Perinatal Institute, Division of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine (CCHMC and UC), Cincinnati, OH, USA.
Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Commun Biol. 2021 May 12;4(1):568. doi: 10.1038/s42003-021-02083-4.
The transcription factor NKX2-1/TTF-1 is involved in lung pathophysiology, including breathing, innate defense and tumorigenesis. To understand the mechanism by which NKX2-1 regulates genes involved in such pathophysiology, we have previously performed ChIP-seq and identified genome-wide NKX2-1-binding sites, which revealed that NKX2-1 binds to not only proximal promoter regions but also multiple intra- and inter-genic regions of the genes regulated by NKX2-1. However, the roles of such regions, especially non-proximal ones, bound by NKX2-1 have not yet been determined. Here, using CRISPRi (CRISPR/dCas9-KRAB), we scrutinize the functional roles of 19 regions/sites bound by NKX2-1, which are located in genes involved in breathing and innate defense (SFTPB, LAMP3, SFTPA1, SFTPA2) and lung tumorigenesis (MYBPH, LMO3, CD274/PD-L1). Notably, the CRISPRi approach reveals that a portion of NKX2-1-binding sites are functionally indispensable while the rest are dispensable for the expression of the genes, indicating that functional roles of NKX2-1-binding sites are unequally yoked.
转录因子 NKX2-1/TTF-1 参与肺生理病理过程，包括呼吸、先天防御和肿瘤发生。为了了解 NKX2-1 调节此类生理病理过程中相关基因的机制，我们先前进行了 ChIP-seq 分析，鉴定了 NKX2-1 的全基因组结合位点，结果表明 NKX2-1 不仅结合于近端启动子区域，还结合于 NKX2-1 调控的基因的多个内含子和基因间区域。然而，NKX2-1 结合的这些区域（尤其是非近端区域）的作用尚未确定。在此，我们使用 CRISPRi（CRISPR/dCas9-KRAB）方法仔细研究了位于呼吸和先天防御（SFTPB、LAMP3、SFTPA1、SFTPA2）和肺肿瘤发生（MYBPH、LMO3、CD274/PD-L1）相关基因中 19 个由 NKX2-1 结合的区域/位点的功能作用。值得注意的是，CRISPRi 方法表明，一部分 NKX2-1 结合位点对于基因表达是必需的，而其余的则是可有可无的，这表明 NKX2-1 结合位点的功能作用是不均衡的。
