![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, November 2004, p. 9601-9609, Vol. 24, No. 21
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.21.9601-9609.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky
Received 24 May 2004/ Returned for modification 21 June 2004/ Accepted 11 August 2004
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The tight linkage, similar pattern of expression, and reciprocal imprinting suggest that the H19 and Igf2 genes share a common set of transcriptional regulatory elements. In support of this notion, multiple enhancers that regulate endodermal and mesodermal Igf2 and H19 expression have been identified. Two enhancers, called E1 and E2, are located 5 and 6.5 kb downstream, respectively, of the H19 gene polyadenylation site and were identified by transient transfections in Hep3B human hepatoma cells (55). When these enhancers were deleted by homologous recombination in embryonic stem cells, a dramatic reduction of H19 and Igf2 expression was seen in endodermal tissues, although mesodermal expression remained normal (32). Additional transgenic studies, genomic comparisons, and characterization of the radiation-induced minute (Mnt) mutation identified mesodermal enhancers located further downstream of E1 and E2 and in the Igf2-H19 intergenic region (1, 12, 14, 21, 26, 55).
The regulatory sequences of numerous genes expressed in the liver contain binding sites for endoderm-enriched transcription factors, including FoxA, C/EBP, hepatocyte nuclear factor 1 (HNF1), HNF6, and orphan nuclear receptors (reviewed in reference 10). FoxA proteins (previously called HNF3) were originally identified as liver-enriched factors that regulated the rat transthyretin (TTR) gene (9). Three FoxA isoforms (FoxA1, FoxA2, and FoxA3) are encoded by distinct genes (28, 29) and are defined by a novel "winged-helix" DNA binding motif (6) which also is found in related Fox proteins (27). FoxA proteins are among the earliest factors to be expressed during hepatogenesis (24, 35) and are known to play a role in regulating the transcription of many hepatic genes, including those for albumin and alpha-fetoprotein (AFP) (7). C/EBPs comprise a family of transcription factors that regulate the differentiation of hematopoietic cells, adipocytes, and hepatocytes as well as additional target genes in these cell lineages (reviewed in reference 49).
Despite numerous studies that have helped elucidate aspects of imprinting in the Igf2-H19 locus, little is known about developmental and tissue-specific regulation of these genes. Since H19 enhancers E1 and E2 govern endoderm (liver)-specific expression of H19-Igf2, it is likely that liver-enriched transactivating factors interact with these enhancers. As a rapid in vivo screen to test the binding of liver-enriched factors to H19 control regions, we performed chromatin immunoprecipitation (ChIP) (53) assays with antibodies to FoxA and C/EBP proteins. This analysis revealed no interactions between C/EBP and E1 or E2 but showed that FoxA proteins associate with E1. Electrophoretic mobility shift assays (EMSAs) identified two FoxA binding sites within E1. The importance of these FoxA sites for full E1 activity was confirmed using transient transfections. Finally, ChIP assays in fetal and adult liver, as well as F9 embryonal carcinoma cells, showed an absolute correlation among FoxA binding, H19 expression, and acetylation of histone H4 proteins in the H19 promoter. These findings indicate that FoxA proteins help govern H19 expression in endodermal cell lineages.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ChIP assays.
