1. Volume 138, Issue 2, Pages 705-714.e4 (February 2010)
MeCP2 Controls an Epigenetic Pathway That Promotes Myofibroblast Transdifferentiation and Fibrosis 
Jelena Mann, David C.K. Chu, Aidan Maxwell, Fiona Oakley, Nian–Ling Zhu, Hidekazu Tsukamoto, Derek A. Mann 
Gastroenterology 
Volume 138, Issue 2, Pages e4 (February 2010)
DOI: /j.gastro
Copyright © 2010 AGA Institute Terms and Conditions

2. Figure 1
Diminution of PPARγ in myofibroblasts is regulated by MeCP2. (A) PPARγ mRNA levels were quantified by qRT-PCR in rat qHSCs and day 10–transdifferentiated myofibroblasts. (B) Cross-linked chromatin obtained from rat HSCs and myofibroblasts was incubated with 10 μg anti-RNAPolII phosphoSer2; ChIP assay was carried out, and exon A2 of rat PPARγ was amplified. (C) ChIP assay was carried as in panel B with the use of 10 7μg anti-MeCP2 antibody; PPARγ exons and promoter were amplified by qPCR. (D) Rat myofibroblasts (5 × 106) were electroporated with 2 μg total siRNA designed to target rat MeCP2, and knockdown was confirmed by a Western blot. (E) Total RNA was isolated from control or rat MeCP2 siRNA transfected cells and used as a template in qRT-PCR reactions with primers specific for rat PPARγ. *P = (F) Quiescent HSCs were isolated from Wt C57Bl6 or Mecp2−/y mice and transdifferentiated in vitro for 14 days. Total RNA was prepared from both cell populations, and PPARγ mRNA was quantified by qRT-PCR; ∔P = Error bars indicate standard error of the mean.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
MeCP2 knockouts are attenuated for fibrogenesis. (A) Whole-cell protein extract (200 μg) isolated from frozen CCl4-injured Wt or Mecp2−/y liver was separated by SDS-PAGE; proteins were transferred onto membrane and immunoblotted for MeCP2. (B) RNA was isolated from frozen CCl4-injured Wt or Mecp2−/y liver and used in qRT-PCR reactions with primers specific for mouse PPARγ; *(P = .0025), collagen I (P = .036), TIMP-1 (P = .036), and smooth muscle α-actin (α-SMA) (P = .036). (C) Sirius Red immunostaining on sections cut from CCl4-injured Wt or Mecp2−/y liver (magnification, ×5). (D) Sirius Red immunostained sections from 5 Wt and Mecp2−/y CCl4-injured livers were blinded and scored for the grade of fibrosis based on the Metavir scale (P = .0015) and (E) quantified by morphometry (P = .0029). Averaged results are for 5 animals. (F) qHSCs were isolated from Wt or Mecp2−/y livers and transdifferentiated in vitro for 14 days. Total RNA was prepared from both cell populations and used for qRT-PCR with primers specific for mouse collagen1 (P = .032). Error bars indicate standard error of the mean.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
MeCP2 expression in HSCs. (A) Whole-cell extract (30 μg) from freshly isolated HSCs harvested at days 1, 2, 3, and 7 of culture were separated by SDS-PAGE and immunoblotted for MeCP2 and β-actin. (B) RNA isolated from qHSCs (culture day 0) and myofibroblasts (culture day 7) was used for semiquantitative RT-PCR analysis of MeCP2 and β-actin or (C) interrogated with primers specific for short/long MeCP2 transcript.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
MeCP2 expression is regulated by miR132. (A) Micro RNAs were isolated from rat qHSCs and myofibroblasts with the use of the miRNeasy mini kit and were reverse transcribed with the use of the miScript Reverse Transcription Kit. miR132 was detected with miScript primer assay (B) Micro RNA was purified from HSCs isolated from livers of mice after BDL (or sham control) and CCl4 (or olive control) injuries, and miR132 was detected as described above. (C to E) Myofibroblasts (5 × 106) were mixed with 2 μg miR132 mimic or control siRNA and electroporated. Cells were harvested 48 hours later for RNA and whole-cell protein extract. (C) mIR132 was quantified by miScript primer assay (D) Whole-cell protein extracts were separated by SDS-PAGE and immunoblotted for MeCP2 and β-actin. (E) cDNA obtained in panel B was further used as template for qRT-PCR analysis of PPAR7γ. (F) Native chromatin was prepared from in vitro transdifferentiated Wt or Mecp2−/y myofibroblasts. Native chromatin (100 μg) was incubated with 10 μg anti-dimethyl H3K27, H3K9, or H3K4, and ChIP assays were carried out. Immunoprecipitated DNA was used as a template in qPCR reactions using mouse PPARγ promoter-specific primers. Error bars indicate the standard error of the mean.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
MeCP2 regulates HP1α recruitment, expression of EZH2 and H3K27 methylation, (A) Cross-linked chromatin (100 μg) from rat myofibroblasts was incubated with 10 μg of anti-HP1α, and ChIP assay was carried out. Immunoprecipitated DNA was used as a template in qPCR reactions by using primers specific for PPAR7γ promoter/exons in the rat gene. (B) Native chromatin (100 μg) was prepared from rat HSCs, or myofibroblasts were incubated with 10 μg anti-dimethyl H3K27 (top) or anti-trimethyl H3K4 (bottom), and ChIP assay was carried out. Immunoprecipitated DNA was used as a template in qPCR reactions by using primers specific for rat PPARγ promoter/exons. (C) Whole-cell extract (30 μg) from freshly isolated HSCs or myofibroblasts harvested at days 1, 2, 3, and 7 of culture was separated by SDS-PAGE and immunoblotted for EZH2 and β-actin. (D) qRT-PCR analysis of EZH2 mRNA was performed by using RNA extracted from HSCs isolated from rat liver injured by BDL (or sham operated) and CCl4 (or olive oil) (n = 5; *P = .0184). (E) RNA isolated from myofibroblasts transfected with 2 μg control or MeCP2 siRNA (from Figure 1E) was for qRT-PCR analysis of EZH2 *(P = .0152). (F) RNA from Wt or Mecp2−/y myofibroblasts (from Figure 1F) was used for qRT-PCR analysis of EZH2 expression *(P = .0478). Error bars indicate standard error of the mean.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

