单核染色质可及性确定了 TWIST1 在特发性肺纤维化肌成纤维细胞活性中的关键作用,European Respiratory Journal

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单核染色质可及性确定了 TWIST1 在特发性肺纤维化肌成纤维细胞活性中的关键作用
European Respiratory Journal ( IF 24.3 ) Pub Date : 2023-07-07 , DOI: 10.1183/13993003.00474-2022
Eleanor Valenzi _{1,

2} , Harinath Bahudhanapati _{2,

3} , Jiangning Tan ₃ , Tracy Tabib ₄ , Daniel I Sullivan ₃ , Mehdi Nouraie ₃ , John Sembrat ₅ , Li Fan ₅ , Kong Chen ₅ , Silvia Liu ₆ , Mauricio Rojas ₇ , Audrey Lafargue ₈ , Dean W Felsher ₉ , Phuoc T Tran ₈ , Daniel J Kass _{2,

3} , Robert Lafyatis _{2,

4}

Affiliation

Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
These authors contributed equally to this work.
Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Division of Pulmonary, Critical Care and Sleep Medicine, The Ohio State University College of Medicine, Columbus, OH, USA.
Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD, USA.
Division of Oncology, Departments of Medicine and Pathology, Stanford University School of Medicine, Stanford, CA, USA.

背景

在特发性肺纤维化（IPF）中，肌成纤维细胞是纤维化和细胞外基质过度沉积及其获得性收缩能力导致结构扭曲的关键效应器。单细胞 RNA 测序 (scRNA-seq) 精确定义了 IPF 肌成纤维细胞转录组，但通过这种方法识别关键转录因子活性并不精确。

方法

我们对 IPF 患者 (n=3) 和供体对照 (n=2) 的外植肺进行单核测定，进行转座酶可及的染色质测序，并将其与更大的 scRNA-seq 数据集（10 个 IPF，8 个对照）整合，以识别肺细胞群内差异可及的染色质区域和富集的转录因子基序。我们对博莱霉素损伤的Twist1过表达 COL1A2 Cre-ER 小鼠的肺成纤维细胞进行 RNA 测序，以检查胶原蛋白生成细胞中Twist1过表达后纤维化相关通路的变化。

结果

与 IPF 非肌源性（log ₂倍变化 (FC) 8.909，调整后的 p 值 1.82 x 10 ^–35）和对照成纤维细胞（log ₂ FC 8.975，调整后的 p 值 3.72 x 10 ^–28 )。TWIST1表达在 IPF 肌成纤维细胞中选择性上调（log ₂ FC 3.136，调整后的 p 值 1.41 x 10 ^–²⁴），其中TWIST1的两个区域在 IPF 肌成纤维细胞中的可及性显着增加。在博莱霉素损伤小鼠的表达 COL1A2 的成纤维细胞中过度表达 Twist1，导致 IPF 肌成纤维细胞中胶原蛋白合成增加和染色质可及性丰富的基因上调。

结论

我们利用人类多组学单细胞分析结合体内小鼠疾病模型的研究证实了 TWIST1 在纤维化肺中 IPF 肌成纤维细胞活性中的关键调节功能。了解打开 TWIST1 和其他控制肌成纤维细胞分化的 E-box 转录因子基序的整体过程可能会确定纤维化肺部疾病的新治疗干预措施。

"点击查看英文标题和摘要"

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity

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 activity by this approach is imprecise.

Methods

We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor 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 transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log₂ fold change (FC) 8.909, adjusted p-value 1.82x10^–35) and control fibroblasts (log₂FC 8.975, adjusted p-value 3.72x10^–28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log₂FC 3.136, adjusted p-value 1.41x10^–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 utilising 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 transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

更新日期：2023-07-08

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