ChIP assays were performed according to the protocol provided by Upstate Biotechnology with several modifications. Briefly, tissues (liver and yolk sac) were diced into small pieces using a scalpel blade and resuspended in phosphate-buffered saline (PBS) whereas undifferentiated and differentiated F9 cells were collected by centrifugation and resuspended in PBS. Cells (from tissues or cell culture) were fixed for 15 min by adding formaldehyde to a final concentration of 1%, followed by the addition of glycine to a final concentration of 0.125 M. Cells were then washed three times with ice-cold PBS containing protease inhibitors (1 µg of aprotinin/ml, 1 µg of pepstatin A/ml, 1 µg of leupeptin/ml, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) and then lysed in cell lysis buffer (5 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid); pH 8.0], 85 mM KCl, 0.5% NP-40, and proteinase inhibitors). After 10 min on ice, the cells were homogenized in a glass Dounce homogenizer and centrifuged at 2,500 x g rpm. The nuclear pellet was resuspended in sodium dodecyl sulfate lysis buffer containing proteinase inhibitors for 20 min on ice and was sonicated on ice to fragment the genomic DNA. The mixture was then centrifuged to remove debris, and aliquots (one 100-µl aliquot equaled 107 cells or 0.03 g of tissue) were stored at –80°C. For ChIP, aliquots were diluted in ChIP dilution buffer containing proteinase inhibitors. After preclearance with salmon sperm DNA-protein A agarose, samples were incubated overnight at 4°C with rabbit polyclonal antibodies to FoxA1 and FoxA2 (gifts from Robert Costa, University of Illinois at Chicago), C/EBP
, C/EBP
proteins (obtained from Santa Cruz Biotechnology), or acetylated histone H4 (Upstate Biotechnology). Immune complexes were precipitated by the addition of salmon sperm DNA-protein A agarose slurry and low-speed spinning. Precipitates were sedimented at 100 x g for 2 min and were washed twice (5 min each at 4°C) with low-salt buffer, once (5 min at 4°C) with high-salt buffer, once (5 min at 4°C) with LiCl buffer, and three times (5 min each at 4°C) with Tris-EDTA. The DNA-protein-antibody complexes were eluted from the beads by vortexing in 250 µl of 0.1 M NaHCO3-1% sodium dodecyl sulfate and were pooled with a second extraction. NaCl was added to the eluates to a final concentration of 0.3 M, and the samples were incubated at 65°C for 4 h to reverse formaldehyde-induced cross-linking. After deproteinization, the DNA was extracted with phenol-chloroform followed by ethanol precipitation. The DNA pellet was resuspended in 50 µl of sterile H2O, and 4 µl of DNA solution was used as a template for PCR with an annealing temperature of 65°C. The linear range for each primer pair was determined empirically to be between 31 and 37 cycles. The following primer combinations were used for amplification of precipitated DNA: H19 enhancer E1, 5'-TCTAGAGTCCATGCATCTGAGG and 5'-AAGCTGCAGACATGGTGG; H19 enhancer E2, 5'-TGTTAACCTGGCTCTGCCCTGC and 5'-CTGCAGGTACAAACACGTAGG; H19 promoter, 5'-CTGTGGATCCGTAGGCTGTTCATACTCC and 5'-ATTAGATCTCCCACACCCGGTGCTTCG; TTR promoter, 5'-AGCGAGTGTTCCGATACTC and 5'-ACCCCCTCCTTCCAACCCA; albumin promoter, 5'-CTCCAGATGGCAAACATACG and 5'-TCTGTGTGCAGAAAGACTCG. The PCR products were separated on a 1.5% agarose gel, visualized by Syber Green staining, and quantified using ImageQuant.
Plasmids and oligonucleotides.
The fragment corresponding to the mouse Igf2-H19 enhancer E1 was generated by PCR amplification, with the primers 5'-TCTAGAGTCCATGCATCTGAGG-3' and 5'-AAGCTGCAGACATGGTGG-3' and cloned into pGEM-T (Promega, Madison, Wis.). This fragment was confirmed by restriction digest and DNA sequencing and then excised from pGEM-T and inserted into the BamHI site of the 
