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Abstract: SA-PO293

TGF-β1 Promotes Fibrotic Gene Expression through Induction of Histone Variant H3.3 and Histone Chaperone HIRA

Session Information

Category: Cell Biology

  • 204 Extracellular Matrix Biology, Fibrosis, Cell Adhesion

Authors

  • Shindo, Toshihiro, Hiroshima university, Hiroshima city, Japan
  • Doi, Shigehiro, Hiroshima university, Hiroshima city, Japan
  • Sasaki, Kensuke, Hiroshima university, Hiroshima city, Japan
  • Nakashima, Ayumu, Hiroshima university, Hiroshima city, Japan
  • Masaki, Takao, Hiroshima university, Hiroshima city, Japan
Background

Recent studies show that histone variants and their chaperones serve as epigenetic marks that regulate transcriptional activity. In this study, we investigated transforming growth factor (TGF)-β1-induced histone variant H3.3 and its histone chaperone, HIRA, on fibrotic genes in vivo and in vitro.

Methods

Male C57BL/6J mice underwent unilateral ureteral obstruction (UUO) and were sacrificed on day 7. In UUO mice, expression of H3.3, HIRA, and α-smooth muscle actin (αSMA) were evaluated by western blotting (WB) and immunohistochemistry (IHC) with or without administration of a TGF-β1-neutralizing antibody. For in vitro experiments, rat renal tubular cells (NRK52E) and rat kidney fibroblasts (NRK49F) were used. TGF-β1-iduced expression of H3.3, HIRA and αSMA was assessed by WB with pretreatment of Smad3 or HIRA siRNAs. Furthermore, chromatin immunoprecipitation (ChIP) assays were carried out using primers for fibrotic genes, which were designed to include in a Smad-binding element, in TGF-β1-stimulated NRK52E cells.

Results

Expression of H3.3 and HIRA was increased in UUO mice, and the TGF-β1 -neutralizing antibody suppressed their expression. Smad3 siRNA treatment inhibited expression of H3.3 and HIRA in TGF-β1-stimulated NRK52E cells. HIRA siRNA treatment attenuated expression of H3.3 and αSMA in TGF-β1-stimulated NRK52E cells. In ChIP assays, TGF-β1 increased promoter activities of collagen 1 (Col1a1), connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1 (PAI-1) in NRK-52E cells, which were colocalized with H3.3 and suppressed by the TGF-β1 neutralizing antibody .

Conclusion

TGF-β1-induced H3.3 and HIRA play an important role in expression of fibrotic genes.