The Methyl Trasnferase PRMT1 Functions As Co-Activator of FXR/RXR and Regulates Transcription of FXR Responsive Gene In Hepatocytes.

1. The 2004 AASLD Annual
Meeting Speaker Disclosure
Slide
I have no conflict of interest

2. The Methyl Trasnferase PRMT1 Functions As Co-
Activator of FXR/RXR and Regulates
Transcription of FXR Responsive Gene In
Hepatocytes.
Stefano Fiorucci1, Giovanni Rizzo1, Barbara Renga1,
Elisabetta Antonelli1, Eleonora Distrutti1, Mark
Pruzanski2; Antonio Morelli1, Roberto Pellicciari3
1
Departement of Internal Medicine,
Gastrointestinal and Liver Unit and
3
Department of Drug Technology,
University of Perugia, Italy
2
Intercept Pharmaceuticals, New York, NY

3. FARNESOID X RECEPTOR (FXR)
FXR is a ligand
Role of FXR in bile acid biosynthesis
activated NR that
regulates bile acid
synthesis and
excretion and lipid
homeostasis
FXR is expressed in
liver,kidney and
intestine
Target genes:
CYP7A1, SHP,
BSEP, MRP2,
MDR3, I-BABP
Mikishima M., et al. Science 1999; 284: 1362-65; Parks DJ., et al. Science 1999; 284: 1365-68
Goodwin B., et al. Molecular cell 2000; 6: 507-515

4. Background
FXR is an obligate partner of nuclear
RXR (Retinoid X Receptor)
In the presence of FXR ligands, the
FXR/RXR complex recruits members
of the p160 coactivator family that
acetylate histones

5. Background
In addition to acetylation, histone methylation
is critically involved in chromatin remodelling in
eukaryotic cells
PRMT(Protein aRginine MethylTransferase)-1
is the predominant, if not exclusive, H4
Arg3-methyltransferase in mammalian cells
PRMT1 functions as a coactivator for NRs
(thyroid, estrogen and androgen receptors..)
Kouzarides T. Current Opinion in Genetics & Develop 2002; 12: 198-209

6. FXR Crystal Structure In Complex
With 6-ECDCA
CO2H
FXR -LBD H
GW4064
6-ECDCA
150
+
Relative Luciferase/βGal
CDCA
HO OH π
100
50
0
-10 -9 -8 -7 -6 -5 -4
Agonist concentration (Log M)
6-
ECDCA
Mi L-Z., et al. Molecular Cell 2003; 11:1093-1100

7. Aims
Activation of FXR/RXR complex
recruits PRMT1
PRMT1 mediates the effects of FXR/RXR
ligands on target genes

8. Results

9. FXR/RXR activation
leasds to methylation of Histone 4

10. In vitro methylation
293T cells (kidney fibroblast) were transiently
transfected with FXR, RXR and PRMT1
expression vectors and then induced
with FXR ligands
Cell lysates were Immunoprecipitated
with anti-FXR antiboby.
The immunoprecipitated were then used for
in vitro methylation by incubating them with
methyl 3H-methionine followed by
SDS page and fluorography

11. FXR/RXR posses H4 specific histone methylation activity
6-ECDCA - + - - + + +
In vitro H4
methylation
GST- IP:
IP anti-: PAS CD3 FXR CD3 FXR
PRMT1 anti-FXR
- + 6-ECDCA
WB: anti FXR
WB: anti RXR
WT
FXR/RXR

12. Is FXR/RXR-induced histone
methylation mediated
by PRMT1?

13. FXR Interacts In Vivo With PRMT1
6-ECDCA + + - + -
WB: PRMT1
(42 KDa)
IP: IP:
αCD3 α FXR LT 293T cells
FXR/RXR/PRMT1
GW4064 + - + - + -
WB: PRMT1
(42 KDa)
Mks
IP: IP:
LT
α CD3 α FXR 293T cells
FXR/RXR/PRMT1

14. HepG2 cells
PRMT1 Functions As A Coactivator of
FXR/RXR Heterodimer
4000000 DMSO
6-ECDCA * 150000 FXR/RXR/PRMT1
*
3000000
Rlu/ β Gal
FXRE-Mut 100000
rlu/β-gal
2000000
*
* ** FXR/RXR
1000000 50000
0
0
PRMT1 FXR/RXR FXR/RXR/PRMT1
0 0.1 1 10 100
6-ECDCA (µM)

15. PRMT1 Induces Transcriptional Activation
of BSEP
FXR/RXR FXR/RXR/PRMT1

16. PRMT1 Induces Transcriptional Activation
of SHP
FXR/RXR FXR/RXR/PRMT1

17. PRMT1 Enhances FXR Activation of The
BSEP Promoter by H4 Methylation
Immunochromatin precipitation (ChIP) was used to analyze
the promotorial context of FXR responsive gene promoter
in HepG2 cells cotransfected with FXR, RXR, PRMT1
expression vectors
ChIPs was performed with anti: FXR, PRMT1, dimethyl-
H4 (Arg3) and CytC. Immunoprecipitates were analyzed
by qRT-PCR to BSEP gene promoter (binds FXR)*
Ananthanarayanan M., et al., JBC 2001:276:28857

18. PRMT1 ENHANCES FXR ACTIVATION OF THE BSEP PROMOTER BY
H4 METHYLATION.
- + - + - + - + 6-ECDCA
BSEP Promoter
Met-H4
IP anti: FXR PRMT1 CytC
(Arg3)
HepG2 cells
FXR/RXR/PRMT1
IP anti: FXR Met-H4
PRMT1 CytC
(arg3)

19. Inhibition of Arginine Methyltransferase Activity
Decreases FXR-mediated Transcription
6E
MTA 6E MTA
+MTA
BSEP
β-actin
6E GW
HepG2 cells
NT MTA 6E GW
+MTA +MTA FXR/RXR/PRMT1

20. Conclusion
PRMT1 and histone methylation
pathways are implicated in the activation
of FXR responsive
genes with biological relevance
in the homeostasis
of bile acids and cholesterol

21. COACTIVATOR COMPLEX RECRUITED
BY FXR ACTIVATION
NR NR

22. FXR/RXR Transactivation Induced By PRMT1
Is Enhanced by RXR Ligand 9-CIS RA
HepG2 cells
Rlu/βgal
NT 9-cis 6E 6E NT 9-cis 6E 6E
9-cis 9-cis
FXR/RXR Is a Permissive Heterodimer.
RXR ligand 9-cisRA induces
transactivation by PRMT1