The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling
Fabienne Gally, Sarah K. Sasse, Jonathan S. Kurche, Margaret A. Gruca, Jonathan H. Cardwell, Tsukasa Okamoto, Hong W. Chu, Xiaomeng Hou, Olivier B. Poirion, Justin Buchanan, Sebastian Preissl, Bing Ren, Sean P. Colgan, Robin D. Dowell, Ivana V. Yang, David A. Schwartz, Anthony N. Gerber
Fabienne Gally, Sarah K. Sasse, Jonathan S. Kurche, Margaret A. Gruca, Jonathan H. Cardwell, Tsukasa Okamoto, Hong W. Chu, Xiaomeng Hou, Olivier B. Poirion, Justin Buchanan, Sebastian Preissl, Bing Ren, Sean P. Colgan, Robin D. Dowell, Ivana V. Yang, David A. Schwartz, Anthony N. Gerber
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Research Article Pulmonology

The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling

Abstract

The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this –3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the –3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.

Authors

Fabienne Gally, Sarah K. Sasse, Jonathan S. Kurche, Margaret A. Gruca, Jonathan H. Cardwell, Tsukasa Okamoto, Hong W. Chu, Xiaomeng Hou, Olivier B. Poirion, Justin Buchanan, Sebastian Preissl, Bing Ren, Sean P. Colgan, Robin D. Dowell, Ivana V. Yang, David A. Schwartz, Anthony N. Gerber

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Figure 1

The MUC5B –3 kb region exhibits enhancer activity that is regulated by STAT3 and SPDEF.

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The MUC5B –3 kb region exhibits enhancer activity that is regulated by S...
(A) Location of MUC5B Short (red, 766 bp) and Long (blue, 4532 bp) regions cloned into reporter constructs relative to the transcription start site (black arrow). (B–F) Mean (±SD) normalized luciferase activity of indicated Short (red; top) and Long (blue; bottom) MUC5B reporter constructs and respective empty vector (EV) control in A549 cells. (B) WT constructs under basal culture conditions (n = 4/group, *P ≤ 0.05 versus EV via t test). (C) WT versus mutant constructs with binding site mutations for STAT3, ETS1, or both (n = 4/group, *P ≤ 0.0001 versus WT via 1-way ANOVA with Bonferroni correction). (D) WT constructs cotransfected with SPDEF or STAT3 cDNA expression constructs or empty expression vector control (Ctrl; n = 4/group, *P ≤ 0.003 versus WT + Ctrl via 1-way ANOVA with Bonferroni correction). (E and F) WT constructs cotransfected with siRNA targeting SPDEF (E), STAT3 (F), or control (siCtrl; n = 4/group, *P ≤ 0.001 versus WT + siCtrl via 1-way ANOVA with Bonferroni correction). (G) ChIP-qPCR analysis of mean (±SD) relative occupancy of SPDEF and IgG across the MUC5B –3 kb region in A549 cells (n = 3 (IgG) or 4 (SPDEF) per group; *P ≤ 0.05 versus geometric mean of IgG occupancy at 3 negative control regions via t test. Schematic indicates locations of putative STAT/ETS1 binding sites and regions targeted by ChIP-qPCR primers. (H) Mean (±SD) normalized luciferase activity of WT and mutant MUC5B Short reporter constructs with G or variant T at the G/T transversion site, respectively (n = 4/group). (I) qPCR analysis quantifying relative MUC5B expression in untransfected WT control (Ctrl-GG) and CRISPR-Cas9–edited WT (GG) and variant (TT) A549 clones (n = 3/group). Data in each panel are representative of at least 3 independent experiments, except for I, which was performed in 2 independent clones for each genotype.