44-lacZ reporter vector (45). PCR-directed mutagenesis was performed as described previously (20) to change site 1 and site 2, either alone or together, in the context of the entire E1. For site 1, the sequence was changed from GTGTACACAAAGCCCTC to AGACTCGAGCCAATAGA; for site 2, the sequence was changed from CACTCCTGTGTCAAC to AGTGAAGCTTCAGCT. Mutated constructs were inserted into the BamHI site of the 44-lacZ reporter vector. The pGL2-promoter luciferase plasmid was obtained from Promega. The cytomegalovirus-based rat FoxA1 and FoxA2 expression vectors (provided by Robert Costa) and empty vector (EV) were described previously (20, 40, 41). Oligonucleotides used for PCR amplification of DNA fragments for EMSAs were as follows: H19 promoter, 5'-CTGTGGATCCGTAGGCTGTTCATACTCC and 5'-TATTAGATCTCCCACACCCGGTGCTTCG; H19E1, 5'-TCTAGAGTCCATGCATCTGAGG and 5'-AAGCTGCAGACATGGTGG; H19E2, 5'-TGTTAACCTGGCTCTGCCCTGC and 5'-CTGCAGGTACAAACACGTAGG; H19E1A, 5'-TCTAGAGTCCATGCATCTGAGG and 5'-AGCACTACCCTGAGAGG; H19E1B, 5'-AGGGTAGTGCTGTGAGG and 5'-AACCAGTCTTTCCCAGC; H19E1C, 5'-GACTGGTTTATTCAGG and 5'-TTTGTGTACACCTCCC; H19E1D, 5'-TGTACACAAAGCCCTC and 5'-AAGCTGCAGACATGGTGG. Oligonucleotides that were annealed to generate transcription factor binding sites for use in EMSAs were as follows: FoxA-TTR, 5'-GATCGTTGACTAAGTCAATAATCAGAATCA and 5'-GATCTGATTCTGATTATTGACTTAGTCAAC; C/EBP-Alb, 5'-GATCCTATGATTTTGTAATGGGGC and 5'-GATCGCCCCATTACAAAATCATAG; E1D-a (FoxA site 1), 5'-GGGAGGTGTACACAAAGCCCTC and 5'-GAGGGCTTTGTGTACACCTCCC; E1D-a (FoxA site1mut), 5'-GGAGAGACTCGAGCCAATAGAAATA and 5'-TATTTCTATTGGCTCGAGTCTCTCC; E1D-b, 5'-CACAAAGCCCTCAATAAGTT and 5'-AACTTATTGAGGGCTTTGTG; E1D-c, 5'-CCTCAATAAGTTCCCCATTG and 5'-CAATGGGGAACTTATTGAGG; E1D-d, 5'-TCCCCATTGGCTACTGGTGA and 5'-TCACCAGTAGCCAATGGGGA; E1D-e, 5'-GCTACTGGTGACTTCACTCCTG and 5'-CAGGAGTGAAGTCACCAGTAGC; E1D-f (FoxA site 2), 5'-TTCACTCCTGTGTCAACACC and 5'-GGTGTTGACACAGGAGTGAA; E1D-f (FoxA site2mut), 5'-TTAGTGAAGCTTCAGCTACC and 5'-GGTAGCTGAAGCTTCACTAA; E1D-g, 5'-TGTCAACACCACCATGTCTGCA and 5'-TGCAGACATGGTGGTGTTGACA.
Cell transfection.
Transient transfections were carried out in 6-cm-diameter plates by the calcium phosphate method with a total of 7.5 µg of DNA consisting of 5 µg of the 44-lacZ reporter construct and 2.5 µg of the pGL2 luciferase expression vector. For HeLa-synthesized FoxA proteins, cells in 10-cm-diameter plates were transfected with 15 µg of FoxA vectors by using Lipofectamine. Forty-eight hours after the addition of DNA, cells were washed three times in PBS, scraped from plates into 1.5 ml of PBS, and transferred to 1.5-ml microcentrifuge tubes. Cells were pelleted by centrifugation and used for ß-galactosidase (ß-Gal)-luciferase assays or the preparation of nuclear extracts as described below.
ß-Gal-luciferase assays. Cell pellets from transient transfections were resuspended in 400 µl of reporter lysis buffer (Promega). Cells were lysed by one cycle of freeze-thawing with the use of liquid nitrogen, followed by centrifugation at 14,000 x g for 1 min. The ß-Gal activity was measured using the colorimetric substrate chlorophenol red-ß-D-galactopyranoside (CPRG; Boehringer Mannheim, Indianapolis, Ind.) as described previously (45). Luciferase assays were performed in triplicate according to the Promega protocol with 20 µl of cell extract and 100 µl of the luciferase assay reagent. The ß-Gal activity was normalized to luciferase to control for variations in transfection efficiency.
Preparation of nuclear extracts-EMSAs. Nuclear extracts were prepared from Hep3B or HeLa cells. Briefly, monolayers of untransfected or transfected cells were harvested by scraping as described above and resuspended in buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM PMSF. After a 10-min incubation on ice, nuclei were collected by centrifugation at 14,000 x g for 2 min. The nuclear pellet was resuspended in buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% (vol/vol) glycerol, 0.5 mM dithiothreitol, and 0.2 mM PMSF. After a 20-min incubation on ice, samples were centrifuged at 14,000 x g for 2 min. The supernatant was removed, and nuclear extracts were stored in aliquots at –80°C until use. Protein concentrations were determined using the BCA assay kit (Pierce Biochemicals, Rockford, Ill.).
