SAT-175
Recruitment of HBx on HBV DNA depends on FXR and is inhibited
by FXR agonist
Benoît Lacombe1, Camille Ménard1, Vincent Lotteau1, Patrice Andre1,
Christophe Ramière1. 1INSERM U1111-CNRS UMR5308-Université Lyon
1 and ENS de Lyon, CIRI, Lyon, France
Email: [email protected]
Background and aims: HBV infection and bile acids metabolism are
interdependent. HBV infection increases expression of the BA nuclear
receptor FXR in infected patients. Reciprocally, FXR is a proviral factor
for HBV whose activity is inhibited by FXR agonists, in vitro and in
vivo. Presence of FXR favors cccDNA completion as well as viral
mRNAs transcription, likely through its binding to two HBV genome
sites (FXRE). Since FXR and HBx interact,we aimed at deciphering the
respective role of HBx and FXR, and ligand, in controlling HBV
transcription.
Method: Expression of FXR was studied in HBV-permissive differentiated
HepaRG (dHepaRG) and in hepatocarcinoma cell line Huh7.
dHepaRG were infected HBVWT or ΔHBx-HBV. ChIP were realized in
Huh7 transfected with Enh2/Cp regulatory region of HBV containing
the two FXRE. FXR KO Huh7 cell line was generated through FXRshRNA
to study the impact of HBx on FXR fixation at FXRE. FXR
agonist GW4064 (GW) was used at 10μM.
Results: HBV infection with either wild type or ΔHBx-HBV increased
FXR expression at the protein level in agreement with in vivo
observations, when treatment with FXR agonist decreased this
expression. Down regulation of FXR by agonists renders difficult
studying the effect of that molecule on FXR binding to FXRE in
dHepaRG. On the opposite, agonist increased FXR protein expression
and/or stabilization in Huh7. We thus took advantage of this
particular FXR expression regulation to perform FXR and HBx-ChIP
in Huh7 transfected with Enh2/Cp promoter. FXR-ChIP revealed FXR
presence in the Enh2/cp in a dose dependent manner. GWtreatment
decreased FXR presence at the viral promoter in contrast to FXR
stabilization at the BSEP promoter. RXR was also recruited at the
Enh2/Cp and released after GW treatment, again at odds with the
stabilization at the BSEP promoter. When HBx was cotransfected in
Huh7 or shFXR-Huh7 cells with Enh2/Cp region, HBxwas recruited to
the viral promoter but not in the shFXR-Huh7 cells or in presence of
agonist.
Conclusion: Altogether, data suggest that FXR proviral activity
depends on its binding to the viral genome at the Enh2/Cp region.
Presence of FXR might recruit cellular factors for cccDNA completion
and HBx for efficient transcription. Inhibition of FXR proviral activity
by agonist might result from repression of FXR expression and of the
release of FXR from its viral targets. This later effect seems to be DNA
sequence dependent since agonist stabilize FXR on cellular promoter.作者: fuke 时间: 2019-4-19 11:49