology (Supp Fig. 9B). Following bleomycin injury we saw no difference in the ratio of wet-to-dry lung mass, suggesting a comparable degree of acute lung injury between genotypes (Figure 5F). In contrast, we observed increased collagen content in Twist1-LUC mice injured with bleomycin compared to Twist1-WT mice (Figure 5G). Histologically, we observed comparable acute lung injury in both Twist1-WT and Twist1-LUC mice injured with bleomycin, but increased collagen deposition in Twist1-LUC mice (Figure 5H). We incidentally noted airspace multinucleated giant cells in bleomycin-treated Twist1-LUC mice. As expected Twist1 mRNA was overexpressed in whole lungs of Twist1-LUC mice (Supp Fig 10A). Bleomycin induced the inflammatory mediators Tnfa, Il1, Il6, Cxcl12, and Ccl7, however Twist1 overexpression in Col1a2+ cells did not alter expression of these mediators except for Cxcl12 and Il6, which were slightly decreased (Supp Fig 10A–F). Taken together, these data show that increased expression of Twist1 in collagen-producing cells is associated with increased collagen synthetic activity in both in vitro and in vivo models.

Timing of Twist1 expression

To investigate the timing of Twist1 expression in lung injury and fibrosis, we analyzed publicly available whole lung scRNA-seq data collected at days 3, 7, 10, 14, 21, and 28 of the murine intratracheal bleomycin injury model [27]. Similar to our human IPF scRNA-seq studies, Twist1 was primarily expressed in myofibroblasts (Supp Fig 11A, B). Within the mesenchymal compartment, no Twist1 expression was detected in the uninjured control, day 3, or day 7 animals. Twist1 expression was present in the highest number of cells at day 10 of bleomycin injury, with a lower percentage of cells expressing it at days 14, 21, and 28 (Supp Fig 11C). Day 10 Twist1 expression correlates with early extracellular matrix deposition in the bleomycin injury model (typically peaking at day 14) [28], as well as peak alveolar Krt8 expression reflecting a transitional alveolar cell present in lung injury that persists within the fibrotic lung [27].

Twist1 overexpression induces dysregulation of multiple profibrotic genes

To further explore genes regulated by TWIST1, we compared gene expression in cultured lung fibroblasts from three Twist1-LUC mice and three Twist1-WT mice in vitro by RNA-seq (Figure 6). On clustering by DEGs between Twist1-LUC and Twist1-WT fibroblasts, one of the three Twist1-WT fibroblasts clustered more closely to Twist1-LUC fibroblasts. By immunoblotting, this line spontaneously expressed higher TWIST1 protein than the other two Twist1-WT lines but less than the Twist1-LUC fibroblasts, thus representing an intermediate phenotype (Figure 6A). Several significantly up-regulated genes (Figure 6B–D) have been associated with TGFβ signaling and pulmonary fibrosis including Ltbp1, Tbx, Tnc and Thbs4. Based on IPA dysregulated canonical pathways included “Systemic Lupus Erythematosus in B cell Signaling” (Tnfsf11, and Tnfsf15) and “Role of Hypercytokinemia in the Pathogenesis of Influenza” as well as HIPPO signaling—implicated in pulmonary fibrosis [29]—and pulmonary and hepatic fibrosis pathways (Ptch2, Il1rap, Itgb3, and Flt3). Downregulated signaling for xenobiotic metabolism, glutathione-mediated detoxification, and NRF2-mediated oxidative stress response (Gsta3, Nqo1, Cyp1a1, and Acta1) support previous data suggesting loss of these pathways is a component of fibrosis (Figure 6E) [30, 31]. In the heat map based on genes showing the highest coefficient of variation, fibrotic genes such as Col1a1, Col1a2, Col3a1, Tnc, Thbs1, and Lox were highly upregulated in Twist1-LUC fibroblasts (Supp Fig12). Using qRT-PCR, we validated the top-most differentially expressed genes that are upregulated, down-regulated, as well as ECM genes (Supp Fig 10). In comparing our snATAC-seq data with the mouse lung fibroblasts, we observed that MMP8 and TNFRSF9, amongst the top upregulated genes in Twist1-LUC fibroblasts (Figure 6C), have significantly increased chromatin accessibility in IPF myofibroblasts and a trend towards increased accessibility in IPF vs control fibroblasts (Supp Fig 13–14). This finding demonstrates that downstream targets of TWIST1 have altered chromatin in myofibroblasts.

FIGURE 6.

