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Identification of Functional Non-Coding Regions Influencing Lung Inflammation
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A2302 - Identification of Functional Non-Coding Regions Influencing Lung Inflammation
Author Block: Y. Maeda1, M. Guo2, K. Tomoshige1, I. Fink-Baldauf1, J. P. Clancy3; 1Cincinnati Childrens Hosp Med Ctr, Cincinnati, OH, United States, 2Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States, 3MLC 2021, Cincinnati Childrens Hosp, Cincinnati, OH, United States.
Rationale: Recent technological advances using ChIP-seq and CRISPR/Cas9 allow us to identify functional non-coding regions regulating the expression of genes influencing lung diseases. Such technologies led to our previous finding that rs35705950 (SNP associated with Idiopathic Pulmonary Fibrosis) is located at the non-coding enhancer region upstream of MUC5B, which is bound by the pro-mucous transcription factor SPDEF. In this study, we aim to identify the role of non-coding regulatory regions bound by the anti-mucous transcription factor NKX2-1 (also known as TTF-1) influencing the expression of genes associated with lung inflammation.
Methods: Using ChIP-seq datasets, we identified non-coding regions bound by NKX2-1 (anti-mucous transcription factor) in lung epithelial cells. Subsequently using CRISPR/Cas9, we deleted such a region in the genome bound by NKX2-1 in lung epithelial cells and determined whether the deletion influenced the expression of genes associated with lung inflammation, including mucous genes such as MUC5AC.
Results: We identified that NKX2-1 bound non-coding regions at close proximity to genes associated with mucus production and inflammation. Previously, deletion of the non-coding region ~7kb upstream of MUC5AC bound by the mucous activator SPDEF suppressed the expression of MUC5AC but not that of other mucous genes including AGR2, FOXA3, MUC5B and SPDEF, indicating that the region is a specific enhancer inducing the expression of MUC5AC. Unexpectedly, the deletion of the non-coding region ~6kb upstream of MUC5AC bound by the mucous repressor NKX2-1 also suppressed the expression of MUC5AC, indicating that the region is also an enhancer but not a silencer. These results suggest that NKX2-1 represses mucous genes by blocking the activity of the enhancer region but not by recruiting a co-repressor complex such as HDAC.
Conclusions: The approach of deleting non-coding regions associated with disease-linked SNPs, transcription factors or histone marks using CRISPR/Cas9 provides functional knowledge as to whether such non-coding regions function as enhancers, silencers or are redundant in regulating genes that influence lung diseases.
Author Block: Y. Maeda1, M. Guo2, K. Tomoshige1, I. Fink-Baldauf1, J. P. Clancy3; 1Cincinnati Childrens Hosp Med Ctr, Cincinnati, OH, United States, 2Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States, 3MLC 2021, Cincinnati Childrens Hosp, Cincinnati, OH, United States.
Rationale: Recent technological advances using ChIP-seq and CRISPR/Cas9 allow us to identify functional non-coding regions regulating the expression of genes influencing lung diseases. Such technologies led to our previous finding that rs35705950 (SNP associated with Idiopathic Pulmonary Fibrosis) is located at the non-coding enhancer region upstream of MUC5B, which is bound by the pro-mucous transcription factor SPDEF. In this study, we aim to identify the role of non-coding regulatory regions bound by the anti-mucous transcription factor NKX2-1 (also known as TTF-1) influencing the expression of genes associated with lung inflammation.
Methods: Using ChIP-seq datasets, we identified non-coding regions bound by NKX2-1 (anti-mucous transcription factor) in lung epithelial cells. Subsequently using CRISPR/Cas9, we deleted such a region in the genome bound by NKX2-1 in lung epithelial cells and determined whether the deletion influenced the expression of genes associated with lung inflammation, including mucous genes such as MUC5AC.
Results: We identified that NKX2-1 bound non-coding regions at close proximity to genes associated with mucus production and inflammation. Previously, deletion of the non-coding region ~7kb upstream of MUC5AC bound by the mucous activator SPDEF suppressed the expression of MUC5AC but not that of other mucous genes including AGR2, FOXA3, MUC5B and SPDEF, indicating that the region is a specific enhancer inducing the expression of MUC5AC. Unexpectedly, the deletion of the non-coding region ~6kb upstream of MUC5AC bound by the mucous repressor NKX2-1 also suppressed the expression of MUC5AC, indicating that the region is also an enhancer but not a silencer. These results suggest that NKX2-1 represses mucous genes by blocking the activity of the enhancer region but not by recruiting a co-repressor complex such as HDAC.
Conclusions: The approach of deleting non-coding regions associated with disease-linked SNPs, transcription factors or histone marks using CRISPR/Cas9 provides functional knowledge as to whether such non-coding regions function as enhancers, silencers or are redundant in regulating genes that influence lung diseases.
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