Tian WF et al: Brahma related gene 1 (Brg1) bridges epigenetic regulation of proinflammatory cytokine production to steatohepatitis
Supplemental Materials and Methods
Supplementary figures: 5
Supplementary tables: 1
Materials and Methods
Plasmids and RNA interference
Promoter luciferase fusion constructs for IL-6, MCP-1, IL-8, iNOS, Brg1 and Brm expression
constructs, and short hairpin RNA (shRNA) construct targeting Brg1 or Brm have been described
previously (1-5). Small interfering RNA (siRNA) sequences were as follows: for NF-B/p65,
UGACGUAAAGGGAUAGGGC; for Brg1, GCCCATGGAGTCCATGCAT; and for Brm,
AAGTCCTGGACCTCCAAGTGT.
Animals
To induce steatohepatitis, 8 week-old male db/db mice (Jackson Laboratory) were fed a
methionine- and choline-deficient (MCD) diet (A02082002B, Research Diets) for 4 consecutive
weeks. In certain experiments, these mice were injected via tail vein purified lentiviral particles
(1X109 MOI) that carry short hairpin RNA (shRNA) targeting both Brg1 and Brm
(GCUGGAGAAGCAGCAGAAG) at week 1 and week 3. Alternatively, 8 week-old male
C57/BL6 mice were fed a high fat high carbohydrate (HFHC) diet (D12492, Research Diets) for
16 consecutive weeks (6). Blood triglyceride (TG) and alanine aminotransferase (ALT) levels
were measured as described before (7). A third NASH model was made in which 6-8 week-old
male Apoe-/- mice (Jackson Laboratory) were fed with a high fat diet (D12079, Research Diets)
for 12 weeks.
Histology
Paraffin sections were stained with hematoxylin and eosin (Sigma), oil red O (Sigma), or
picrosirius red (Sigma) according to standard procedures. Parallel sections were stained for cell
surface markers. Briefly, the sections were blocked with 10% normal goat serum for 1 hour at
room temperature and then incubated with anti-CD3 (BD Bioscience), anti-CD45 (Abcam), or
anti-F4/80 (Abcam) antibodies. For ROS measurement, frozen liver sections were stained with
dihydroethidium (DHE, Sigma). Slides were visualized under and photographed by an Olympus
IX-70 microscope. Histology data were analyzed using Image Pro (Media Cybernetics). Hepatic
pathology was scored by two independent investigators as described before (8).
B
A
VEC
5
PA
OA 4.5
HepG2
2.5
HepaRG
Brg1
Brg1
Brm
Brm
-actin
-actin
4
Relative mRNA levels
Relative mRNA levels
2
HepG2
HepaRG
1.5
1
3.5
VEC OA
3
PA
VEC OA
PA
2.5
2
1.5
0.5
1
0.5
0
Brg1
0
Brm
Brg1
Brm
C
HepG2
HepaRG
1.5
1
Relative mRNA levels
Relative mRNA levels
2
2.5
2
1.5
1
0.5
0.5
0
Glu (h)
Mannitol (h)
0

3

0
Glu (h)
Manitol (h)
6 12 24 
   24
0

6 12 24 
   24
3

Brg1 mRNA
1.8
3
2.5
Relative mRNA levels
Brm mRNA
3.5
1.8
1.6
1.6
1.4
1.4
Relative mRNA levels
Brg1 mRNA
3
1.2
1
0.8
0.6
1.2
1
0.8
0.6
0.4
0.4
0.2
0.2
0
Glu (h) 0
3
6
Brm mRNA
0
Glu (h) 0
12
3
6
12
D
Brm
-actin
Glucose (h)


3
0h
3h
6h
12h
2.5
2
HepaRG
Brg1
1.5
Brm
1
-actin
0.5
0
Glucose (h)
Brg1
Brm
E


F
HepG2
2.5
3
1.4 Primary murine hepatocyte
1.2
HepG2
2.5
1.5
1
1
Relative mRNA levels
Relative mRNA levels
Relative mRNA levels
2
0.8
0.6
0.4
0.5
 

 

Brg1
Brm
0
PA
E2
1.5
1
0.5
0.2
0
PA
E2
2




Brg1
Brm
0
Glucose
E2
 

 

