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Supplementary Information
Supplementary Figure 1.
Liver sections from 45 days wild type (WT P45), or HNF-4lox/lox /Alb-Cre (HNF-4
KO P45) mice and from 18.5 day wild type embryos (WT E18.5), or HNF-4lox/lox
/Alfp-Cre (HNF-4 KO E18.5) mouse embryos were immunostained with HNF-1β
(A), HNF-1α (B), HNF-3β (C), HNF-4α (D) and HNF-6 (E) antibodies. Fluorescent
images (Left panels), nuclear staining with DAPI (Middle panels) and merged
pictures (Right panels) are shown.
Merged images with HNF-4α antibody allowed rough estimation of the proportion of
hepatocytes in the total cell population. Examination of 8-10 fields positively stained
nuclei varied between 40-60% in wild type E14.5 and E18.5 livers and 60-70% in
wild type P45 livers. The identity of non-hepatocyte cell populations was not
examined directly. In the adult liver correspond to epithelial and sinusoid cells as
judged by morphology, while in fetal livers they must be mainly hemopoetic cell
types.
With HNF-1α and HNF-4α antibodies we detect nuclear staining only in hepatocytes
in both fetal and adult liver sections. In P45 and E18.5 HNF-4 KO livers we observe
background cytoplasmic staining for HNF-4α. In E18.5 HNF-4 KO livers HNF-1α
signal is also diminished, while in P45 HNF-4 KO samples nuclear HNF-1α staining
is still detectable.
In adult livers of wild type animals’ HNF-1β immunosignal is restricted to biliary
epithelium. In P45 HNF-4-KO livers nuclear staining is evident in both hepatocytes
and biliary epithelial cells. In embryonic livers positive nuclear HNF-1β staining is
detected in both hepatocytes and in the developing biliary epithelium. Embryonic
inactivation of HNF-4α causes a disorganized liver architecture and weaker HNF-1β
staining.
HNF-3β and HNF6 antibodies gave positive nuclear staining in both hepatocytes and
cholangiocytes of embryonic and adult liver. In E18.5 HNF-4-KO sections mainly
background cytoplasmic staining is observed for both proteins, while in P-45 HNF-4
KO livers nuclear staining in hepatocytes is somewhat reduced.
Large hypertrophic hepatocytes in P45 HNF-4a KO livers are evident and stained
positive for HNF-1α, HNF-1β, HNF-3β and HNF-6.
αααα-HNF-1ββββ DAPI Merged
WT
P45
HN
F-4
KO
P45
WT
E18
.5H
NF
-4K
O E
18.5
Supplementary Figure 1 A
αααα-HNF-1αααα DAPI Merged
WT
P45
HN
F-4
KO
P45
WT
E18
.5H
NF
-4K
O E
18.5
Supplementary Figure 1 B
WT
P45
HN
F-4
KO
P45
WT
E18
.5H
NF
-4K
O E
18.5
αααα-HNF-3ββββ DAPI Merged
Supplementary Figure 1 C
αααα-HNF-4αααα DAPI Merged
WT
P45
HN
F-4
KO
P45
WT
E18
.5H
NF
-4K
O E
18.5
Supplementary Figure 1 D
WT
P45
HN
F-4
KO
P45
WT
E18
.5H
NF
-4K
O E
18.5
αααα-HNF-6 DAPI Merged
Supplementary Figure 1 E
20
40
60
80
100
120
HNF-4αααα - - + +HNF-1αααα - + - +
Nor
mal
ized
luc
acti
vity
(%
of
cont
rol)
Supplementary Figure 2.
The upstream regulatory region (nt -3876 to +5) of the mouse HNF-1β gene was subcloned
into pGl3 luciferase vector. 1 µg of the HNF-1b-luc plasmid, together with 0.5 µg pCMV-
lacZ, and 0.5 µg of pCMV-HNF-4α, or pCMV-HNF-1α expression vectors was used for
transfections of the mouse Hepa 1-6 cell line. The graph shows lucifearse reporter activitiesnormalized to LacZ and expressed as a percentage of values obtained with the HNF-1β-luc
reporter plasmid alone. Bars correspond to average values and standard errors from three
independent experiments.
