Supplementary Info legend - Genes &...

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


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


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.


RNA pol-II

wt E14.5

wt E18.5

wt P-2

wt P-45

HNF4 KOE18.5

HNF4 KOP 45


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


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


RNApol-II

wt E14.5

wt E18.5

wt P-2

wt P-45

HNF4 KOE18.5

HNF4 KOP 45


RNApol-II

wt E14.5

wt E18.5

wt P-2

wt P-45

HNF4 KOE18.5

HNF4 KOP 45


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


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


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

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