FIGURE 6

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Twist1 overexpression in mouse lung fibroblasts is associated with dysregulation of several pulmonary fibrosis genes and pathways. Bulk RNA-sequencing (seq) was performed on fibroblasts isolated from lungs of wild-type (WT) and Twist1-LUC mice (n=3). Estimation of differential gene expression using CLC Genomics Workbench was performed comparing fibroblasts from knock-in with normal lungs. a) Hierarchical clustering heatmap of significant differentially expressed genes was generated using CLC Genomics Workbench using minimum absolute fold change of 3.0 and false discovery rate (FDR) p-value threshold of 0.05. Immunoblotting is shown for the individual lines subjected to RNA-seq. Densitometry normalised to β-actin is shown beneath. b) Volcano plot shows comparative analysis of differentially expressed genes between the WT and Twist1-LUC. c) List of dysregulated genes by FDR that are upregulated ranked by −log10 (FDR p-value) with p<0.05 cut-off. d) List of top downregulated genes ranked by −log10 (FDR p-value) with p<0.05 cut-off downregulated in Twist1-LUC fibroblasts compared to Twist1-WT fibroblasts. e) Ingenuity pathway analysis (IPA) of dysregulated canonical pathways between WT and Twist1-LUC (n=3) by z-score and FDR.

Discussion

Our study demonstrates that the differentiation of myofibroblasts—the central effector cells in IPF—is characterized by a significant shift in chromatin accessibility, dominated by opening of E-box TF binding sites. We utilized single-cell sequencing platforms of ex vivo IPF lungs to obviate the distortion of signals across heterogeneous populations and the changes in chromatin accessibility and gene expression that may occur with expansion in culture. Of the E-box TFs, we identified TWIST1 as the most highly enriched regulator of myofibroblast activity. We then confirmed a critical regulatory role for TWIST1 by demonstrating that overexpression of Twist1 in the fibroblast compartment, in vitro and in vivo, led to increased expression of collagen I and α-SMA.

Previous studies have identified epigenetic changes in IPF lungs via methylation profiling [32–34], however knowledge of cell-type specific epigenetic alterations remains limited. A recent study by Hanmandlu et al. utilized bulk ATAC-seq of cultured fibroblasts to investigate chromatin accessibility in IPF upper lobe fibroblasts [35]. They similarly identified enrichment of the E-box TFs TWIST1 and ZBTB18 motifs in IPF fibroblasts, further supporting an important role for TWIST1 and the E-box TFs in IPF myofibroblasts. However, other motifs implicated by their analysis including FOXA1 and FOXP1 were significantly less enriched in our analyses, while CBFB was enriched in IPF fibroblasts in the bulk analyses only, indicating accessibility may be altered by in vitro culture, though heterogeneity amongst samples cannot be ruled out. The E-box TF MYF5, a known regulatory factor critical to myogenic differentiation, was also enriched in our snATAC-seq, but not the cultured fibroblasts.

The consistent enrichment of E-box motifs in IPF myofibroblasts, suggests modulation of their accessibility as a critical step within or resulting from activation of the aberrant myofibroblast program. While we focus in this work on TWIST1, the complex mechanism of myofibroblast activation undoubtedly involves the coordinated activation of multiple TFs. Defining such shifts in a cell population’s epigenetic state now opens the door for novel molecular and computational approaches to therapeutic development in IPF. For instance, the rapid advancement of small molecule therapeutics including those inhibiting DNA-protein binding and TF complexes supports identifying and targeting key dimerization pairs or other coordinated binding partners of TWIST1 [36]. Investigating chromatin alterations in the context of therapeutics may identify agents halting (and ideally reversing) such epigenetic changes, potentially as an early signal for therapeutic response.

In contrast to previous studies [26, 37–39] examining TWIST1 in fibroblasts, to our knowledge, we are the first to report that fibroblast-specific overexpression of Twist1 in vivo is associated with increased collagen synthesis following bleomycin injury. This corroborates our observation that increased expression of TWIST1 in IPF is associated with worse gas exchange [26]. It was striking that one Twist1-WT line of fibroblasts, through experimental variation, mapped more closely with the Twist1-LUC fibroblasts, suggesting a very narrow dynamic range of expression of TWIST1 in unstimulated cells. Deviations from that narrow range of expression can lead to pronounced differences in fibroblast phenotypes [26]. Taken together, these data support a unique model whereby TWIST1 serves as a critical “rheostat” in IPF. Cellular levels of TWIST1 are tightly regulated and even small changes can significantly impact the fibrotic phenotype. It is clear that the “good/bad” paradigm does not suffice in describing the role of TWIST1 in pulmonary fibrosis. Downstream studies which will require much greater depth include: (1) What are TWIST1’s binding partners[40]?, and (2) Do other E-Box transcription factors compensate for the loss of TWIST1?

