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. 2021 Feb;160(3):889-905.e10.
doi: 10.1053/j.gastro.2020.10.008. Epub 2020 Oct 12.

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-β Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis

Sayed Obaidullah Aseem  1 , Nidhi Jalan-Sakrikar  1 , Cheng Chi  1 , Amaia Navarro-Corcuera  1 , Thiago M De Assuncao  1 , Feda H Hamdan  1 , Shiraj Chowdhury  1 , Jesus M Banales  2 , Steven A Johnsen  3 , Vijay H Shah  3 , Robert C Huebert  4
Affiliations

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-β Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis

Sayed Obaidullah Aseem et al. Gastroenterology. 2021 Feb.

Abstract

Background & aims: 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.

Methods: Chromatin immunoprecipitation (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 used 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.

Results: TGFβ stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFβ-mediated transcription. In contrast, 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). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFβ-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFβ-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis.

Conclusions: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.

Keywords: Epigenetics; FN; PAI1.

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Figures

Figure 1:
Figure 1:
ChIP-seq analysis of cholangiocytes shows selective, TGFβ-mediated H3K9ac. A) H3K27ac is increased with TGFβ stimulation, whereas H3K9ac is seen in fewer sites. SMAD3 binding is increased following TGFβ stimulation (top panels show mean) (N=2). Sites were arranged in the same descending order in all panels. B) Overlap of ChIP-seq sites that were significantly increased with TGFβ stimulation (Fold change >/= 2, FDR 0.05). C) The TSS and surrounding 3 Kbps of each ChIP-seq target was averaged for all TGFβ-upregulated genes identified by RNA-seq, showing an increase in H3K27ac and SMAD3 binding, but relatively fewer genes gaining H3K9ac. D) IPA was performed on the TGFβ-regulated genes identified by RNA-seq. E) IPA was performed on the H3K27ac and H3K9ac associated genes that were also upregulated in the RNA-seq analysis. F) In the hepatic fibrosis pathway, all upregulated genes associated with increased H3K27ac, and 71% associated with increased H3K9ac.
Figure 2:
Figure 2:
TGFβ-mediated expression of FN1 and SERPINE is associated with H3K9ac in cholangiocytes. A) Upregulation of canonical HSC activating genes identified by RNA-seq were confirmed by RT-PCR (N=4). B) ChIP-seq tracks showing increased H3K9ac, H3K27ac and SMAD3 binding with TGFβ (red tracks) compared to vehicle (blue tracks) in the FN1 and SERPINE1 promoters. C) ChIP-qPCR shows significant TGFβ-mediated increase in H3K9ac in the FN1 and SERPINE1 promoters. H3K27ac is also significantly increased but H3K4me3 is not (N=3). D) qPCR analysis normalized to GAPDH shows KAT2A transcript is expressed >10 fold compared to KAT2B in H69 cells and >4 fold in NHC (N=3). E) Cholangiocytes from WT and Mdr2−/− mice along with the non-cholangiocyte liver cells were subjected to immunoblot analysis (N=3). F) Immunohistochemistry confirms Kat2a localization in cholangiocytes of Mdr2−/− mice (N=3–4).
Figure 3:
Figure 3:
KAT2A is essential for the TGFβ-mediated expression of FN1 and SERPINE1. A) siRNA resulted in >70% KAT2A knockdown, which significantly inhibited FN and PAI1. B) TGFβ stimulation results in increased binding of KAT2A to the promoters of FN1 and SERPINE1, which is significantly inhibited by KAT2A siRNA knockdown. C) The TGFβ-mediated increase in H3K9ac in the promoters of FN1 and SERPINE1 is inhibited by KAT2A knockdown. D) The TGFβ-mediated increase in FN1 and SERPINE1 is inhibited by CPTH6. E) CPTH6 (50 μM) inhibited the TGFβ-mediated increase in H3K9ac in the promoters of FN1 and SERPINE1. (N=3).
