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A2285 - Transcriptional Regulation of MUC5B and the Enhancer Harboring the IPF-Associated Variant rs35705950
Author Block: A. N. Gerber1, F. Gally2, S. K. Sasse1, B. A. Helling3, C. M. Evans4, I. V. Yang5, S. P. Colgan4, D. A. Schwartz4; 1Dept of Med, National Jewish Health, Denver, CO, United States, 2National Jewish Hlth, Denver, CO, United States, 3University of Chicago, Chicago, IL, United States, 4Dept of Med, Univ of Colorado, Aurora, CO, United States, 5Dept of Med, Univ of Colorado Denver, Aurora, CO, United States.
Rationale: The MUC5B variant, rs35705950, a G-to-T transversion ~3kb upstream of the MUC5B start site has been strongly associated with the risk of developing idiopathic pulmonary fibrosis (IPF) in numerous studies. Moreover, we have previously shown that MUC5B expression is increased in IPF in GG, GT, and TT genotype patients relative to non-IPF controls, with the highest expression associated with GT and TT genotypes. Thus, both MUC5B regulation and the molecular basis for the regulatory effect of the G-to-T transversion on MUC5B expression are implicated in the pathogenesis of IPF and may represent novel targets for therapeutic manipulation. However, the mechanisms underpinning the impact of the G-to-T transversion on MUC5B expression and the general regulatory control of MUC5B expression are not well understood.
Methods: We used cloning and reporter assays to evaluate a ~700 bp region centered at the rs35705950 variant for enhancer activity and to interrogate other conserved regions within the MUC5B locus for regulatory function. We applied bioinformatics-based approaches to identify putative controlling transcription factors and their associated binding sites within the 5’-enhancer that encompasses the rs35705950 variant. We applied site directed mutagenesis, gene knockdown and chromatin immunoprecipitation (ChIP) to ascribe regulatory activity to specific transcription factors.
Results: Within the 700 bp 5’-enhancer, we identified highly conserved sequences that align with the consensus binding site preferences for STAT, ETS, FOXA, and ZIC transcription factor families, among others. Site directed mutagenesis revealed a functional role for several of these binding sites, including FOXA2, which was corroborated using ChIP and gene knockdown. We also identified a novel intronic regulatory region that functions as a repressor of transcription, whereas enhancer activity was identified adjacent to the MUC5B 3’-UTR.
Conclusion: MUC5B expression is controlled by multiple regions that are widely separated across genomic space, implicating a role for chromatin looping in MUC5B regulation. Within the 5’ enhancer that harbors the rs35705950 variant, multiple transcription factors from different families, including the FOXA and STAT families, regulate MUC5B expression. The proximity of the binding sites for these factors with the rs35705950 variant suggests that the G-T transversion may influence the activity of a STAT-FOXA multi-protein MUC5B regulatory complex. Ongoing studies are targeted at further defining these interactions within the context of the 700 bp 5’-enhancer, the rs35705950 G-to-T transversion, and the greater MUC5B locus.