Our studies have focused specifically on the behavior of Twist1 expression in fibroblasts. In our hands, Twist1 expression did not protect fibroblasts from apoptosis in vitro. An important remaining question would be the persistence of Twist1-LUC fibroblasts as fibrosis “resolves” after bleomycin injury in mice[41, 42]. We also did not fully assess the effect of Twist1 overexpression on other cell lineages in the lung as well as the more complex question of modeling human pulmonary fibrosis. This is clearly important as we surprisingly observed multinucleated giant cells (MGC) in bleomycin-injured Twist1-LUC mice. This phenotype in macrophages may be driven by increased expression of receptor activator of NF-κB Ligand (RANKL) signaling—which is essential for MGC formation in bone[43]—in mouse lung fibroblasts following overexpression of Twist1. This finding suggests that Twist1 overexpression impacts other cell types in the lung and may have important effects on epithelial cell function[40, 44]. Future studies should include testing Twist1-LUC mice in other models of pulmonary fibrosis and would include opportunities for single cell transcriptomics.

As the demonstrated changes in TWIST1 motif enrichment were consistently observed despite the modest sample size and TWIST1 binds in accessible chromatin in IPF fibroblasts, it merited further mechanistic investigation. Despite small snATAC-seq sample numbers, we demonstrate consistency of DARs and motif activity between cell types and IPF and control fibroblasts across individual samples. All IPF samples were from patients with end-stage disease receiving care at a tertiary medical center and may not reflect the comprehensive IPF population.

In summary, our analysis utilizes human multiomic single-cell analyses combined with in vivo murine disease models to investigate TF networks critical to IPF myofibroblasts. Comparison of in vivo IPF myofibroblasts to non-myogenic and control fibroblasts identified a dynamic opening of E-box TF binding sites, with the E-box TF TWIST1 particularly implicated as a positive regulator of myofibroblast activity. Both low [26] and high expression of Twist1 in fibroblasts is associated with increased collagen deposition in the lung, confirming its role as a critical regulator in the fibrotic lung. Future studies delineating the global mechanism modulating E-box TF motif accessibility may identify crucial therapeutic targets for deactivating the aberrant myofibroblast program.

Supplementary Material

Supplementary material
Supplementary Table S1
Supplementary Table S2
Supplementary Table S3
Supplementary Table S4
Supplementary Table S5
Supplementary Table S6
Supplemental Figures

Take-home message:

Multiomic single-cell analyses on human IPF lungs identify a global opening of TWIST1 and other E-box motifs in IPF myofibroblasts, with in vivo murine models confirming a critical regulatory function for TWIST1 in IPF myofibroblast activity.

Acknowledgements

The authors would like to acknowledge the Center for Organ Recovery & Education (CORE) as well as organ donors and their families for the generous donation of tissues used in this study. CLC Genomics Workbench software licensed through the Molecular Biology Information Service of the Health Sciences Library System, University of Pittsburgh was used for data analysis. This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483.

Support Statement

Support for the studies was provided by R01 HL 126990 to DJK, P50 AR 060780-06A1 to RL and DJK, K08 HL 161258 to EV, and from the Pulmonary Fibrosis Foundation and National Scleroderma Foundation to EV.

Footnotes

Conflict of interest: R. Lafyatis reports the following conflicts of interest outside the scope of work of this manuscript: R. Lafyatis has served as a consultant for Pfizer, Bristol Myers Squibb, Boehringer Ingelheim, Formation, Sanofi, Boehringer-Mannheim, Merck and Genentech/Roche, and holds or recently had research grants from Corbus, Formation, Moderna, Regeneron, Pfizer and Kiniksa, and holds equity in Thirona. All other authors report no relevant conflicts of interest.

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Supplementary Materials

Supplementary material
NIHMS1916053-supplement-Supplementary_material.pdf (303.9KB, pdf)
Supplementary Table S1
NIHMS1916053-supplement-Supplementary_Table_S1.xlsx (91.5KB, xlsx)
Supplementary Table S2
NIHMS1916053-supplement-Supplementary_Table_S2.xlsx (5.2MB, xlsx)
Supplementary Table S3
NIHMS1916053-supplement-Supplementary_Table_S3.xlsx (4.4MB, xlsx)
Supplementary Table S4
NIHMS1916053-supplement-Supplementary_Table_S4.xlsx (449.1KB, xlsx)
Supplementary Table S5
NIHMS1916053-supplement-Supplementary_Table_S5.xlsx (377.6KB, xlsx)
Supplementary Table S6
NIHMS1916053-supplement-Supplementary_Table_S6.xlsx (55.2KB, xlsx)
Supplemental Figures
NIHMS1916053-supplement-Supplemental_Figures.pdf (47.1MB, pdf)

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