EMSAs were carried out as described previously (20). Briefly, 1 µg of extract and 0.25 µg of poly(dI:dC) were incubated with indicated probes (labeled with [
-32P]ATP by using T4 polynucleotide kinase) with or without cold competitors. Reaction mixtures were resolved on nondenaturing 8% polyacrylamide gels in 1x TBE (2.2 mM Tris, 2.2 mM boric acid, 0.5 mM EDTA) running buffer. Gels were dried and subjected to autoradiography by using phosphorimaging screens. Oligonucleotides used as probes or cold competitors in EMSAs were either chemically synthesized and annealed oligonucleotides (IDT, Coralville, Iowa) or PCR-amplified products. Supershift antibodies used in EMSAs were obtained from Santa Cruz Biotechnology (anti-FoxA2, p-19; anti-C/EBP, c-22).
Reverse transcription-PCR (RT-PCR) assays. Total RNA was isolated from undifferentiated or differentiated F9 cells (from tissues-cell culture) with Trizol according to the instructions of the manufacturer (Invitrogen); total RNA from liver tissues was prepared using the LiCl-urea method (36). One micromolar oligo(dT) primers were used to synthesize first-strand cDNA from 1 to 2 µg of total RNA with the Ominiscript RT kit (Qiagen). PCRs were carried out using 2.5 µl of the RT reaction mixture and the following primers designated to amplify transcripts of H19, TTR, Igf2, AFP, and ß-actin: H19, 5'-CGTTCTGAATCAAGAAGATGCTGC-3' and 5'-TTTGAGTCTCTCAAGCAAGGAAGG-3'; TTR, 5'-ACAAGCTCCTGACAGGATGG-3' and 5'-CAGCATCCAGGACTTTGACC-3'; AFP, 5'-AAAACTCGTGATGCTTTGGGCG-3' and 5'-GGACATCTTCACCATGTGG-3'; Igf2, 5'-TCAGTTTGTCTGTTCGGACC-3' and 5'-ATTGGAAGAACTTGCCCACG; ß-actin, 5'-GTGGGCCGCTCTAGGCACCA-3' and 5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3'.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ) precipitated DNA from the positive-control albumin promoter (33, 34), anti-C/EBP antibodies did not bind any of the H19 control regions tested. These data demonstrated an in vivo association between FoxA proteins and H19 E1.
|
|
|
44-lacZ expression vector that we have used previously to monitor the transcriptional activity of enhancer fragments (45). Constructs were transiently transfected into HepG2 human hepatoma cells along with a luciferase vector to normalize for variations in transfection efficiency. The wild-type E1 dramatically activated the 44 promoter (Fig. 4B). The site 1 mutation reduced enhancer activity about 30%, indicating that this FoxA site contributes to full E1 activity. A much more dramatic effect was seen with the site 2 mutation, which reduced enhancer activity to roughly 10% of wild-type levels. The double mutant had the same activity as the site 2 mutant. These data confirm the importance of these sites for full E1 activity and indicate that site 2 is more important than site 1 in HepG2 cells. Similar results were seen when transfections were performed in Hep3B cells (data not shown).
|
|
|
Changes in H19 promoter acetylation during liver development. Histone proteins are normally hyperacetylated in the promoters of actively transcribed genes (reviewed in reference 47). Since H19 transcription changes during liver development and F9 cell differentiation, we examined whether H4 acetylation within the H19 promoter correlates with changes in H19 transcription. Chromatin was isolated from fetal and adult liver and F9, VE, and PE cells and immunoprecipitated with antibodies to acetylated histone H4 (Fig. 7). The extent of histone H4 acetylation at the H19 promoter is higher in fetal liver than in adult liver (Fig. 7A and B). In contrast, H4 acetylation of the TTR promoter is slightly higher in the adult liver than the fetal liver. When similar assays were performed using F9 cells, increased H4 acetylation of the H19 promoter was seen only in cells differentiated into VE, where H19 is activated; this change was evident at day 6 and more pronounced by day 8 (Fig. 7C). In contrast, H4 histones on the TTR promoter were acetylated in all cells examined, including undifferentiated F9 cells. H4 hyperacetylation in the TTR promoter in undifferentiated F9 cells might explain why this gene is activated earlier than H19 in the VE pathway and in the PE pathway.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using ChIP assays as a rapid in vivo screen for factors bound to the H19 regulatory regions, we have shown here that FoxA proteins bind to H19 enhancer E1. The data also indicate that FoxA proteins do not bind to the H19 promoter or E2 and that C/EBP proteins bind none of these three regulatory regions. This is the first identification of a tissue-specific factor involved in H19 regulation. Interestingly, our computer analysis of H19 regulatory regions failed to reveal FoxA sites within E1. Thus, while computer-based screening offers one strategy to identify potential transcription factor binding sites (37, 38), ChIP assays provides a valuable alternative to in silico approaches for identifying factor binding sites. Furthermore, ChIP assays provide a direct in vivo measure of transcription factor occupancy in a control region, whereas factor binding sites based on computer-based predictions must be confirmed using in vivo or in vitro strategies.