Figure 4:
Figure 4:
ATAC-seq demonstrates that the TGFβ-mediated chromatin acessibility of HSC-activating genes is H3K9ac-dependent in H69 cholangiocytes. A) Heat map of ATAC-seq shows that TGFβ leads to a large number of chromatin sites undergoing changes to an open conformation (bottom panels), and relatively fewer sites undergoing changes to a closed conformation (middle panels). B) The chromatin sites significantly reduced by CPTH6 co-treatment compared to TGFβ alone were taken to be H3K9ac-dependent. These sites were mapped to the nearest genes, which were used to perform IPA. C) The ATAC-seq sites that underwent significant increase in accessibility with TGFβ were overlayed with ChIP-seq sites showing a significant TGFβ-mediated increase. D) Analysis of the H3K9ac-dependent ATAC-seq sites for transcription factor binding sites.
Figure 5:
Figure 5:
SMADs form a complex with KAT2A and recruit the complex to the promoter of FN1 and SERPINE1. A) Immunoprecipiating with KAT2A antibodies shows pull down of SMAD2/3, and conversely, immunoprecipitating with SMAD2/3 shows the presence of KAT2A (N=3). B) Proximity ligation assays using anti-KAT2A and anti-SMAD2/3 antibodies shows the presence of KAT2A-SMAD2/3 complexes that are significantly increased with TGFβ (N=3). C) The TGFβ-mediated increase in promoter binding of KAT2A is inhibited by SMAD4 siRNA knockdown. Similarly, the TGFβ-mediated increase in SMAD3 binding at the FN1 and SERPINE1 promoters is attenuated by SMAD4 or KAT2A siRNA knockdown (N=4). D) siRNA SMAD4 knockdown inhibited the TGFβ-mediated increase in expression of FN1 and PAI1 (N=3). E) The SMAD3 inhibitor, SIS3, inhibited the TGFβ-mediated increase in expression of FN1 and PAI1 (N=3).
Figure 6:
Figure 6:
Pharmacologic inhibition of H3K9ac with CPTH6 prevents biliary fibrosis in Mdr2−/− mice. A-B) Picrosirius Red and Trichrome stained liver sections from Mdr2−/− mice treated with vehicle or CPTH6 (N=11 vehicle, 12 CPTH6). C) Hydroxyproline assays show reduced levels with CPTH6 treatment of Mdr2−/− mice compared to vehicle. D) Col1a1, α-Sma and Fn1 mRNA expression were reduced in whole liver of the Mdr2−/− mice treated with CPTH6. E-F) Isolated cholangiocytes from CPTH6-treated Mdr2−/− mice show reduced H3K9ac in the promoters and mRNA expression of Fn1 and Serpine1 (N=4).
Figure 7:
Figure 7:
Cholangiocyte-selective genetic knockdown of Kat2a prevents biliary fibrosis in mice. A) Mice carrying a Krt19-CreERT allele and Kat2afl/fl mice served as Controls. Kat2afl/+ carrying a Krt19-CreERT allele are denoted as Kat2a Het. All mice received Tamoxifen for 5 days after which they received either normal chow or DDC diet for 3 weeks. B) Immunohistochemistry shows Kat2a immunostaining in the portal regions of Control and Kat2a Het, DDC diet-fed mice (N=4 each). C) qPCR showed that Kat2a mRNA levels are increased with DDC diet in Control mice, but this affect is blunted in Kat2a Het mice. Col1a1, α-Sma and Fn1 mRNA expression are increased with DDC but this affect is attenuated in Kat2a Het mice (N=9 Control; 5 Het, normal chow; 10 Control, DDC; 10 Het, DDC). D) Hydroxyproline assays show increased levels in Control mice on DDC diet compared to Normal diet. This was significantly reduced in Kat2a Het mice on DDC diet (N=6 Control; 8 Het, normal chow; 10 Control, DDC; 10 Het, DDC). E) Picrosirius Red stained liver (N=3 Control; 3 Het, normal chow; 10 Control, DDC; 10 Het, DDC).
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