CRISPRi-mediated functional analysis of NKX2-1-binding sites in the lung. 2021

William D Stuart, and Iris M Fink-Baldauf, and Koichi Tomoshige, and Minzhe Guo, and Yutaka Maeda
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.

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.

UI MeSH Term Description Entries
D008168 Lung Either of the pair of organs occupying the cavity of the thorax that effect the aeration of the blood. Lungs
D008175 Lung Neoplasms Tumors or cancer of the LUNG. Cancer of Lung,Lung Cancer,Pulmonary Cancer,Pulmonary Neoplasms,Cancer of the Lung,Neoplasms, Lung,Neoplasms, Pulmonary,Cancer, Lung,Cancer, Pulmonary,Cancers, Lung,Cancers, Pulmonary,Lung Cancers,Lung Neoplasm,Neoplasm, Lung,Neoplasm, Pulmonary,Pulmonary Cancers,Pulmonary Neoplasm
D009687 Nuclear Proteins Proteins found in the nucleus of a cell. Do not confuse with NUCLEOPROTEINS which are proteins conjugated with nucleic acids, that are not necessarily present in the nucleus. Nucleolar Protein,Nucleolar Proteins,Nuclear Protein,Protein, Nuclear,Protein, Nucleolar,Proteins, Nuclear,Proteins, Nucleolar
D011401 Promoter Regions, Genetic DNA sequences which are recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Highly conserved sequences within the promoter include the Pribnow box in bacteria and the TATA BOX in eukaryotes. rRNA Promoter,Early Promoters, Genetic,Late Promoters, Genetic,Middle Promoters, Genetic,Promoter Regions,Promoter, Genetic,Promotor Regions,Promotor, Genetic,Pseudopromoter, Genetic,Early Promoter, Genetic,Genetic Late Promoter,Genetic Middle Promoters,Genetic Promoter,Genetic Promoter Region,Genetic Promoter Regions,Genetic Promoters,Genetic Promotor,Genetic Promotors,Genetic Pseudopromoter,Genetic Pseudopromoters,Late Promoter, Genetic,Middle Promoter, Genetic,Promoter Region,Promoter Region, Genetic,Promoter, Genetic Early,Promoter, rRNA,Promoters, Genetic,Promoters, Genetic Middle,Promoters, rRNA,Promotor Region,Promotors, Genetic,Pseudopromoters, Genetic,Region, Genetic Promoter,Region, Promoter,Region, Promotor,Regions, Genetic Promoter,Regions, Promoter,Regions, Promotor,rRNA Promoters
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D002471 Cell Transformation, Neoplastic Cell changes manifested by escape from control mechanisms, increased growth potential, alterations in the cell surface, karyotypic abnormalities, morphological and biochemical deviations from the norm, and other attributes conferring the ability to invade, metastasize, and kill. Neoplastic Transformation, Cell,Neoplastic Cell Transformation,Transformation, Neoplastic Cell,Tumorigenic Transformation,Cell Neoplastic Transformation,Cell Neoplastic Transformations,Cell Transformations, Neoplastic,Neoplastic Cell Transformations,Neoplastic Transformations, Cell,Transformation, Cell Neoplastic,Transformation, Tumorigenic,Transformations, Cell Neoplastic,Transformations, Neoplastic Cell,Transformations, Tumorigenic,Tumorigenic Transformations
D005818 Genetic Engineering Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc. Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000074482 Thyroid Nuclear Factor 1 A homeobox protein and transcription factor that localizes to the cell nucleus where it activates expression of thyroid specific genes such as THYROGLOBULIN and the THYROTROPIN RECEPTOR. It is critical for maintaining thyroid tissue in a differentiated state and also plays a role in lung development. Mutations in the NKX2-1 gene are associated with CHOREA, BENIGN HEREDITARY. Homeobox Protein Nkx-2.1,NK2 Homeobox 1 Protein,TITF-1 Protein,TITF1 Protein,TTF-1 Thyroid Nuclear Factor,Thyroid Transcription Factor 1,Thyroid-Specific Enhancer-Binding Protein,Enhancer-Binding Protein, Thyroid-Specific,Homeobox Protein Nkx 2.1,Nkx-2.1, Homeobox Protein,TITF 1 Protein,TTF 1 Thyroid Nuclear Factor,Thyroid Specific Enhancer Binding Protein
D000081204 Chromatin Immunoprecipitation Sequencing A technique for identifying the BINDING SITES on nucleic acid sequences that are associated with binding proteins. ATAC-Seq,Assay for Transposase-Accessible Chromatin Using Sequencing,CLIP-Seq,ChIA-PET,ChIP Sequencing,ChIP-Chip,ChIP-Exo,ChIP-Exonuclease,ChIP-PET,ChIP-Seq,Chromatin Immuno-Precipitation Paired-End Tag,Chromatin Immuno-precipitation Sequencing,Chromatin Immunoprecipitation Paired-End Tag,Chromatin Immunoprecipitation Sequencing-Chip,Cross-Linking and Immunoprecipitation Followed by Deep Sequencing,HITS-CLIP,High-Throughput Sequencing of RNA Isolated by Crosslinking Immunoprecipitation,Assay for Transposase Accessible Chromatin Using Sequencing,ChIP Exonuclease,Chromatin Immuno Precipitation Paired End Tag,Chromatin Immuno precipitation Sequencing,Chromatin Immunoprecipitation Paired End Tag,Chromatin Immunoprecipitation Sequencing Chip,Chromatin Immunoprecipitation Sequencing-Chips,Cross Linking and Immunoprecipitation Followed by Deep Sequencing,High Throughput Sequencing of RNA Isolated by Crosslinking Immunoprecipitation,Sequencing, ChIP,Sequencing, Chromatin Immuno-precipitation,Sequencing, Chromatin Immunoprecipitation

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