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNA pol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HN
F-1αααα
pro
mot
erH
NF
-1ββββ
pro
mot
erH
NF
-3ββββ
pro
mot
erSupplementary Table 1
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
Supplementary Table 1A
Tabular presentation of promoter occupancy patterns. The table was drawn according
to the data presented in Figure 2,3,5 and 6. ChIP signals corresponding to more than
3-fold enrichment in all experiments are indicated by filled circles.
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNA pol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HN
F-4αααα
1en
hanc
erH
NF
-4αααα
1 p
rom
oter
HN
F-6
pro
mot
erSupplementary Table 1B
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HN
F-4αααα
7 p
rom
oter
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
FX
R αααα
1αααα2
prom
oter
PX
Rpr
omot
erG
AT
A-6
prom
oter
Supplementary Table 1C
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
LR
H-1
pro
mot
er
HNF-1αααα HNF-1ββββ HNF-3ββββ HNF-4αααα HNF-6 LRH-1 C/EBPαααα COUP-TFII
RNApol-II
wt E14.5
wt E18.5
wt P-2
wt P-45
HNF4 KOE18.5
HNF4 KOP 45
Supplementary Table 2.
Conserved binding site sequences on the regulatory regions of hepatic transcription factorsidentified by Genomatix MatInspector search. Reference is included for the cases when previous invitro binding and/or transfection data in cultured cells exist on mouse genes, or on conservedregions of human and rat genes.
HNF-1α promoter
HNF-3β (-31) CTCTGTTTACATTG (- strand) (Ref 1.) (-67) TCATATTTATCCGT (- strand) (Ref. 1)
HNF-4α (-105) CTGAACTTTGGACTTCAGCCT (- strand) (Ref. 1,2,3)LRH-1 (-45) TCTCCAAGGTTCA (+ strand) (-95) TCAGCCTTGCAAG (- strand)C/EBPα (-173) ACTCCCAATTGCA (+,- strand) (Ref. 1.) (-517) ATGCAATACAT (+ strand)
HNF-1β promoter
HNF-4α (-396) GCACCCCAACTTTGTCCAGCT (- strand) (Ref. 4)ΗNF-6 (-705) CGAATCGGCGTC (+ strand)C/EBPα (-558) CCCTCCAATTCGTTG (+ strand)
HNF-3β promoter
ΗΝF-1 (-165) GGTTACTTTTCAGTTA (+ strand)HNF-3β (-210) CTAAAACAAACAGGGCA (+ strand) (Ref. 5) (-512) ACAAAACAAACATGAAA (+ strand)HNF-4α (-32) GTTACCTCAGTCCA (+ strand)ΗNF-6 (-249) AAAAAAAATCAATAATG (+ strand) (Ref. 6)LRH-1 (-178) ATCCAAGGTGCC (+ strand) (Ref. 16)C/EBPα (-195) AGCATTTCGTAACTAA ( +,- strand) (Ref. 7.)