Our studies identified two FoxA sites in E1. FoxA proteins can bind both sites as judged by EMSA analysis, although these factors bound site 2 with a higher avidity than they bound site 1 (Fig. 3). Functional analysis of these two sites by transient transfection revealed that site 2 contributed more to enhancer activity than did site 1 (Fig. 4). Thus, by two criteria, site 2 is more important than site 1 for FoxA binding and enhancer activity. Multiple FoxA binding sites are found in other regulatory regions, including the TTR promoter, which also has both a strong and a weak FoxA binding site (8). In addition, site 2 binds FoxA2 more strongly than it binds FoxA1 as judged by EMSA (Fig. 3B) and ChIP analysis of fetal liver (Fig. 5). Taken together, these data suggest that FoxA2 may be more important than FoxA1 in regulating E1. FoxA1 and FoxA2 proteins, though structurally similar, have distinct activities. These two proteins have different patterns of expression in various tissues during fetal life, although their patterns of expression in the developing liver are the same (35). FoxA2-knockout mice die early in development and lack an organized node and notochord (2, 52), whereas FoxA1-knockout mice die shortly after birth and have impaired glucose homeostasis (25). Furthermore, nuclear localization of FoxA2, but not FoxA1, is regulated by phosphorylation via the Akt signaling pathway (54).
FoxA proteins possess a forkhead or winged-helix DNA binding domain, which structurally resembles the globular domain of linker histones H1 and H5 (5). FoxA proteins are able to open and reposition chromatin by displacing linker histones in an ATP-independent manner (42). Elegant studies investigating albumin activation during hepatogenesis indicate that FoxA proteins bind to the albumin enhancer prior to albumin activation (15). This has led to the concept of genetic potentiation, whereby FoxA binding facilitates the binding of additional factors that subsequently activate albumin transcription at a later developmental stage (56). Using F9 cells as a tissue culture model of differentiation, we tested whether FoxA binding to E1 occurred prior to H19 activation. In contrast to what Zaret and colleagues saw with albumin, our data with differentiated F9 cells suggest that FoxA binding to H19 E1 occurs concomitantly with H19 activation (Fig. 6). This would indicate that FoxA binding does not act to potentiate H19 transcription but instead is more directly involved in gene activation. This raises the interesting possibility that other factors exist to potentiate the H19 gene prior to transcriptional activation. In contrast to H19, we found that the Igf2 gene was activated in both the VE and PE pathways. This suggests that additional mechanisms can facilitate Igf2 activation in the PE pathway that cannot act upon the H19 promoter.
Histone modification plays an important role in gene regulation by remodeling chromatin structure (23, 47). Previous studies showed that parental origin-specific acetylation exists at the promoters of the Igf2-H19 genes and correlates with the parental origin-specific expression of the Igf2-H19 genes (16). This suggested a role for histone acetylation in regulating monoallelic H19 and Igf2 expression. Our studies indicate a decrease in histone H4 acetylation and H19 gene expression during the transition from fetal to adult liver (Fig. 7A and B) and increased histone H4 acetylation and H19 gene expression during F9 cell differentiation (Fig. 7C). Whether proteins that bind the H19 enhancers, including FoxA, influence promoter acetylation, or whether histone modifications in the H19 promoter region influence factor binding to E1 or E2, will require additional studies.
