Significant progress has been made towards new therapies for Cystic Fibrosis (CF). This devastating inherited disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a multifunctional protein (CFTR) with a pivotal role in regulating anion transport across cell membranes. CFTR is particularly well characterized in airway, pancreas, intestine and male genital duct epithelia. Current pharmacological approaches to restore normal activity to defective CFTR focus on correcting cellular trafficking of misfolding mutants and on potentiating activity of ion channels with faulty conductance. For both classes of mutation increasing CFTR gene expression and hence the amount of CFTR protein substrate, would likely enhance in vivo drug efficacy. Moreover, existing therapeutic goals will not benefit the ~15% of patients lacking sufficient functional CFTR due to mutations that disrupt gene expression. A detailed understanding of the normal transcriptional mechanisms controlling the gene is a prerequisite for successful approaches to modulate CFTR expression. In the previous R01 funding period we achieved our goal to elucidate the tissue-specific control pathways of the CFTR gene in airway and intestinal epithelial cells. The CFTR locus lies within a topologically associating domain (TAD) established by CCCTC-binding factor (CTCF) insulator elements. Within this environment, distal cis-acting enhancers are brought into close association with the gene promoter by a looping mechanism that is stabilized by the cohesin complex. The enhancers are cell- type specific and associate with different activating or repressing transcription factors (TFs). Building on our greatly improved understanding of CFTR regulation, we will pursue three specific aims addressing the overarching hypothesis that targeted modulation of CFTR gene expression will increase functional CFTR protein at the cell surface. Increasing transcript levels, alone or in combination with pharmacological approaches, will alleviate disease phenotype. Further, we hypothesize that an enhanced understanding of the cis-elements and interacting factors coordinating cell-type specific gene expression will reveal new ways to augment CFTR transcription. In the first aim we will determine how cell-specific cis-regulatory elements coordinate expression of the endogenous CFTR locus. Experiments will combine CRISPR/Cas9 targeting of these elements with gene expression assays and analysis of locus architecture. In the second aim we will decipher the transcriptional network that regulates CFTR expression in primary airway epithelial cells. Candidate TFs will be examined by genome-wide methods to reveal their impact on the locus. In the third aim we will identify and characterize pathways that activate CFTR gene expression to enhance CFTR mRNA and functional CFTR protein expression. These results will enable novel approaches to increase CFTR in the airway for therapeutic benefit and will inform new therapies for other complex genetic diseases.

Public Health Relevance

Airway epithelial dysfunction has a pivotal role in the pathology of cystic fibrosis, a devastating inherited disorder in search of new therapies. Here we build on our substantial progress in elucidating regulatory mechanisms for the causative CFTR gene, to develop novel therapeutic approaches that enhance its expression.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL094585-08
Application #
9903422
Study Section
Genetics of Health and Disease Study Section (GHD)
Program Officer
Lachowicz-Scroggins, Marrah Elizabeth
Project Start
2009-01-01
Project End
2021-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
8
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Genetics
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Strug, Lisa J; Stephenson, Anne L; Panjwani, Naim et al. (2018) Recent advances in developing therapeutics for cystic fibrosis. Hum Mol Genet 27:R173-R186
Gillen, Austin E; Yang, Rui; Cotton, Calvin U et al. (2018) Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells. J Cyst Fibros 17:444-453
Mutolo, Michael J; Leir, Shih-Hsing; Fossum, Sara L et al. (2018) A transcription factor network represses CFTR gene expression in airway epithelial cells. Biochem J 475:1323-1334
Fossum, Sara L; Mutolo, Michael J; Tugores, Antonio et al. (2017) Ets homologous factor (EHF) has critical roles in epithelial dysfunction in airway disease. J Biol Chem 292:10938-10949
Yang, Rui; Kerschner, Jenny L; Harris, Ann (2016) Hepatocyte nuclear factor 1 coordinates multiple processes in a model of intestinal epithelial cell function. Biochim Biophys Acta 1859:591-8
Yang, Rui; Kerschner, Jenny L; Gosalia, Nehal et al. (2016) Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus. Nucleic Acids Res 44:3082-94
Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared et al. (2015) Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating. J Physiol 593:2427-46
Zhang, Zhaolin; Leir, Shih-Hsing; Harris, Ann (2015) Oxidative stress regulates CFTR gene expression in human airway epithelial cells through a distal antioxidant response element. Am J Respir Cell Mol Biol 52:387-96
Fossum, Sara L; Mutolo, Michael J; Yang, Rui et al. (2014) Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells. Nucleic Acids Res 42:13588-98
Browne, James A; Harris, Ann; Leir, Shih-Hsing (2014) An optimized protocol for isolating primary epithelial cell chromatin for ChIP. PLoS One 9:e100099

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