GATA-6 promoter
HNF-1 (-832) CATTAACACACATTAAATTCTT (- strand)HNF-4α /COUP-TFII (-1273) CGATCTGGCCTTTCCCCTGCTGGGG (- strand) (-1145) GGACGTGAGGTTTTGCCCATT (- strand)C/EBPα (-1293) TGACCAATTCC (+ strand)
PXR promoter
HNF-3β (-167) AAGTGTTTGCTCTA (- strand) (-193) CAATGTTTGCCTCT (- strand)HNF-4α /COUP-TFII (-144) TCCCAAGTCCCAAGTCCAGCA (+ strand) (Ref.8) ΗNF-6 (-510) ACATCATCGGGGT (+ strand)LRH-1 (-720) ATACAAAGGTCTCT (+ strand)C/EBPα (-188) GGGCCAATGTT (+ strand)
HNF-4α7 promoter
ΗΝF-1α (-79) GGTTACTGTTTAACGTA (+ strand) (Ref. 9)ΗNF-6 (-44) TAGAAGAATCA (+ strand) (Ref. 9)
HNF-4α1 promoter
ΗΝF-1α (-118) GTGATTAACCATTAACT (- strand) (Ref. 10, 11)ΗNF-6 (-406) GATAGAAGTCAATGATC (+ strand) (Ref. 11)
HNF-4α1 enhancer
ΗΝF-1α (-6365) AGTTAATTTTTTAAAGC (+ strand) (Ref.12)HNF-3β (-6566) AGAGCAAATATACT (+ strand) (Ref.12)HNF-4α/COUP-TFII (-6522) TTGACTCTTGAGCAAAGTCTTC (+strand) (Ref.12)C/EBPα (-6452) GCCTCTTGCATAACCCAGGAG (+ strand) (Ref. 12)
HNF-6 promoter
ΗΝF-1α (-217) AGTCTGCAACAGTAACC (- strand)HNF-4α (-660) GCGAGTGGGCTTTGGGCCATG (- strand) (Ref. 13)ΗNF-6 (-340) GTGCTGGGACGTCACGG (+ strand) (Ref. 14)C/EBPα (-202) CCGAGCCATGGCT (+ strand) (Ref. 15) (-216) GTCTGCAACAGTAA (+ strand)LRH-1 promoter
ΗΝF-1α (-158) CAAAGAAATGATTAACT (- strand)HNF-3β (-468) GGGTATTTACTTAC (- strand) (-514) GTATGTTTGTTAGA (- strand)HNF-4α ( +37) TCCGTGTTCTCCTGGACTCTGC (- strand) (Ref. 16)LRH-1 (-264) TCAAGTCCA (- strand) (Ref. 16)COUP-TFII (-357) TCTTCTGACCTCTGCAAATAC (- strand)
FXR α1α2 promoter
ΗΝF-1α (-80) CAGATTAGTCATTAACAGT (+,-strands)HNF-3β (-590) GGATGTGTTGACAGTCA (- strand)HNF-4α/ COUP-TFII (-61) TGCCCCATGGACA (- strand) (Ref. 17)LRH-1 (-134) AGAGCCTTGGAAT (- strand)C/EBPα (-606) TAGCTCATTGCTTCC (- strand)
Supplementary Table 2 References
1. Kuo, C. J., Conley, P. B., Chen, L., Sladek, F. M., Darnell, J. E., Jr., and Crabtree, G. R.(1992) Nature 355, 457-461
2. Kritis, A. A., Ktistaki, E., Barda, D., Zannis, V. I., and Talianidis, I. (1993) Nucleic AcidsRes. 21, 5882-5889
3. Ktistaki, E., and Talianidis, I. (1997) Science 277, 109-1124. Power, S. C., and Cereghini, S. (1996) Mol. Cell. Biol. 16, 778-7915. Pani, L., Quian, X. B., Clevidence, D., and Costa, R. H. (1992) Mol. Cell. Biol. 12, 552-5626. Samadani, U., and Costa, R. H. (1996) Mol. Cell. Biol. 16, 6273-62847. Samadani, U., Porcella, A., Pani, L., Johnson, P. F., Burch, J. B., Pine, R., and Costa, R. H.
(1995) Cell Growth Differ. 6, 879-8908. Kamiya, A., Inoue, Y., and Gonzalez, F. J. (2003) Hepatology 37, 1375-13849. Briancon, N., Bailly, A., Clotman, F., Jacquemin, P., Lemaigre, F. P., and Weiss, M. C.
(2004) J. Biol. Chem. 279, 33398-3340810. Zhong, W., Mirkovitch, J., and Darnell, J. E., Jr. (1994) Mol. Cell. Biol. 14, 7276-728411. Hatzis, P., and Talianidis, I. (2001) Mol. Cell. Biol .21, 7320-733012. Bailly, A., Torres-Padilla, M. E., Tinel, A. P., and Weiss, M. C. (2001) Nucleic Acids Res.
