CYP2C9 Transactivation assays. HepG2 cells were cultured in the Eagles minimal essential medium supp

1
Mediator subunit-25 (Med25) enhances the
synergistic effect of CAR-HNF4a on CYP2C9 gene
regulation Waynekid Kam, Ritu Rana, Sailesh
Surapureddi, Joyce A. Goldstein Laboratory of
Pharmacology, National Institute of Environmental
Health Sciences, NIH, DHHS, Research Triangle
Park, NC 27709

Results
CYP2C9 is an important liver enzyme which
metabolizes many clinically important
therapeutics as well as endogenous compounds.
The CYP2C enzymes can metabolize about 20 of
clinical drugs and over-the-counter remedies.
Drug metabolism, the degradation of chemicals
serves as an essential role to foreign compound
clearance, a bodys way to inactivate and clear
drugs. Based on the important pharmacological
and physiological function of CYP2C9 in human
liver, it is necessary to understand the
mechanism of the various factors that influence
its transcriptional regulation. Recent studies
have shown that NCOA6, an important coactivator,
mediates the cross talk between the CAR binding
site and the HNF4a binding site in the CYP2C9
promoter that results in a synergistic activation
of CYP2C9 by CAR and HNF4a. The present study
shows that Med25, as an important subunit of
mammalian mediator complex, is involved in CYP2C9
gene regulation by bringing the CAR-HNF4a complex
to the basal transcriptional machinery.
Gene Regulation
Introduction

Figure 1.
Protein Interactions
A.
Med25 enhances CAR-HNF4a synergistic
transactivation of CYP2C9 reporter expression.
GST -
Input
CAR -
PXR -
HNF4a -
Beads (containing bound GST-fusion protein)
radiolabeled protein
3 hours, RT, agitation
Med25
Pellet beads by centrifugation
3 Washes
Washed Beads
Beads pelleted by centrifugation
PGC-1
Transcription
Supernatant discarded
Bead samples analysed by SDS-PAGE
B.
Med25 enhances CAR-HNF4a synergistic upregulation
of CYP2C9 gene expression.
GST-pull down shows that Med25 specifically
interacts with HNF4a

Two-Hybrid Screening
Med25, as coactivator of HNF4a, recruits
Polymerase II for transcription
Confocal Scans

• In the present study, we proposed to establish
  the role of Med25 on the human CYP2C9 promoter,
  providing strong evidence that it contributes to
  the regulation of the metabolism of many clinical
  drugs and endogenous compounds. Through previous
  studies, HNF4a has already been known to
  upregulate the CYP2C9 transcription. We herein
  identify Med25 as a coactivator and a new
  interacting partner of HNF4a. GST-pull down
  assay indicated that Med25 specifically interacts
  with HNF4a but not with CAR or PXR.
  Coimmunoprecipitations confirmed the Med25 and
  HNF4a interaction in a mammalian cell system.
  Promoter expression and mRNA induction studies
  involving overexpression of Med25 in HepG2 cells
  resulted in an increase in the CAR-HNF4a mediated
  transcriptional upregulation of CYP2C9 gene
  expression. Conversely, silencing of Med25
  abolished HNF4a mediated and reduced CAR-HNF4a
  mediated synergistic activation of the CYP2C9
  gene expression. Furthermore, coimmunoprecipitati
  on with HNF4a antibody showed that Med25
  associates with RNA polymerase II along with
  HNF4a binding proteins and silencing of Med25
  diminishes this association. These results
  suggest that Med25 may have a crucial role in
  bringing the CAR-HNF4a complex to the
  polymerase-II based transcriptional machinery.
  The interaction of Med25 with the activation
  domain of HNF4a and the large subunit of RNA
  polymerase II would form the pre-initiation
  complex (PIC) on the CYP2C9 promoter.

HNF4a
Discussion
Flag-Med25
HNF4a
Figure 2.
Med25
siRNA against Med25 specifically downregulates
Med25 in HepG2 cells
DAPI
Merged
Confocal images show colocalization between HNF4a
and Med25
The yeast two-hybrid screening with Myriad
Genetics indicates Med25 is a new interacting
partner of HNF4a.

In Vivo Interactions
CYP2C9 Transactivation assays. HepG2 cells
were cultured in the Eagles minimal essential
medium supplemented with 10 fetal bovine serum.
Transfections with outlined plasmid constructs
were performed with Lipofectamine 2000 as
suggested. Medium was replaced 24 h later .
After an additional 24 h, they were subsequently
lysed with 100 µl of passive lysis buffer for 15
min at room temperature with gentle rocking.
Firefly and renilla luciferease activity were
assayed with Promega dual Glo luciferase system.
Protein-Protein Interactions. HNF4a, CAR and
PXR were expressed as GST fusion proteins in
bacterial systems and were immobilized on
GSH-spharose beads. In vitro synthesized (35S
Methionine) Med25 was allowed to interact with
immobilized nuclear receptors as GST fustion
proteins with appropriate controls. Interactions
were analyzed on SDS-PAGE and auto radiography.
Immunoblotting and Co-Immunoprecipitations.
HepG2 cells were infected with Adeno virus
expressing HNF4a or Flag tagged Med25
individually or in combination. Nuclear extracts
were prepared and immunorprecipitated with HNF4a
antibody. The immunoprecipitates were probed with
Anti-Flag antibody in order to determine the
presence of Med25 and RNA polymerase II antibody
to detect RNA Polyermase II along with HNF4a
antibody. The westerns were probed with
Streptavidin-HRP conjugate and the interaction
detected with ECL detection systems
Methods
Silencing Med25
a-Flag Ab Flag-Med25 HNF4a
HNF4a Ab
Med25 Immunoblot
HNF4a Immunoblot
CO-IP with a-Flag Ab
N C N C
N C N C

• Chen Y, Kissling G, Negishi M and Goldstein JA
  (2005) The nuclear receptors constitutive
  androstane receptor and pregnane X receptor
  cross-talk with hepatic nuclear factor 4alpha to
  synergistically activate the human CYP2C9
  promoter. J Pharm Exp Ther. 314(3)1125-33.
• Surapureddi S, Rana R, Reddy JK, Goldstein JA
  (2008) The coactivator NCOA6 mediates the
  synergistic activation of human cytochrome P-450
  2C9 by the constitutive androstane receptor and
  hepatic nuclear factor-4alpha. Mol Pharmacol.
  

Input H M HM
References
Flag-Med25
HNF4a
GFP
HNF4a
Med25 SiMed25 Med25 SiMed25
Silencing of Med25 does not have an effect on the
expression of HNF4a
CO-IP with HNF4a Ab
HNF4aFlag-Med25
HNF4aSiMed25
Input H M HM
Pol II
Flag-Med25
Flag-Med25
HNF4a
Input IgG HNF4aAb Input IgG
HNF4aAb
Med25 recruits RNA polymerase II to the HNF4a
binding complex
Immunoblots showing interaction of Med25 with
HNF4a in AD-293