Although FoxA proteins are equally abundant in the fetal and adult livers, our data show that FoxA proteins are bound to H19 enhancer E1 at higher levels in the fetal liver than in the adult liver. This reduction in binding correlates with postnatal H19 repression in the liver. This is specific for the H19 gene; FoxA binding to the TTR promoter continues to be high in the adult liver, which correlates with continued TTR transcription after birth. The decrease in FoxA binding to E1 in the adult liver raises the possibility that loss of enhancer activity may contribute to postnatal H19-Igf2 repression. Furthermore, our studies with F9 cells also show that FoxA binding occurs only in VE cells, in which H19 is active, but not in PE cells, which do not express H19, even though FoxA proteins are activated in both cell lineages (this study with TTR and reference 22). These data in both liver and F9 cells provide in vivo evidence that the presence of FoxA proteins, by itself, is not sufficient for FoxA binding to H19 E1. Additional mechanisms must restrict the accessibility of FoxA proteins to this enhancer during gene activation (F9 
VE but not F9 PE) and repression (fetal to adult liver). The presence of negative regulators, or inaccessibility due to chromatin modification, could block FoxA binding to E1 in adult liver and PE cells. Further studies will be needed to investigate these possibilities.
|
ACKNOWLEDGMENTS |
|---|
This research was supported by Public Health Service grant DK-51600 from the National Institute of Diabetes and Digestive and Kidney Diseases.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Ang, S.-L., A. Wierda, D. Wong, K. A. Stevens, S. Cascio, J. Rossant, and K. Zaret. 1993. The formation and maintenance of the definitive endoderm lineage of the mouse: involvement of HNF3-forkhead proteins. Development 119:1301-1315.[Abstract]
3. Arney, K. L. 2003. H19 and Igf2—enhancing the confusion? Trends Genet. 19:17-23.[CrossRef][Medline]
4. Bartolomei, M., S. Zemel, and S. M. Tilghman. 1991. Parental imprinting of the mouse H19 gene. Nature 351:153-155.[CrossRef][Medline]
5. Burley, S. K., X. Xie, K. L. Clark, and F. Shu. 1997. Histone-like transcription factors in eukaryotes. Curr. Opin. Struct. Biol. 7:94-102.[CrossRef][Medline]
6. Clark, K. L., E. D. Halay, E. Lai, and S. K. Burley. 1993. Cocrystal structure of the HNF3/fork head DNA-recognition motif resembles histone H5. Nature 364:412-418.[CrossRef][Medline]
7. Costa, R. H. 1994. Hepatocyte nuclear factor 3/forkhead protein family: mammalian transcription factors that possess divergent cellular expression patterns and binding specificities, p. 183-205. In R. Tronche and M. Yaniv (ed.), Liver gene expression. R. G. Landes Company, New York, N.Y.
8. Costa, R. H., and D. R. Grayson. 1991. Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region. Nucleic Acids Res. 19:4139-4145.
9. Costa, R. H., D. R. Grayson, and J. E. Darnell, Jr. 1989. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and 1-antitrypsin genes. Mol. Cell. Biol. 9:1415-1425.
10. Costa, R. H., V. V. Kalinichenko, A. X. Holterman, and X. Wang. 2003. Transcription factors in liver development, differentiation, and regeneration. Hepatology 38:1331-1347.[CrossRef][Medline]
11. Costa, R. H., T. A. Van Dyke, C. Yan, F. Kuo, and J. E. Darnell, Jr. 1990. Similarities in transthyretin gene expression and differences in transcription factors: liver and yolk sac compared to choroid plexus. Proc. Natl. Acad. Sci. USA 87:6589-6593.
12. Davies, K., L. Bowden, P. Smith, W. Dean, D. Hill, H. Furuumi, H. Sasaki, B. Cattanach, and W. Reik. 2002. Disruption of mesodermal enhancers for Igf2 in the minute mutant. Development 129:1657-1668.
13. DeChiara, T., E. Robertson, and A. Efstratiadis. 1991. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64:849-859.[CrossRef][Medline]
14. Drewell, R. A., K. L. Arney, T. Arima, S. C. Barton, J. D. Brenton, and M. A. Surani. 2002. Novel conserved elements upstream of the H19 gene are transcribed and act as mesodermal enhancers. Development 129:1205-1213.
15. Gauldi, R., P. Bossard, M. Zheng, Y. Hamada, J. R. Coleman, and K. S. Zaret. 1996. Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control. Genes Dev. 10:1670-1682.
16. Grandjean, V., L. O'Neill, T. Sado, B. Turner, and A. Ferguson-Smith. 2001. Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain. FEBS Lett. 488:165-169.[CrossRef][Medline]
17. Grover, A., R. Oshima, and E. Adamson. 1983. Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells. J. Cell Biol. 96:1690-1696.
18. Hogan, B. L. M., D. P. Barlow, and R. Tilly. 1983. F9 teratocarcinoma cells as a model for the differentiation of parietal and visceral endoderm in the mouse embryo. Cancer Surv. 2:115-140.
19. Hogan, B. L. M., A. Taylor, and E. Adamson. 1981. Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature 291:235-237.[CrossRef][Medline]
20. Huang, M.-C., K. K. Li, and B. T. Spear. 2002. The mouse alpha-fetoprotein promoter is repressed in HepG2 hepatoma cells by hepatocyte nuclear factor-3 (FOXA). DNA Cell Biol. 21:561-569.[CrossRef][Medline]
21. Ishihara, K., N. Hatano, H. Furuumi, R. Kato, T. Iwaki, K. Miura, Y. Jinno, and H. Sasaki. 2000. Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in Igf2/H19 imprinting. Genome Res. 10:664-671.
22. Jacob, A., S. Budhiraja, X. Qian, D. Clevidence, R. Costa, and R. R. Reichel. 1994. Retinoic acid-mediated activation of HNF3 during EC stem cell differentiation. Nucleic Acids Res. 22:2126-2133.
23. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science 293:1074-1080.
24. Kaestner, K. H., H. Hiemisch, B. Luckow, and G. Schutz. 1994. The HNF-3 gene family of transcription factors in mice: gene structure, cDNA sequence, and mRNA distribution. Genomics 20:377-385.[CrossRef][Medline]
25. Kaestner, K. H., J. Katz, Y. Liu, D. J. Drucker, and G. Schutz. 1999. Inactivation of the winged helix transcription factor HNF3alpha affects glucose homeostasis and islet glucagon gene expression in vivo. Genes Dev. 13:495-504.
26. Kaffer, C. R., A. Grinberg, and K. Pfeifer. 2001. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol. Cell. Biol. 21:8189-8196.
27. Lai, E., K. L. Clark, S. K. Burley, and J. E. Darnell, Jr. 1993. Hepatocyte nuclear factor 3/fork head or "winged helix" proteins: a family of transcription factors of diverse biologic function. Proc. Natl. Acad. Sci. USA 90:10421-10423.
28. Lai, E., V. R. Prezioso, E. Smith, O. Litvin, R. H. Costa, and J. E. J. Darnell. 1990. HNF-3A, a hepatocyte enriched transcription factor of novel structure is regulated transcriptionally. Genes Dev. 4:1427-1436.
29. Lai, E., V. R. Prezioso, W. Tao, W. S. Chen, and J. E. Darnell. 1991. Hepatocyte nuclear factor 3 belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev. 5:416-427.
30. Lee, J. E., J. Pintar, and A. Efstratiadis. 1990. Pattern of the insulin-like growth factor II gene expression during early mouse embryogenesis. Development 110:151-159.[Abstract]
31. Leighton, P. A., R. S. Ingram, J. Eggenschwiler, A. Efstratiadis, and S. M. Tilghman. 1995. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 375:34-39.[CrossRef][Medline]
32. Leighton, P. A., J. R. Saam, R. S. Ingram, C. L. Stewart, and S. M. Tilghman. 1995. An enhancer deletion affects both H19 and Igf2 expression. Genes Dev. 9:2079-2089.
33. Lichtsteiner, S., J. Waurin, and U. Schibler. 1987. The interplay of DNA-binding proteins on the promoter of the mouse albumin gene. Cell 51:963-973.[CrossRef][Medline]
34. Milos, P. M., and K. S. Zaret. 1992. A ubiquitous factor is required for C/EBP-related proteins to form stable transcription complexes on an albumin promoter segment in vitro. Genes Dev. 6:991-1004.
35. Monaghan, A. P., K. H. Kaestner, E. Grau, and G. Schutz. 1993. Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 , ß and genes in determination of the definitive endoderm, chordamesoderm, and neuroectoderm. Development 119:567-578.[Abstract]
36. Pachnis, V., A. Belayew, and S. M. Tilghman. 1984. Locus unlinked to -fetoprotein under the control of the murine raf and Rif genes. Proc. Natl. Acad. Sci. USA 81:5523-5527.
37. Pennacchio, L. A., and E. M. Rubin. 2003. Comparative genomic tools and databases: providing insights into the human genome. J. Clin. Investig. 111:1099-1106.[CrossRef][Medline]
38. Pennacchio, L. A., and E. M. Rubin. 2001. Genomic strategies to identify mammalian regulatory sequences. Nat. Rev. Genet. 2:100-106.[CrossRef][Medline]
39. Poirier, F., C. T. Chan, P. M. Timmons, E. J. Robertson, M. J. Evans, and P. W. Rigby. 1991. The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo. Development 113:1105-1114.[Abstract]
40. Qian, X., and R. H. Costa. 1995. Analysis of hepatocyte nuclear factor-3b protein domains required for transcriptional activation and nuclear targeting. Nucleic Acids Res. 23:1184-1191.
41. Qian, X., U. Samadani, A. Porcella, and R. H. Costa. 1995. Decreased expression of hepatocyte nuclear factor 3 during the acute phase response influences transthyretin gene transcription. Mol. Cell. Biol. 15:1364-1376.[Abstract]
42. Shim, E. Y., C. Woodcock, and K. S. Zaret. 1998. Nucleosome positioning by the winged helix transcription factor HNF3. Genes Dev. 12:5-10.
43. Soprano, D., K. Soprano, M. Wyatt, and D. Goodman. 1988. Induction of the expression of retinol-binding protein and transthyretin in F9 embryonal carcinoma cells differentiated into embryoid bodies. J. Biol. Chem. 263:17897-17900.
44. Spear, B. T., and A. W. Ellis. 1995. Endogenous and transfected mouse alpha-fetoprotein genes in undifferentiated F9 cells are activated in transient heterokaryons. Somat. Cell Mol. Genet. 21:19-31.[CrossRef][Medline]
45. Spear, B. T., T. Longley, S. Moulder, S. L. Wang, and M. L. Peterson. 1995. A sensitive lacZ-based expression vector for analyzing transcriptional control elements in eukaryotic cells. DNA Cell Biol. 14:635-642.[Medline]
46. Spear, B. T., and S. M. Tilghman. 1990. Role of -fetoprotein regulatory elements in transcriptional activation in transient heterokaryons. Mol. Cell. Biol. 10:5047-5054.
47. Spencer, V. A., and J. R. Davie. 1999. Role of covalent modifications of histones in regulating gene expression. Gene 240:1-12.[CrossRef][Medline]
48. Strickland, S., and V. Mahdavi. 1978. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15:393-403.[CrossRef][Medline]
49. Takiguchi, M. 1998. The C/EBP family of transcription factors in the liver and other organs. Int. J. Exp. Pathol. 79:369-391.[CrossRef][Medline]
50. Vernucci, M., F. Cerrato, N. Besnard, S. Casola, P. V. Pedone, C. B. Bruni, and A. Riccio. 2000. The H19 endodermal enhancer is required for Igf2 activation and tumor formation in experimental liver carcinogenesis. Oncogene 19:6374-6385.
51. Verona, R. I., M. R. Mann, and M. S. Bartolomei. 2003. Genomic imprinting: intricacies of epigenetic regulation in clusters. Annu. Rev. Cell Dev. Biol. 19:237-259.[CrossRef][Medline]
52. Weinstein, D., A. Altaba, W. Chen, P. Hoodless, V. Prezioso, T. Jessell, and J. E. Darnell, Jr. 1994. The winged-helix transcription factor HNF-3 is required for notochord development in the mouse embryo. Cell 78:575-588.[CrossRef][Medline]
53. Wells, J., and P. J. Farnham. 2002. Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26:48-56.[CrossRef][Medline]
54. Wolfrum, C., D. Besser, E. Luca, and M. Stoffel. 2003. Insulin regulates the activity of forkhead transcription factor Hnf-3beta/Foxa-2 by Akt-mediated phosphorylation and nuclear/cytosolic localization. Proc. Natl. Acad. Sci. USA 100:11624-11629.
55. Yoo-Warren, H., V. Pachnis, R. S. Ingram, and S. M. Tilghman. 1988. Two regulatory domains flank the mouse H19 gene. Mol. Cell. Biol. 8:4707-4715.
56. Zaret, K. 1999. Developmental competence of the gut endoderm: genetic potentiation by GATA and HNF3/fork head proteins. Dev. Biol. 209:1-10.[CrossRef][Medline]
57. Zemel, S., M. S. Bartolomei, and S. M. Tilghman. 1992. Physical linkage of two mammalian imprinted genes, H19 and insulin-like growth factor 2. Nat. Genet. 2:61-65.[CrossRef][Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
