Epigenomic Evaluation of Cholangiocyte TGFβ Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis.

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Transforming growth factor β (TGFβ) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFβ that mediate transcription of fibrogenic signals.ChIP-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFβ stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was utilized to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) fed mice were used as animal models.TGFβ stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFβ-mediated transcription. In contrast, histone 3 lysine 9 acetylation (H3K9ac) was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFβ. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). siRNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFβ-mediated increase in FN1 and SERPINE. SMAD3 ChIP-seq and ATAC-seq suggested that TGFβ-mediated H3K9ac occurs through SMAD signaling, which was confirmed using co-localization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis.Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.


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