Brg1
Brm
Relative protein levels
(random units)
Brg1
Relative protein levels
(random units)
3
HepG2
0h
3h
6h
12h
2.5
2
1.5
1
0.5
0
Brg1
Brm
G
H
1.4
Apoe-/- AL (N=5)
Apoe-/- HFD (N=5)
4
*
AL
Brm
1
-actin
0.8
0.6
Apoe-/- AL (N=4)
Apoe-/- HFD (N=4)
*
3.5
Brg1
1.2
Relative mRNA levels
HFD
Relative protein levels
(random units)
1.6
3
2.5
2
*
1.5
1
0.5
0
Brg1
0.4
Brm
0.2
0
Brg1
Brm
Figure S1: (A, B) HepG2 and HepaRG cells were treated with a vehicle (VE), palmitate
(0.4mM), or oleate (0.4mM) for 6 hours. mRNA (A) and protein (B) levels of Brg1 and
Brm were assessed by qPCR and Western. (C, D) HepG2 and HepaRG cells were treated
with or without glucose (35mM). Cells were harvested at indicated time points to probe
for mRNA (A) and protein (B) levels of Brg1 and Brm. (E, F) HepG2 cells or Primary
murine hepatocytes were treated with PA (E) or glucose (F) in the presence or absence of
17-estradiol (E2, 10-8~10-6M). Expression of Brg1 and Brm was measured by qPCR. (G,
H) 6-8 week-old male Apoe-/- mice were fed with a high fat diet (HFD) for 12 weeks.
Liver homogenates were probed for mRNA (G) and protein (H) expression of Brg1 and
Brm.
A
AL
HFHC
Brg1
IL-6 promoter
Brm
Brg1
MCP-1 enhancer
Brm
B
IL-1 ChIP
IL-6 ChIP
8
VEC
PA
OA
4.5
7
10
6
5
4
3
4
Relative enrichment
Relative enrichment
Relative enrichment
MCP-1 ChIP
5
12
8
6
4
3.5
3
2.5
2
1.5
2
1
2
1
0.5
0
Brg1
0
Brm
Brg1
0
Brm
Brg1
Brm
C
2
IL-1 intronic ChIP
3
IL-6 intronic ChIP
0h
3h
6h
9h
1.8
2.5
1.4
Relative enrichment
Relative enrichment
1.6
1.2
1
0.8
0.6
0.4
2
1.5
1
0.5
0.2
0
0
Brg1
Brm
Brg1
Brm
D
5
5
IL-1 ChIP
9
IL-6 ChIP
MCP-1 ChIP
8
4
3
2
3
2
1
1
0
0
VEC
PA
PA+E2 low
PA+E2 high
7
Relative enrichment
Relative enrichment
Relative enrichment
4
6
5
4
3
2
1
Brg1
Brm
0
Brg1
Brm
Brg1
Brm
Figure S2: (A) Nuclear proteins were extracted from the livers of NASH mice. DNA
affinity pull-down assay was performed as described under Materials and Methods. (B)
HepG2 cells were treated with a vehicle (VE), palmitate (0.4mM), or oleate (0.4mM) for
6 hours. ChIP assays were performed with indicated antibodies. (C) HepG2 cells were
treated with or without palmitate. ChIP assays were performed with indicated antibodies.
Precipitated DNA was amplified by primers spanning the intronic region of the indicated
gene. (D) HepG2 cells were treated with palmitate in the presence or absence of 17estradiol (E2 low, 10-7M; E2 high, 10-6M). ChIP assays were performed with indicated
antibodies.
A
Relative mRNA levels
1
sip65
p65 mRNA
SCR
1.2
p65
0.8
-actin
0.6
0.4
0.2
0
SCR sip65
B
siBrm
Relative mRNA levels
1
siBrg1
SCR
siBrg1
siBrm
1.2
SCR
1.4
Brg1
0.8
Brm
0.6
-actin
0.4
0.2
0
Brg1
Brm
Figure S3: (A) HepG2 cells were transfected with indicated siRNA. NF-B/p65 levels
were measured by qPCR and Western. (B) HepG2 cells were transfected with indicated
siRNA. Brg1 and Brm levels were measured by qPCR and Western.
1.4
1.2
*
*
*
*
Relative mRNA levels
*
SCR+V
SCR+PA
siBrg1/Brm+PA
1
0.8
0.6
0.4
0.2
0
Brg1
Brm
IL-1
IL-6
TNF-
Figure S4: Murine primary hepatic cells were infected with lentivirus carrying shRNA
targeting Brg1/Brm or SCR followed by treatment with palmitate (PA) or vehicle (V).
Expression levels of indicated genes were measured by qPCR.
A
AL+SCR
MCD+SCR
MCD+shBrg1/Brm
B
0.7
Relative mRNA levels
0.6
db/db+SCR MCD
db/db+shBrg1/Brm MCD
*
*
0.5
0.4
0.3
0.2
0.1
0
Col1a1
Col1a2
Figure S5: Lentiviral particles carrying shRNA targeting Brg1/Brm or SCR were
injected via tail vein as described under Materials and Methods. (A) ROS levels were
measured by DHE staining. (B) Hepatic collagen type I levels were measured by qPCR.
N=3
Table I: ChIP Real-time qPCR primers
Gene name
Human IL-1
Human IL-6
Human MCP-1
Mouse Il-1
Mouse Il-6
Mouse Mcp-1
Primer sequences
Forward: 5’-CTGTGTGTCTTCCACTTTGTCCC-3’
Reverse: 5’-TGCATTGTTTTCCTGACAATCG-3’
Forward: 5’-CTTCGTGCATGACTTCAGCTTT-3’
Reverse: 5’-CGTCCTTTAGCATGGCAAGAC-3’
Forward: 5’-CCCATTTGCTCATTTGGTCTCAGC-3’
Reverse: 5’-GCTGCTGTCTCTGCCTCTTATTGA-3’
Forward: 5’-TTCGATGCTGCCCTTTATCT-3’
Reverse: 5’-AGGCAGGTGGGTACTCATGTA-3’
Forward: 5’-GACATGCTCAAGTGCTGAGTCAC-3’
Reverse: 5’-AGATTGCACAATGTGACGTCG-3’
Forward: 5’-CGTGGGAAAATCCAGTATTTTAATG-3’
Reverse: 5’-CAAATGTATCACCATGCAAATATGC-3’
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