29, 3495-350513. Lahuna, O., Rastegar, M., Maiter, D., Thissen, J. P., Lemaigre, F. P., and Rousseau, G. G.
(2000) Mol. Endocrinol. 14, 285-29414. Rastegar, M., Szpirer, C., Rousseau, G. G., and Lemaigre, F. P. (1998) Biochem. J. 334,
565-56915. Rastegar, M., Rousseau, G. G., and Lemaigre, F. P. (2000) Endocrinology 141, 1686-169216. Pare, J. F., Roy, S., Galarneau, L., and Belanger, L. (2001) J. Biol. Chem. 276, 13136-1314417. Zhang, Y., Castellani, L. W., Sinal, C. J., Gonzalez, F. J., and Edwards, P. A. (2004) Genes
Dev. 18, 157-169
Supplementary Table 3A.
Nucleotide sequence of primers used in chromatin immunoprecipitation assays.Positions relative to the transcription start sites are shown.
mHNF-4α1 gene: -400 5’ tctgggacgtgattggcttag +2 5’ tcccttctctgccttcctctc
mHNF-4α1 gene: -6576 5’ ccagctgcctttatctccct -6308 5’ cattgctgagcctgttggtc
mHNF-1α gene: -290 5’ aagttctcctgtgccaggct +8 5’ ccctgcctgctctgtttaca
mHNF-1β gene: -274 5’ ccgcttgctttaccagtcca -49 5’ gtgggaagggctcagctttc
mHNF-3β gene: -346 5’ cctcctgaagtcatcccaca -117 5’ ctttctggctacccacctca
mHNF- 6 gene: -225 5’ gcagccacagtctgcaacag +31 5’ ttgccttctctcttgctccc
mHNF-4α7 gene: -467 5’ ccctcaaactcctttggctc +17 5’ caggaaggcagtgagcacag
mGATA-6 gene: -1043 5’ gcctttcctggcatctcact -867 5’ aatgctgtctcaccctgcct
mLRH-1: -293 5’ gccatgtcacaagctgcagt +72 5’ gaaaggctccagtccgtgtt
mFXR-α1α2 gene: -225 5’ accctacaagacagccagca -27 5’ agcatctctcccttggctct
mPXR gene: -190 5’ ctcagagaggcaaacattggc +76 5’ ggctatgttgtccacaggcat
Supplementary Table 3B.
Nucleotide sequence of primers used in quantitative RT-PCR assays.
mHNF-1α mRNA: 5’ tatcagcagcctctcatgcc 5’ tgaggtgaagacctgcttgg
mHNF-1β mRNA: 5’ tccacccaacaagatgtcagg 5’ tcctcccgacactgtgatctg
mHNF-3β mRNA: 5’ ctccgtgtcaggagcacaag 5’ gtggctgtggtgatgttgct
mHNF-4α1 mRNA : 5’ ggcatggatatggccgactac (exon 1) 5’ cgccattgatcccagagatg (exon 5) 5’ tgtctaccacacattgtcggctaaac (exon 3)
mHNF-4α7 mRNA: 5’ gtcatggtcagtgtgaacg (exon 1) 5’ cgccattgatcccagagatg (exon 5) 5’ tgtctaccacacattgtcggctaaac (exon 3)
mHNF-6 mRNA: 5’ acagacgtccaacgtcgaact 5’ gctcgatgaggacgatgaact
mGATA-6 mRNA: 5’ caccaccaccatcaccatcac 5’ ctctccgacaggtcctccaac
mLRH-1 mRNA: 5’ tggtggaaggtgtccaagag 5’ gcagcatctcaatgaggagg
mFXR mRNA: 5’ cgatcgtcatcctctctcca 5’ atcagcatctcagcgtggtg
mPXR mRNA: 5’ ccactgcatgctgaagaagc 5’ gttgatgcttcgcagctcag
mGAPDH mRNA: 5’ ggtcatcatctccgccccttctgc 5’ gactgggagttgctgttgaagtcg