7. Figure 6
EZH2 regulates PPARγ, myofibroblast transdifferentiation, and fibrogenesis. (A and B) Rat myofibroblasts (5 × 106) were electroporated with 2 μg control or EZH2 siRNA. Total RNA was isolated and used for qRT-PCR detection of EZH2 (A) or PPARγ (P =.0012) (B). (C) Myofibroblasts were treated with 1μM 3-deazaneplanocin A or vehicle for 72 hours; total RNA was prepared and used for qRT-PCR detection of PPARγ. (D) Freshly isolated rat HSCs were cultured for 12 hours in standard media (bottom photomicrograph) or media containing 1μM 3-deazaneplanocin A (top photomicrograph). Cultures were washed and replaced with standard media, and cells were incubated for a further 10 days. (E) RNA obtained in Figure 5C was used for qRT-PCR detection of collagen I and TIMP-1. (F) qRT-PCR analysis of type 1 collagen and TIMP-1 transcripts from RNA isolated from CCl4-injured livers from control or mice were pretreated for 1 hour with 3-deazaneplanocin A (n = 4). Error bars indicate standard error of the mean.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

8. Figure 7
mIR132, MeCP2, and EZH2 operate in a regulatory epigenetic relay pathway that culminates in transcriptional repression of PPARγ. In qHSCs, miR132 exerts a translational block on MeCP2 transcript. On transdifferentiation, mIR132 expression is down-regulated, enabling translation of MeCP2 protein and binding to the 5′ end of the PPARγ gene where it promotes H3K9 methylation and recruitment of the transcription repressor HP1α. In addition, MeCP2 stimulates expression of EZH2, which methylates H3K27 to form a binding site for the Polycomb repressor 1 complex, which promotes chromatin condensation in the 3′ PPARγ exons. The culmination of the pathway is transcriptional repression of the master antifibrogenic gene PPARγ.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

9. Supplementary Figure 1
PPARγ gene structure-gene is composed of 9 exons in total A1 and A2 exons are only found in PPARγ, whereas exan B is only found in PPARγ2 transcript. A CpG island is present is 5‵ end of the gene; it starts -372 from the start of exon A1, extends over the entire A1 and 626bp into intron 1. 5CG=59.8 with total length of the island being 1412bp.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

10. Supplementary Figure 2
DNA Methylation at PPARγ -500 ng of genomic DNA from both hepatic stellate cells (HSC) and transdifferentitated myofibroblasts (MFB) were enzymatically digested to produce around 600bp fragments. DNA was then incubated with His-tagged MBD2 protein and captured by applying a magnet. Bound complexes were washed and methylated DNA eluted which was used as template in PCR reactions using rat PPARγ exons A1, A2, and 1-6 specific primers.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

11. Supplementary Figure 3
Mecp2-/y and wt mice have same level of injury following CCI4 injection-Alkaline phosphatase (Alk P), aspartate aminotransferase (AST), alanine aminotransferase (ALT) are serum biomarkers that reflect liver status. Administration of CCI4 caused liver damage and in turn elevation in levels of these markers. Even rise in ALT and AST in serum of CCI4 injured wt and Mecp2-/y mice suggests equivalent level of injury in both animal groups.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

12. Supplementary Figure 4
Western blot analysis of MeCP2 protein was performed using 100μg whole cell extract obtained from HSCs isolated from acutely CCI4 injured livers or olive oil controls (48 hours after the IP injection). Representative gel of two separate experiments.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

13. Supplementary Figure 5
(A) qRT-PCR analysis of MeCP2 mRNA was performed using RNA extracted from HSC isolated from BDL (or sham operated) and CCI4 (or olive oil) injured rat liver (n = 5). (B) The amount of MeCP2 mRNA species with long 3‵UTR were quantitated by qRT-PCR in samples in A.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

14. Supplementary Figure 6
5 million rate myofibroblast were electroporated with 2μg of control or EZH2 siRNA. Total RNA was isolated using qRT-PCR detection of TIMP1 and Collagen Ia.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions