SEMINARS IN LIVER DISEASE-VOL.
12, NO. 3 , 1992
Regulation of Hepatic Gene Expression and Development
The liver carries out a wide range of vital functions, regulating carbohydrate and lipid metabolism, detoxification of exogenous and endogenous compounds, and producing most serum proteins. These functions are attributable to one cell type, the hepatic parenchymal cell, which comprises over 90% by mass of the liver. In order to perform these specialized functions, the hepatocyte expresses a large number of genes that are not expressed in most other cells. It is the specific expression of these genes that distinguishes the hepatocyte from all other cell types. Because gene expression is controlled primarily at the level of gene transcription, this review will focus on recent advances in our understanding of the proteins that regulate transcription in the hepatocyte. In addition to maintaining the differentiated function of the hepatocyte, recent evidence suggests that some of these factors may play important roles in the development of the liver. Transcription of genes encoding proteins is performed by RNA polymerase 11. The transcription rate of a gene is determined by the rate of the initiation of RNA synthesis, which in turn is controlled by actions of multiple proteins that interact with the RNA polymerase and with the regulatory region of the gene. Figure 1 shows a schematic diagram of this region of a typical gene. The TATA site, usually found 25-35 nucleotides upstream of the transcription start site binds the basic transcription factor TFIID, which nucleates the transcription complex.' Other factors that bind at sites termed "enhancers" regulate transcription by stimulating the formation of transcription complex formation. These sites can be near the TATA site (promoter proximal) or up to several kilobases away (distal) either upstream or downstream .~ recently, it has been found that of the p r ~ m o t e r More factors that repress transcription can also bind to some of these same sites.3 Cell-specific gene expression requires factors that are present in all cells plus additional factors that function only in certain cell types. For the hepatocyte, these cell-specific factors would be expected to control the expression of one or more liver-specific
proteins, such as the serum proteins albumin and transthyretin. A combination of advances in recombinant DNA technology, sensitive assays to measure proteinDNA interaction, and improved affinity chromatography methods have made it possible to identify, purify, and clone the genes for several liver-specific transcription factors.
IDENTIFICATION OF LIVER-ENRICHED TRANSCRIPTION FACTORS Most of the proteins that participate in hepatocytespecific transcription have been identified through the analysis of the genes for liver-specific products, such as the serum proteins (albumin, transthyretin) or liver-enriched enzymes (phosphoenolpyruvate carboxykinase). Techniques had been developed to introduce or transfect DNA into mammalian cells in ~ u l t u r e .This ~ DNA is transported into the nucleus and if the appropriate sequences are present in the transfected DNA, it will be transcribed and then translated. By assaying for expression of the transfected DNA, early studies confirmed that liver-specific genes with their associated regulatory sequences were expressed in cells derived from hepatocytes and not in other cell types. Human hepatoma cells, HepG2, and human HeLa cells have been most frequently used for these comparative studies. It was then possible to localize DNA sequences essential for hepatoma-specific expression by testing successive series of deletions and mutations of the genomic DNA. These sequences were known to function as sites of DNA binding by proteins that regulate transcription." Consequently, they could be used to identify the transcription factors that regulate liver-specific gene expression. The analysis of the regulatory sequences of transthyretin and a,-antitrypsin promoter reveals an organization that is representative of many liver-specific genes. Figure 2 illustrates the known sites for both liver-
From the Division of Endocrinology and Program of Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York. Reprint requests: Dr. Lai, Division of Endocrinology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
246
Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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ESENG LAI, M.D. Ph.D.
REGULATION OF HEPATIC GENE EXPRESSION-LA1
==P site
Promoter-proximal sites
Distal sites
1
TATA
I
hy II
FIG. 1. Regulatory regions of a typical gene. A TATA site is found 25-35 nucleotides upstream of the transcription start (cap) site. Sites for DNA binding proteins that regulate transcription can be found near the cap site (proximal) or up to several kilobases away (distal).
I
could then be greatly enriched by chromatography over this matrix. Purified protein can then be partially sequenced and the information used to design probes to isolate the cDNA clones.
TRANSCRIPTION FACTOR FAMILIES REGULATING LIVER GENE EXPRESSION Four transcription factor families that are expressed in the liver and have a limited cellular distribution, C1 EBP, HNF-1, HNF-3, and HNF-4, will be described. Members of these protein families are expressed in embryonic liver as well as the adult, evidence that these factors may act during development and cell differentiation. This survey is representative of the factors that are important in liver-specific expression and is not meant to be exhaustive. Table 1 lists the transcription factors to be discussed.
CIEBP CIEBP (now called CIEBPa) was identified because of its ability to bind to virus enhancer sequences and because it was originally thought to be a CCAAT binding protein. Subsequently, sequences that were shown to drive TTR and albumin transcription in hepatoma cells were identified as CIEBP binding sites. These results coupled with the limited cellular distribution of CIEBP, as assayed by gel shift experiments, suggested
0 0 -1 00
C/EBP
HNF-4
'
r,
TATA
'
TTR
0CCAAT
FIG. 2. Regulatory regions of transthyretin (TTR) and a,-antitryptsin (a,-AT). Binding sites for several liver-specific factors as well as ubiquitous factors are essential for the tissue-specific expression of these genes. (Adapted from Costa et a1.39)
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specific and general transcription factors in the promoters of these genes. Some important generalizations regarding the expression of liver-specific genes can be made from studies of these and other genes: (1) Multiple cis-acting regulatory sequences contribute to the high levels of expression of these genes in the hepatocyte compared with other cells. These are usually found within several hundred nucleotides upstream of the RNA start site. Distant upstream enhancer sequences have also been found for many of the genes that are expressed in the liver. Some of the same proteins bind to both enhancer and to proximal regulatory sequences; (2) the proteins that bind to the regulatory sites include those that are present in most cell types and those that have a limited cell distribution. The latter group are termed "liverspecific," although most of them are also expressed to a greater or lesser extent in a number of other tissues. The high degree of specificity of expression of liver-specific genes appears to be achieved through the combinatorial actions of several of these factors. Once these factors were identified on the basis of their binding to specific DNA sequences, further progress toward understanding their function required their purification and the cloning of their genes. One important technical advance that greatly facilitated the purification of many of these factors was the development of sequence-specific affinity chromatography. Doublestranded oligonucleotides containing the binding site for the protein were covalently coupled to a solid matrix. Proteins that bound specifically to the oligonucleotides
248
SEMINARS IN LIVER DISEASE-VOLUME
12, NUMBER 3, 1992
TABLE 1. Hepatocyte Transcription Factors* Transcri~tionFactor Farnilv'
CIEBP CIEPBa CIEBPP (NF-IL6, LAP, IL-6DBP) HNF- I HNF- I a (LF-B 1, APF) HNF- I P (vHNF- 1) HNF-3 HNF-3a HNF-3P HNF-3y HNF-4
DNA Binding Motif
Turret Genes in Liver'
bZIP
Alb, TTR, a,-AT
POU-homeodomain
a and PFib, @,-AT,alb, TTR, AFP
Undetermined
TTR, a,-AT, Alb AFP, TAT, PEPCK
Zinc finger, nuclear receptor
TTR, apoCIII, a,-AT PyK, GS
its importance in tissue-specific trans~ription.',~Several members of this gene family have now been identified, two of which are expressed at high levels in the liver. C/ EBPa is prominent in liver and in fat cells, and CIEBPP is prominent in these tissues and two additional tissues, lung and inte~tine.~ CIEBPP is identical to cDNA initially identified by several groups from human (NF-IL69), rat (LAP1', IL-6DBP1'), and mouse (AGP-EBPI2) sources. It is a mediator of the acute-phase response in the liver, and has been shown to exhibit an increase in DNA binding activity when cells are exposed to IL- 1 or IL-6.9.1' Members of the CIEBP family form dimers through interacting coiled domains that present leucine residues on one face of the coi1.I3.l4In each monomer, a hinge region connects the coil to a region rich in basic amino acids that is believed to wrap around about one turn of the DNA helix making contacts with the bases in the major groove. This structure has been termed the bZIP binding domain by McKnight and colleagues (for basic region-leucine zipper).15 CIEBP proteins will form heterodimers as well as homodimers, thus greatly expanding the potential number of distinct species arising from this gene family.
HNF-la was detected as a protein binding to sites required for cell-specific expression of fibrinogen and a,-antitrypsin genes. It been shown to be critical for optimal expression of albumin and several other liver-specific genes. In its DNA binding region, the HNF-1 protein is distantly related to the homeobox proteins and contains an adjoining "pou" related sequence.16." Both regions are required for DNA binding. Two activation domains, one serine-threonine rich and one proline rich, have been found by deletion analysi~.'~ HNF-1P is highly homologous in the DNA binding and dimerization domains. It is not capable of transactivation at the fibrinogen promoter and is missing both activation domains of the HNF- 1a protein. HNF- 1P ap-
pears to be identical to the variant form of HNF-I (vHNF-1) whose expression has been correlated with repression of a subset of liver-specific genes. 19,20 HNF1 p will form heterodimers with HNF-la but it does not appear to inhibit the activity of HNF-la in this manner. HNF-la is expressed in a wider range of tissues than initially believed, being present in kidney, intestine, and spleen as well as the liver. It is not ubiquitously expressed, however, being absent in brain, lung, thymus, and skin.17 HNF-1P is expressed in the tissues where HNF-la is expressed and is also found in lung and ovary.21
HNFS The HNF-3 proteins were first identified through their binding to sites in both the TTR and a,-antitrypsin genes, which had been shown to be essential for optimal hepatoma-specific expression. Early studies showed these proteins to be present in liver nuclear extracts and not in extracts from kidney, brain, and spleen. The first member of this group, HNF-3a, was purified and the cDNA cloned using some of the methods already outlined.22Two additional related proteins, HNF-3P and HNF-3y, were cloned soon thereafter by low stringency hybridization screening of a liver cDNA library. Figure 3 shows a schematic representation of these clones. Amino acid identity in a 110-amino acid central domain of these three proteins is extraordinarily high, over 90%. This domain was shown to be essential for binding specifically to DNA.2' Sequence data show that HNF-3 family members do not belong to any already recognized family of DNA binding proteins and thus defines a novel DNA binding structure. Site-directed mutagenesis of the binding domain and physical studies are in progress to identify the mode by which these proteins interact with DNA. Using these cDNA as probes, we were able to determine in detail the distribution of expression of each of these three genes. HNF-3a and P mRNAs are present in liver, lung, intestine, whereas HNF-3y is present in liver
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*Members of transcription factor families are essential for optimal expression in the liver of a number of tissue-specific genes. Three of the four families belong to classes of factors that share a known DNA binding structure. The listing of representative target genes is not meant to be comprehensive. CIEBP: CCAATIenhancer-binding protein; HNF-1,3,4; hepatocyte nuclear factor-1,3,4; bZIP: basic-leucine zipper; AFP:: a-fetoprotein; I AT: ,-antitrypsin; apoCIII: apolipoprotein CHI; Alb: albumin; Fib: fibrinogen; GS: glutamine synthetase; PEPCK: phosphoenolpyruvate carboxykinase; PyK: pyruvate kinase; TAT: tyrosine aminotransferase.
REGULATION OF HEPATIC GENE EXPRESSION-LA1 I
I
HNF- 37 HNF-3a HNF-3P F o r k head
1
I
1186
77
160
1
1269
149
1
1237
200
1
1309
11
I11
LI
El I4
a
kl
El M
fl
FIG. 3. Homologous regions of the HNF-3 proteins and Drosophila fork head. Reg~onI is the DNA bindina domain. The amino acid ~ositionsof this domaln are indicated. Reg~onsII and Ill are short stretches of homology with an as yet unknbwn function
u
HNF-4 was first identified as a protein that bound to the TTR promoter. After the cDNA was cloned, comparison of the sequence with the protein data bank revealed that this protein was a member of the nuclear steroid-thyroid receptor s ~ p e r f a r n i l y .In ~ ~the zinc finger DNA binding domain, there is between 40 and 67% similarity to members of the superfamily. Like some other family members, HNF-4 forms homodimers before binding DNA. HNF-4 does not have a known ligand, as is the case for many proteins in this family.26.27 A recent finding is that HNF-4 has a distinct Drosophila homolog HNF-4 with exceptionally high amino acid conservation in both DNA binding and the ligand binding or dimerization domains (Zhong, Sladek, Darnell: Unpublished observations). The 90% identity in the DNA binding domain shows it to be more closely related to the Drosophila gene than to any mammalian nuclear receptors. Furthermore, preliminary evidence shows that the Drosophila gene is expressed in developing gut tissue and a few other tissues but not in all embryonic tissues.
ROLE OF HNF-1,3,4 AND CEBP IN DIFFERENTIATION AND DEVELOPMENT By in situ hybridization, the earliest expression of CEBP is detected in fetal liver at embryonic day 13 in the mouse.28HNF-1 and HNF-4 have also been detected at this stage of development. Thus, there is evidence for the presence of all four mRNAs during embryogenesis. The role of these factors during the earliest stages of liver determination remains unknown. The most provocative information suggesting that the HNF-3 family and possibly the HNF-4 subfamily of the large nuclear steroidthyroid receptor family have a role in early development arises from the discovery of HNF-3 and HNF-4 homologs in Drosophila. As just described, these homologs are expressed in early embryonic stages in precursors to gut cells. These findings with HNF-3 and 4 suggest that a code of expression of HNF-3 and/or HNF-4 family members helps determine endodermal decisions along the length of the primitive gut in an analogous fashion to the homeobox proteins and the anteroposterior axis.2v That factors that were initially identified to control tissue-specific gene expression in the differentiated cell of the adult animal might play important roles in development and the establishment of differentiated function is not unexpected. Hepatocyte-specific expression of albumin and a-fetoprotein (AFP) is detectable in the 9day-old embryo. Furthermore, the adult liver retains the ability to regenerate. Thus, even though the adult hepatocyte is a highly differentiated cell, it is capable of proliferation and reestablishment of the differentiated phenotype. Extracellular signals play important roles during differentiation and development. The integrity of the organized structure of the liver is apparently required for the optimal differentiated function of the hepatocyte. It has been shown that hepatocytes disaggregated from the liver and placed in tissue culture on a plastic substrate will assume a flattened morphology. Under these conditions, the transcription of liver-specific genes, such as albumin, declines drastically while the transcription of other genes, such as actin and tubulin, remain un~hanged.~O.~' Culture of hepatocytes in defined medium with extracellular matrix components allows maintenance of a more differentiated, cuboidal morphology with preservation of albumin tran~cription.~~ Recent work has demonstrated that the conditions of hepatocyte culture that preserve liver-specific gene expresiion also maintain the DNA-binding activities of at least two liver-
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and intestine but is missing in the lung.2' These tissues are all derived from the embryonic gut. Thus, the pattern of expression of the HNF-3 genes suggested that they might play a role not only in maintaining differentiated function in the mature animal, but also during development. Supporting this hypothesis, we learned that the HNF-3 genes were also highly homologous to the Dro' . ~ region ~ sophila homeotic gene called fork h e ~ d . ~The of greatest homology corresponds precisely to the DNA binding domain of the HNF-3 proteins (Fig. 3). Fork head is produced in the developing Drosophila embryo in the terminal regions (ends) of the embryo. Cells from this region invaginate as development proceeds and give rise to the anterior and posterior gut structures. This striking conservation of both structure and restricted expression pattern suggested a conservation of function We speculate that HNF-3 family members will also be found to be expressed in early gut development in mammals and will play a key role in helping to execute choices that govern endodermal determination. Attempts are now underway to obtain an accurate picture of HNF3a, p, or -y expression in primitive gut. In addition, attempts are underway to find other family members from such cells as pancreas and salivary glands and to examine these tissues for the presently known HNF-3 family members.
specific transcription factors, a member of the CIEBP family and a member of the HNF-3 family.33 Because most of the studies of the function of these transcription factors have been done with purified proteins, cell extracts, or in cultured cells, their physiologic roles in the whole animal remain to be directly demonstrated. A recently developed technique allows the examination of protein-DNA interactions in intact nuclei from tissues. Examination of the TTR gene in adult liver shows that only subsets of the transcription factor binding sites that are identified in vitro are actually occupied.34 HNF-3 sites are prominently protected against both DNAse and chemical attack but protein sequences shown in vitro to be sites for HNF-I, 4, and CIEBP are not. These latter proteins might participate in establishing active transcription sites and may then no longer be required or might sometimes but not always be engaged with DNA.
ROLE OF TRANSCRIPTION FACTORS IN GENETIC DISEASES One recent study of a genetic disease sheds some light on a physiologic role for one of the liver-specific factors. genetically determined diseases are attributable to the absence or diminished levels of essential proteins. Although many of these conditions result from mutations in the coding- sequence and thus affect the pri. mary structure of the protein, an increasing number of examples of mutations that alter the regulation of gene expression are being found. One type of hemophilia has been found to be attributable to mutations in the promoter of the gene that affects the binding of HNF-4.40 Hemophilia B Leyden is associated with low levels of Factor IX during childhood. Patients are found to have one of a number of single point mutations in the promoter of the Factor IX gene. One of these mutations, found in the original family, is a T to A change at position -20. This mutation disrupts a binding site for HNF-4 and greatly diminishes the ability of HNF-4 to activate the actor I k promoter in transfection assays. This study suggests that alteration of the regulatory site for HNF-4 is the basis for this form of hemophilia and provides an example of the function of these transcription factors in the expression of liver-specific genes in vivo.
any
STUDIES FOR THE FUTURE The cloning of the genes for many of the factors involved in liver-specific gene expression will make it possible to examine their function in animals using transgenic and embryonic stem (ES) cell technology. The genes for each of these transcription factors can be mutated by homologous recombination in ES cells. These cells can then be mixed with cells of a blastula stage embryo to generate chimeric mice containing the mutated gene. If the mutation is incorporated into germ cells, animals heterozygous and homozygous for the mutation can be generated and studied for the consequences of these mutations.
12, NUMBER 3, 1992
A number of issues remain unresolved. For example, both HNF- I and HNF-4 are capable in cultured cells of driving transcription of test genes in plasmids containing their binding sites. Both HNF- I and HNF-4 are present in the liver and the k i d n e ~ . ' Although ~ . ~ ~ some of the genes containing HNF-1 and HNF-4 sites are expressed at low levels in the kidney, for example, a,-antitrypsin, others are not. These results suggest that negative-acting factors may also be involved in tissue-specific regulation. Recent evidence with the AFP promoter and enhancer regions has shown clearly that in transgenic animals inappropriate expression of AFP occurs on deletion of certain sites.3s In addition, removal of an 80 nucleotide segment of the retinol-binding protein gene led to expression in HeLa cells as well as hepatoma cells.36The mRNA for a member of the CIEBP family called LAP has been shown to produce another protein, LIP, which binds with a high affinity to the LAP DNA recognition sequence and represses tran~cription.~ Furthermore, negative-acting factors need not bind to DNA to function. Factors that form heterodimers can be subject to dominant negative regulation as exemplified by the helix-loop-helix family of factors some of whose members regulate muscle-specific gene expression. A member of this family called "Id" is unable to bind to DNA and forms heterodimers with positive-acting factors, thereby inhibiting their activity.37 Recently, another member of the CIEBP family called CHOP has been identified, which can inhibit the activity of both CIEBPa and CIEBPP by forming heterodimer~.~'
SUMMARY Tissue-specific expression in the hepatocyte appears to require the actions of multiple factors in specific combinations for each gene. The interactions between two or more positive-acting factors that raise transcription to optimal levels and those between positive and negative factors that enforce strict cell-specific transcription remain to be elucidated. However, recent progress in the identification and cloning of the genes for many of these factors will make it possible to begin to answer these important questions.
REFERENCES Nakajima NM, Horikoshi M, Roeder RG: Factors involved in specific transcription by mammalian RNA polymerase 11: Purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol Cell Biol 8:4028-4040, 1988. Serfling E, Jasin M, Schaffner W: Enhancers and eukaryotic gene transcription. Trends Genet. 1985. Descombes P, Schibler U: A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67:569-579, 1991. Graham FL, Van Der Erb AJ: A new technique for the assay of infectivity of human adenovirus 5' DNA. Virology 52:456467, 1973. Dynan WS, Tjian R: Control of eukaryotic messenger RNA synthesis by sequence-specific DNA binding proteins. Nature 3 16:774-778, 1985.
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Costa RH, Lai E, Grayson DR. Darnell JE Jr: The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor(s) at two other sites. Mol Cell Biol 8:81-90, 1988. Maire P, Wuarin J, Schibler U: The role of cis-acting promoter elements in tissue-specific albumin gene expression. Science 244:343-346, 1989. Cao Z, Umek RM, McKnight SL: Regulated expression of three CIEBP isoforms during adipose conversion of 3T3-LI cells. Genes Dev 5: 1538-1552, 1991. Akira S, Isshiki H, Sugita T, et al: A nuclear factor for IL-6 expression (NF-IL6) is a member of a CIEBP family. EMBO J 9: 1897-1906, 1990. Descombes P, Chojkier M, Lichtsteiner S, et al: LAP, a novel member of the CIEBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev 4: 155 I, 1990. Poli V, Mancini FP, Cortese R: IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to CIEBP. Cell 63:643-653, 1990. Chang CJ, Chen TT. Lei HY, et al: Molecular cloning of a transcription factor, AGPIEBP that belongs to members of the CIEBP family. Mol Cell Biol 10:6642-6653, 1990. Landschulz WH, Johnson PF, McKnight SL: The leucine zipper: A hypothetical structure common to a new class of DNAbinding proteins. Science 240: 1759- 1764, 1988. O'Shea EK, Rutkowski R, Kim PS: Evidence that the leucine zipper is a coiled coil. Science 243:538-542, 1989. Vinson CR, Sigler PB, McKnight SL: Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science 246:911-916, 1989. Frain M, Swart G, Monaci P, et al: The liver-specific transcription factor LF-B I contains a highly diverged homeobox DNA binding domain. Cell 59:145-157, 1989. Baumhueter S, Mendel DB, Conley PB, et al: HNF-I shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes Dev 4:372-379, 1990. Nicosia A, Monaci P, Tomei L, et al: A myosin-like dimerization helix and an extra-large homeodomain are essential elements of the tripartite DNA binding structure of LFBI. Cell 61:1225-1236, 1990. Cereghini S, Blumenfeld M, Yaniv M: A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes Dev 2:957-974, 1988. Baumhueter S, Courtois G, Crabtree GR: A variant nuclear protein in dedifferentiated hepatoma cells binds to the same functional sequences in the P-fibrinogen gene promoter as HNF-1. EMBO J: 1988. Mendel DB, Hansen LP, Graves MK, et al: HNF-la and HNFlP(vHNF-I) share dimerization and homeodomains, but not activation domains and form heterodimers in vitro. Genes Dev 5:1042-1056, 1991. Lai E, Prezioso VR, Smith E, et al: HNF-3A, a hepatocyteenriched transcription factor of novel structure is regulated transcriptionally. Genes Dev 4: 1427-1436, 1990. Lai E, Prezioso VR, Tao W, et al: Hepatocyte nuclear factor 3 a belongs to a gene family in mammals that is homologous
to the Drosophila homeotic genefork heud. Genes Dev 5:416427, 1991. Weigel D, Jackle H: Fork head: A new eukaryotic DNA binding motif? Cell 63:455-456, 1990. Sladek FM, Zhong W, Lai E, Darnell JE Jr: Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev 4:2353-2365, 1990. Nauber U, Pankratz MJ, Kienlin A, et al: Abdominal segmentation of the Drosophila embryo requires a hormone receptorlike protein encoded by the gap gene knirps. Nature 336:489492, 1988. Oro AE, Ong ES, Margolis JS, et al: The Drosophila gene knirps-related is a member of the steroid-receptor gene superfamily. Nature 336:493-496, 1988. Kuo CF, Xanthopoulos KG, Darnell JE Jr: Fetal and adult localization of CIEBP: Evidence for combinatorial action of transcription factors in cell-specific gene expression. Development 109:473-481, 1990. Akam M: Hox and HOM: Homologous gene clusters in insects and vertebrates. Cell 57:347-349, 1989. Clayton DF, Darnell JE Jr: Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol: 1983. Clayton DF, Harrelson AL, Darnell JE Jr: Dependence of liver-specific transcription on tissue organization. Mol Cell Biol: 1985. Caron JM: Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol 10: 12391243, 1990. Liu JK. DiPersio CM, Zaret KS: Extracellular signals that regulate liver transcription factors during hepatic differentiation in vivo. Mol Cell Biol 11:773-784, 1991. Mirkovitch J, Darnell JE Jr: Rapid in vivo footprinting technique identifies proteins bound to the TTR gene in liver. Genes Dev 5: 1991. Vacher J, Tilghman SM: Dominant negative regulation of the mouse wfetoprotein gene in adult liver. Science 250:17321735, 1990. Colantuoni V, Pirozzi A, BIance C. Cortese R: Negative control of liver-specific gene expression: Cloned human retinolbinding protein gene is repressed in HeLa cells. EMBO J 6:63 1-636, 1987. Benezra R, Davis RL, Lockshon D, et al: The protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49-59, 1990. Ron D, Habener J: CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors CIEBP and LAP and functions as a dominant negative inhibitor of gene transcription. Genes Dev 1992. (In press). Costa RH, Grayson DR. Darnell JE Jr: Multiple hepatocytespecific nuclear factors function in the regulation of the transthyretin and a,-antitrypsin genes. Mol Cell Biol 9: 1415-1425, 1989. Reijnen MJ, Sladek FM, Bertina RM, Reitsma, PH: Disruption of a binding site for hepatocytenuclear factor 4 results in Hemophilia B Leyden. Proc Natl Acad Sci, 1992 (in press).
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REGULATION OF HEPATIC GENE EXPRESSION-LA1
12, NO. 3 , 1992
Regulation of Hepatic Gene Expression and Development
The liver carries out a wide range of vital functions, regulating carbohydrate and lipid metabolism, detoxification of exogenous and endogenous compounds, and producing most serum proteins. These functions are attributable to one cell type, the hepatic parenchymal cell, which comprises over 90% by mass of the liver. In order to perform these specialized functions, the hepatocyte expresses a large number of genes that are not expressed in most other cells. It is the specific expression of these genes that distinguishes the hepatocyte from all other cell types. Because gene expression is controlled primarily at the level of gene transcription, this review will focus on recent advances in our understanding of the proteins that regulate transcription in the hepatocyte. In addition to maintaining the differentiated function of the hepatocyte, recent evidence suggests that some of these factors may play important roles in the development of the liver. Transcription of genes encoding proteins is performed by RNA polymerase 11. The transcription rate of a gene is determined by the rate of the initiation of RNA synthesis, which in turn is controlled by actions of multiple proteins that interact with the RNA polymerase and with the regulatory region of the gene. Figure 1 shows a schematic diagram of this region of a typical gene. The TATA site, usually found 25-35 nucleotides upstream of the transcription start site binds the basic transcription factor TFIID, which nucleates the transcription complex.' Other factors that bind at sites termed "enhancers" regulate transcription by stimulating the formation of transcription complex formation. These sites can be near the TATA site (promoter proximal) or up to several kilobases away (distal) either upstream or downstream .~ recently, it has been found that of the p r ~ m o t e r More factors that repress transcription can also bind to some of these same sites.3 Cell-specific gene expression requires factors that are present in all cells plus additional factors that function only in certain cell types. For the hepatocyte, these cell-specific factors would be expected to control the expression of one or more liver-specific
proteins, such as the serum proteins albumin and transthyretin. A combination of advances in recombinant DNA technology, sensitive assays to measure proteinDNA interaction, and improved affinity chromatography methods have made it possible to identify, purify, and clone the genes for several liver-specific transcription factors.
IDENTIFICATION OF LIVER-ENRICHED TRANSCRIPTION FACTORS Most of the proteins that participate in hepatocytespecific transcription have been identified through the analysis of the genes for liver-specific products, such as the serum proteins (albumin, transthyretin) or liver-enriched enzymes (phosphoenolpyruvate carboxykinase). Techniques had been developed to introduce or transfect DNA into mammalian cells in ~ u l t u r e .This ~ DNA is transported into the nucleus and if the appropriate sequences are present in the transfected DNA, it will be transcribed and then translated. By assaying for expression of the transfected DNA, early studies confirmed that liver-specific genes with their associated regulatory sequences were expressed in cells derived from hepatocytes and not in other cell types. Human hepatoma cells, HepG2, and human HeLa cells have been most frequently used for these comparative studies. It was then possible to localize DNA sequences essential for hepatoma-specific expression by testing successive series of deletions and mutations of the genomic DNA. These sequences were known to function as sites of DNA binding by proteins that regulate transcription." Consequently, they could be used to identify the transcription factors that regulate liver-specific gene expression. The analysis of the regulatory sequences of transthyretin and a,-antitrypsin promoter reveals an organization that is representative of many liver-specific genes. Figure 2 illustrates the known sites for both liver-
From the Division of Endocrinology and Program of Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York. Reprint requests: Dr. Lai, Division of Endocrinology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
246
Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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ESENG LAI, M.D. Ph.D.
REGULATION OF HEPATIC GENE EXPRESSION-LA1
==P site
Promoter-proximal sites
Distal sites
1
TATA
I
hy II
FIG. 1. Regulatory regions of a typical gene. A TATA site is found 25-35 nucleotides upstream of the transcription start (cap) site. Sites for DNA binding proteins that regulate transcription can be found near the cap site (proximal) or up to several kilobases away (distal).
I
could then be greatly enriched by chromatography over this matrix. Purified protein can then be partially sequenced and the information used to design probes to isolate the cDNA clones.
TRANSCRIPTION FACTOR FAMILIES REGULATING LIVER GENE EXPRESSION Four transcription factor families that are expressed in the liver and have a limited cellular distribution, C1 EBP, HNF-1, HNF-3, and HNF-4, will be described. Members of these protein families are expressed in embryonic liver as well as the adult, evidence that these factors may act during development and cell differentiation. This survey is representative of the factors that are important in liver-specific expression and is not meant to be exhaustive. Table 1 lists the transcription factors to be discussed.
CIEBP CIEBP (now called CIEBPa) was identified because of its ability to bind to virus enhancer sequences and because it was originally thought to be a CCAAT binding protein. Subsequently, sequences that were shown to drive TTR and albumin transcription in hepatoma cells were identified as CIEBP binding sites. These results coupled with the limited cellular distribution of CIEBP, as assayed by gel shift experiments, suggested
0 0 -1 00
C/EBP
HNF-4
'
r,
TATA
'
TTR
0CCAAT
FIG. 2. Regulatory regions of transthyretin (TTR) and a,-antitryptsin (a,-AT). Binding sites for several liver-specific factors as well as ubiquitous factors are essential for the tissue-specific expression of these genes. (Adapted from Costa et a1.39)
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specific and general transcription factors in the promoters of these genes. Some important generalizations regarding the expression of liver-specific genes can be made from studies of these and other genes: (1) Multiple cis-acting regulatory sequences contribute to the high levels of expression of these genes in the hepatocyte compared with other cells. These are usually found within several hundred nucleotides upstream of the RNA start site. Distant upstream enhancer sequences have also been found for many of the genes that are expressed in the liver. Some of the same proteins bind to both enhancer and to proximal regulatory sequences; (2) the proteins that bind to the regulatory sites include those that are present in most cell types and those that have a limited cell distribution. The latter group are termed "liverspecific," although most of them are also expressed to a greater or lesser extent in a number of other tissues. The high degree of specificity of expression of liver-specific genes appears to be achieved through the combinatorial actions of several of these factors. Once these factors were identified on the basis of their binding to specific DNA sequences, further progress toward understanding their function required their purification and the cloning of their genes. One important technical advance that greatly facilitated the purification of many of these factors was the development of sequence-specific affinity chromatography. Doublestranded oligonucleotides containing the binding site for the protein were covalently coupled to a solid matrix. Proteins that bound specifically to the oligonucleotides
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TABLE 1. Hepatocyte Transcription Factors* Transcri~tionFactor Farnilv'
CIEBP CIEPBa CIEBPP (NF-IL6, LAP, IL-6DBP) HNF- I HNF- I a (LF-B 1, APF) HNF- I P (vHNF- 1) HNF-3 HNF-3a HNF-3P HNF-3y HNF-4
DNA Binding Motif
Turret Genes in Liver'
bZIP
Alb, TTR, a,-AT
POU-homeodomain
a and PFib, @,-AT,alb, TTR, AFP
Undetermined
TTR, a,-AT, Alb AFP, TAT, PEPCK
Zinc finger, nuclear receptor
TTR, apoCIII, a,-AT PyK, GS
its importance in tissue-specific trans~ription.',~Several members of this gene family have now been identified, two of which are expressed at high levels in the liver. C/ EBPa is prominent in liver and in fat cells, and CIEBPP is prominent in these tissues and two additional tissues, lung and inte~tine.~ CIEBPP is identical to cDNA initially identified by several groups from human (NF-IL69), rat (LAP1', IL-6DBP1'), and mouse (AGP-EBPI2) sources. It is a mediator of the acute-phase response in the liver, and has been shown to exhibit an increase in DNA binding activity when cells are exposed to IL- 1 or IL-6.9.1' Members of the CIEBP family form dimers through interacting coiled domains that present leucine residues on one face of the coi1.I3.l4In each monomer, a hinge region connects the coil to a region rich in basic amino acids that is believed to wrap around about one turn of the DNA helix making contacts with the bases in the major groove. This structure has been termed the bZIP binding domain by McKnight and colleagues (for basic region-leucine zipper).15 CIEBP proteins will form heterodimers as well as homodimers, thus greatly expanding the potential number of distinct species arising from this gene family.
HNF-la was detected as a protein binding to sites required for cell-specific expression of fibrinogen and a,-antitrypsin genes. It been shown to be critical for optimal expression of albumin and several other liver-specific genes. In its DNA binding region, the HNF-1 protein is distantly related to the homeobox proteins and contains an adjoining "pou" related sequence.16." Both regions are required for DNA binding. Two activation domains, one serine-threonine rich and one proline rich, have been found by deletion analysi~.'~ HNF-1P is highly homologous in the DNA binding and dimerization domains. It is not capable of transactivation at the fibrinogen promoter and is missing both activation domains of the HNF- 1a protein. HNF- 1P ap-
pears to be identical to the variant form of HNF-I (vHNF-1) whose expression has been correlated with repression of a subset of liver-specific genes. 19,20 HNF1 p will form heterodimers with HNF-la but it does not appear to inhibit the activity of HNF-la in this manner. HNF-la is expressed in a wider range of tissues than initially believed, being present in kidney, intestine, and spleen as well as the liver. It is not ubiquitously expressed, however, being absent in brain, lung, thymus, and skin.17 HNF-1P is expressed in the tissues where HNF-la is expressed and is also found in lung and ovary.21
HNFS The HNF-3 proteins were first identified through their binding to sites in both the TTR and a,-antitrypsin genes, which had been shown to be essential for optimal hepatoma-specific expression. Early studies showed these proteins to be present in liver nuclear extracts and not in extracts from kidney, brain, and spleen. The first member of this group, HNF-3a, was purified and the cDNA cloned using some of the methods already outlined.22Two additional related proteins, HNF-3P and HNF-3y, were cloned soon thereafter by low stringency hybridization screening of a liver cDNA library. Figure 3 shows a schematic representation of these clones. Amino acid identity in a 110-amino acid central domain of these three proteins is extraordinarily high, over 90%. This domain was shown to be essential for binding specifically to DNA.2' Sequence data show that HNF-3 family members do not belong to any already recognized family of DNA binding proteins and thus defines a novel DNA binding structure. Site-directed mutagenesis of the binding domain and physical studies are in progress to identify the mode by which these proteins interact with DNA. Using these cDNA as probes, we were able to determine in detail the distribution of expression of each of these three genes. HNF-3a and P mRNAs are present in liver, lung, intestine, whereas HNF-3y is present in liver
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*Members of transcription factor families are essential for optimal expression in the liver of a number of tissue-specific genes. Three of the four families belong to classes of factors that share a known DNA binding structure. The listing of representative target genes is not meant to be comprehensive. CIEBP: CCAATIenhancer-binding protein; HNF-1,3,4; hepatocyte nuclear factor-1,3,4; bZIP: basic-leucine zipper; AFP:: a-fetoprotein; I AT: ,-antitrypsin; apoCIII: apolipoprotein CHI; Alb: albumin; Fib: fibrinogen; GS: glutamine synthetase; PEPCK: phosphoenolpyruvate carboxykinase; PyK: pyruvate kinase; TAT: tyrosine aminotransferase.
REGULATION OF HEPATIC GENE EXPRESSION-LA1 I
I
HNF- 37 HNF-3a HNF-3P F o r k head
1
I
1186
77
160
1
1269
149
1
1237
200
1
1309
11
I11
LI
El I4
a
kl
El M
fl
FIG. 3. Homologous regions of the HNF-3 proteins and Drosophila fork head. Reg~onI is the DNA bindina domain. The amino acid ~ositionsof this domaln are indicated. Reg~onsII and Ill are short stretches of homology with an as yet unknbwn function
u
HNF-4 was first identified as a protein that bound to the TTR promoter. After the cDNA was cloned, comparison of the sequence with the protein data bank revealed that this protein was a member of the nuclear steroid-thyroid receptor s ~ p e r f a r n i l y .In ~ ~the zinc finger DNA binding domain, there is between 40 and 67% similarity to members of the superfamily. Like some other family members, HNF-4 forms homodimers before binding DNA. HNF-4 does not have a known ligand, as is the case for many proteins in this family.26.27 A recent finding is that HNF-4 has a distinct Drosophila homolog HNF-4 with exceptionally high amino acid conservation in both DNA binding and the ligand binding or dimerization domains (Zhong, Sladek, Darnell: Unpublished observations). The 90% identity in the DNA binding domain shows it to be more closely related to the Drosophila gene than to any mammalian nuclear receptors. Furthermore, preliminary evidence shows that the Drosophila gene is expressed in developing gut tissue and a few other tissues but not in all embryonic tissues.
ROLE OF HNF-1,3,4 AND CEBP IN DIFFERENTIATION AND DEVELOPMENT By in situ hybridization, the earliest expression of CEBP is detected in fetal liver at embryonic day 13 in the mouse.28HNF-1 and HNF-4 have also been detected at this stage of development. Thus, there is evidence for the presence of all four mRNAs during embryogenesis. The role of these factors during the earliest stages of liver determination remains unknown. The most provocative information suggesting that the HNF-3 family and possibly the HNF-4 subfamily of the large nuclear steroidthyroid receptor family have a role in early development arises from the discovery of HNF-3 and HNF-4 homologs in Drosophila. As just described, these homologs are expressed in early embryonic stages in precursors to gut cells. These findings with HNF-3 and 4 suggest that a code of expression of HNF-3 and/or HNF-4 family members helps determine endodermal decisions along the length of the primitive gut in an analogous fashion to the homeobox proteins and the anteroposterior axis.2v That factors that were initially identified to control tissue-specific gene expression in the differentiated cell of the adult animal might play important roles in development and the establishment of differentiated function is not unexpected. Hepatocyte-specific expression of albumin and a-fetoprotein (AFP) is detectable in the 9day-old embryo. Furthermore, the adult liver retains the ability to regenerate. Thus, even though the adult hepatocyte is a highly differentiated cell, it is capable of proliferation and reestablishment of the differentiated phenotype. Extracellular signals play important roles during differentiation and development. The integrity of the organized structure of the liver is apparently required for the optimal differentiated function of the hepatocyte. It has been shown that hepatocytes disaggregated from the liver and placed in tissue culture on a plastic substrate will assume a flattened morphology. Under these conditions, the transcription of liver-specific genes, such as albumin, declines drastically while the transcription of other genes, such as actin and tubulin, remain un~hanged.~O.~' Culture of hepatocytes in defined medium with extracellular matrix components allows maintenance of a more differentiated, cuboidal morphology with preservation of albumin tran~cription.~~ Recent work has demonstrated that the conditions of hepatocyte culture that preserve liver-specific gene expresiion also maintain the DNA-binding activities of at least two liver-
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and intestine but is missing in the lung.2' These tissues are all derived from the embryonic gut. Thus, the pattern of expression of the HNF-3 genes suggested that they might play a role not only in maintaining differentiated function in the mature animal, but also during development. Supporting this hypothesis, we learned that the HNF-3 genes were also highly homologous to the Dro' . ~ region ~ sophila homeotic gene called fork h e ~ d . ~The of greatest homology corresponds precisely to the DNA binding domain of the HNF-3 proteins (Fig. 3). Fork head is produced in the developing Drosophila embryo in the terminal regions (ends) of the embryo. Cells from this region invaginate as development proceeds and give rise to the anterior and posterior gut structures. This striking conservation of both structure and restricted expression pattern suggested a conservation of function We speculate that HNF-3 family members will also be found to be expressed in early gut development in mammals and will play a key role in helping to execute choices that govern endodermal determination. Attempts are now underway to obtain an accurate picture of HNF3a, p, or -y expression in primitive gut. In addition, attempts are underway to find other family members from such cells as pancreas and salivary glands and to examine these tissues for the presently known HNF-3 family members.
specific transcription factors, a member of the CIEBP family and a member of the HNF-3 family.33 Because most of the studies of the function of these transcription factors have been done with purified proteins, cell extracts, or in cultured cells, their physiologic roles in the whole animal remain to be directly demonstrated. A recently developed technique allows the examination of protein-DNA interactions in intact nuclei from tissues. Examination of the TTR gene in adult liver shows that only subsets of the transcription factor binding sites that are identified in vitro are actually occupied.34 HNF-3 sites are prominently protected against both DNAse and chemical attack but protein sequences shown in vitro to be sites for HNF-I, 4, and CIEBP are not. These latter proteins might participate in establishing active transcription sites and may then no longer be required or might sometimes but not always be engaged with DNA.
ROLE OF TRANSCRIPTION FACTORS IN GENETIC DISEASES One recent study of a genetic disease sheds some light on a physiologic role for one of the liver-specific factors. genetically determined diseases are attributable to the absence or diminished levels of essential proteins. Although many of these conditions result from mutations in the coding- sequence and thus affect the pri. mary structure of the protein, an increasing number of examples of mutations that alter the regulation of gene expression are being found. One type of hemophilia has been found to be attributable to mutations in the promoter of the gene that affects the binding of HNF-4.40 Hemophilia B Leyden is associated with low levels of Factor IX during childhood. Patients are found to have one of a number of single point mutations in the promoter of the Factor IX gene. One of these mutations, found in the original family, is a T to A change at position -20. This mutation disrupts a binding site for HNF-4 and greatly diminishes the ability of HNF-4 to activate the actor I k promoter in transfection assays. This study suggests that alteration of the regulatory site for HNF-4 is the basis for this form of hemophilia and provides an example of the function of these transcription factors in the expression of liver-specific genes in vivo.
any
STUDIES FOR THE FUTURE The cloning of the genes for many of the factors involved in liver-specific gene expression will make it possible to examine their function in animals using transgenic and embryonic stem (ES) cell technology. The genes for each of these transcription factors can be mutated by homologous recombination in ES cells. These cells can then be mixed with cells of a blastula stage embryo to generate chimeric mice containing the mutated gene. If the mutation is incorporated into germ cells, animals heterozygous and homozygous for the mutation can be generated and studied for the consequences of these mutations.
12, NUMBER 3, 1992
A number of issues remain unresolved. For example, both HNF- I and HNF-4 are capable in cultured cells of driving transcription of test genes in plasmids containing their binding sites. Both HNF- I and HNF-4 are present in the liver and the k i d n e ~ . ' Although ~ . ~ ~ some of the genes containing HNF-1 and HNF-4 sites are expressed at low levels in the kidney, for example, a,-antitrypsin, others are not. These results suggest that negative-acting factors may also be involved in tissue-specific regulation. Recent evidence with the AFP promoter and enhancer regions has shown clearly that in transgenic animals inappropriate expression of AFP occurs on deletion of certain sites.3s In addition, removal of an 80 nucleotide segment of the retinol-binding protein gene led to expression in HeLa cells as well as hepatoma cells.36The mRNA for a member of the CIEBP family called LAP has been shown to produce another protein, LIP, which binds with a high affinity to the LAP DNA recognition sequence and represses tran~cription.~ Furthermore, negative-acting factors need not bind to DNA to function. Factors that form heterodimers can be subject to dominant negative regulation as exemplified by the helix-loop-helix family of factors some of whose members regulate muscle-specific gene expression. A member of this family called "Id" is unable to bind to DNA and forms heterodimers with positive-acting factors, thereby inhibiting their activity.37 Recently, another member of the CIEBP family called CHOP has been identified, which can inhibit the activity of both CIEBPa and CIEBPP by forming heterodimer~.~'
SUMMARY Tissue-specific expression in the hepatocyte appears to require the actions of multiple factors in specific combinations for each gene. The interactions between two or more positive-acting factors that raise transcription to optimal levels and those between positive and negative factors that enforce strict cell-specific transcription remain to be elucidated. However, recent progress in the identification and cloning of the genes for many of these factors will make it possible to begin to answer these important questions.
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Costa RH, Lai E, Grayson DR. Darnell JE Jr: The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor(s) at two other sites. Mol Cell Biol 8:81-90, 1988. Maire P, Wuarin J, Schibler U: The role of cis-acting promoter elements in tissue-specific albumin gene expression. Science 244:343-346, 1989. Cao Z, Umek RM, McKnight SL: Regulated expression of three CIEBP isoforms during adipose conversion of 3T3-LI cells. Genes Dev 5: 1538-1552, 1991. Akira S, Isshiki H, Sugita T, et al: A nuclear factor for IL-6 expression (NF-IL6) is a member of a CIEBP family. EMBO J 9: 1897-1906, 1990. Descombes P, Chojkier M, Lichtsteiner S, et al: LAP, a novel member of the CIEBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev 4: 155 I, 1990. Poli V, Mancini FP, Cortese R: IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to CIEBP. Cell 63:643-653, 1990. Chang CJ, Chen TT. Lei HY, et al: Molecular cloning of a transcription factor, AGPIEBP that belongs to members of the CIEBP family. Mol Cell Biol 10:6642-6653, 1990. Landschulz WH, Johnson PF, McKnight SL: The leucine zipper: A hypothetical structure common to a new class of DNAbinding proteins. Science 240: 1759- 1764, 1988. O'Shea EK, Rutkowski R, Kim PS: Evidence that the leucine zipper is a coiled coil. Science 243:538-542, 1989. Vinson CR, Sigler PB, McKnight SL: Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science 246:911-916, 1989. Frain M, Swart G, Monaci P, et al: The liver-specific transcription factor LF-B I contains a highly diverged homeobox DNA binding domain. Cell 59:145-157, 1989. Baumhueter S, Mendel DB, Conley PB, et al: HNF-I shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes Dev 4:372-379, 1990. Nicosia A, Monaci P, Tomei L, et al: A myosin-like dimerization helix and an extra-large homeodomain are essential elements of the tripartite DNA binding structure of LFBI. Cell 61:1225-1236, 1990. Cereghini S, Blumenfeld M, Yaniv M: A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes Dev 2:957-974, 1988. Baumhueter S, Courtois G, Crabtree GR: A variant nuclear protein in dedifferentiated hepatoma cells binds to the same functional sequences in the P-fibrinogen gene promoter as HNF-1. EMBO J: 1988. Mendel DB, Hansen LP, Graves MK, et al: HNF-la and HNFlP(vHNF-I) share dimerization and homeodomains, but not activation domains and form heterodimers in vitro. Genes Dev 5:1042-1056, 1991. Lai E, Prezioso VR, Smith E, et al: HNF-3A, a hepatocyteenriched transcription factor of novel structure is regulated transcriptionally. Genes Dev 4: 1427-1436, 1990. Lai E, Prezioso VR, Tao W, et al: Hepatocyte nuclear factor 3 a belongs to a gene family in mammals that is homologous
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REGULATION OF HEPATIC GENE EXPRESSION-LA1
