Mechanisms of liver-specific gene expression Frances M. Sladek and James E. Darnell University of California, Riverside, California, and Rockefeller University, New York, USA. Significant advances in the field of hepatocyte-specific gene expression have been made during the past year. Several new transcription factors have been cloned and partially characterized. Analyses of the promoter regions of several factors have also been initiated and Drosophila homologs for two of these factors have been found, opening the way for studies on development. Current Opinion in Genetics and Development 1992, 2:256-259.
Introduction The unique phenotype of each differentiated cell in an animal arises from the selective expression of genes in a cell- or tissue-specific fashion. Since it was discovered that the expression of the majority of liver-specific proteins is controlled at the level of transcription [1], researchers have avidly sought the regulatory transcription factors that control liver-specific gene expression. There has been considerable success in this field of study and most of the current information on liver-specific gene expression comes from the analysis of these factors. In recent years the genes encoding three positively-acting transcription factors have been cloned. These factors, which have limited tissue distribution and appear to regulate many important liver-specific genes, are all members of gene families and include: C/EBP, the original leucinezipper protein; HNF-1cx, a pou-homeo domain protein; and HNF-3cx, one of a new transcription factor family (reviewed in [2] ). During the past year, isoforms or variants from all three of these families as well as a novel hepatocyte factor belonging to the steroid-hormone-receptor superfamily have been cloned. Studies have also been initiated to elucidate the regulation of these regulators. Homologs of two of these regulatory transcription factors (namely HNF-3 and HNF-4) have been found in Drosophila.
Hepatocyte transcription factors Two groups have isolated cDNA clones corresponding to the 'variant' HNF-1 electrophoretic-mobility shift activity seen in extracts from dedifferentiated hepatoma cell lines [3%4"]. The amino acid sequence of the variant HNF113 protein (also referred to as vHNF-1) encoded by the cDNA is 58% identical to the original HNF-I~ protein, while the homeodomain is 92% identical, the pou-specific box is 66% identical and the dimerization domain is 72% identical [3"]. While HNF-I~ mRNA is found in roughly equal amounts in the liver and kidney, and to a lesser extent in the intestine and spleen, the HNF-1 [3 mRNA is found predominantly in the kidney with lesser amounts occurring in the other tissues, including the liver (Table 1). HNF-la and HNF-I[3 form heterodimers
256
in vitro and in the kidney but no heteroclimers are detectable in liver extracts where HNF-Icx-HNF-10t homodimers are readily found. While the appearance of HNF-I[3 DNA-binding activity has been correlated with the repression of a subset of liver-specific genes, HNF-I~ is capable of stimulating transcription in at least one transfection system [4.*].
Two other groups [5",6"'] have isolated cDNAs encoding isoforms of the transcription factor C/EBP0t: C/EBP[3 (CRP2); C/EBP8 (CRP3); and C/EBPs (CRP1). As with HNF-I~ and HNF-I[3, the amino acid sequences of these proteins vary greatly except in the conserved regions, in this case the basic and leucine-zipper regions, where they are 66-78% and 51--63% identical, respectively. The mRNA's of the isoforms also have distinctive tissue distributions with all of them, in varying degrees, being found in tissues other than the liver (Table 1). The C/EBP isoforms formed heterodimers with each other both in vitro and also intracellularly in transient transfection systems. Finally, all three C/EBP isoforms stimulate transcription from a reporter construct in transient transfection experiments. The effect of the heterodimers on transcription rates has not yet been analyzed. HNF-3]3 and HNF-37 have also recently been cloned [7..]. The HNF-3 family resembles the HNF-1 and C/EBP families in that the DNA-binding domain, but no other lengthy domain, is highly conserved (90% identity). There are also two stretches of about 10 amino acids conserved at the carboxy-termini of the proteins. The three different family members, all of which act as positive activators in transient transfection assays, have distinct tissue distributions with no one factor being expressed solely in the liver (Table 1). In fact, other HNF-3 family members have been found in lymphocytes [8] and brain cells (E Lai, personal communication). Unlike the other two families, there is no evidence for dimerization of the HNF-3 protein, and light-scattering data suggests that HNF-3 binds DNA as a monomer (E tai, S Burley, personal communication). Finally, the purification and cloning of HNF-4 implicated a fourth transcription factor family in liver-specific gene expression - - the steroid-hormone-receptor superfamily [9"]. HNF-4 mRNA is present in roughly equal amounts
Q Current Biology Ltd ISSN 0959-437X
Mechanisms of liver-specific gene expression Sladek, Darnell Table 1. Tissue distribution of hepatocyte transcription factors. Transcription factor
Tissue d i s t r i b u t i o n
Endodermal origin Liver
Intestine
Lung
Stomach
Kidney
HNF-Iot
+ +
HNF'II3
+(--)
C/EBP(z
+ +
C/EBPI~
+ +
+ +
-
+
+ +
-
+
+
+
+ +
.
+
+
ND
+(--)
+
4-
4-
+ +
+ +
4-
+(--)
4-
+(--)
+(--)
.
-
ND
ND
+
ND
4-
+ +
ND
--
4-
+ +
+
+(--)
ND
+
ND
ND
+ +
+
+
+
-
-
-
ND
ND
ND
-
HNF-3~
+ +
+
+
+
--
-
-
ND
NO
ND
+
--
HNF-37
+
+
+
-
+
-
-
-
ND
ND
ND
+
+
HNF-4
+
+
+ +
--
--
--
--
--
NO
-
ND
ND
-,
are the relative
.
+
.
+
.
a m o u n t s of t h e m R N A levels based o n n o r t h e r n b l o t or T2
absent; ND, n o t d e t e r m i n e d . Examples of p r o t e i n levels
that differ from the
4-
Testes
ND
.
+
Ovary
HNF-30~
.
4-
-
Fat
+
.
+
+ .
Thymus
.
.
ND
Heart
C/EBP~:
.
+ +
Spleen
C/EBP~
Shown
+ +
Brain
.
ribonuclease analysis:
.
.
ND --
+ + , m o s t a b u n d a n t ; + , m o d e r a t e ; 4-, marginal;
RNA levels are g i v e n in parentheses.
in liver, kidney and intestine but is absent in all other tissues examined. Although HNF-4 binds to its cognate DNA sites as a dimer and although there is precedence for steroid-hormone-receptor superfamily members forming heterodimers [10], HNF-4 fails to form heterodimers with a number of other family members (F Sladek, unpublished data). Furthermore, while no isoforms or variants of HNF-4 p e r se have been found, the sequence identity between
HNF-4 and known superfamily members ranges from 30-67% amino acid identity in the zinc-finger/DNAbinding domains and up to 37% identity in the dimerization/ligand-binding domain. One of the superfamily members, ARP-1, which appears to be ubiquitous and negative acting, binds many of the same DNA sites as HNF.4 does [11]. Indeed, ARP-1 and HNF-4 homodimers have been shown to compete for binding to the same DNA site in the apoClll gene both in vitro and in in vivo transfections (M Mietus-Snyder, FM Sladek, GS Ginsburg, CF Kuo, JE Darnell, SK Karanthanasis, unpublished data). This is the second recent example of an ubiquitous transcription factor influencing liver-specific gene expression, the first example being Oct-1 (a pou-homeo protein), which aids HNF-10c activation of the hepatitis B vires promoter [12]. Finally, the cloning of HNF-4 raises the possibility of yet another dimension influencing liver-specific gene expression. HNF-4 and ARP-1 are classified as orphan receptors; as yet no ligands have been found for these 'receptors' (see discussion in [13]). ff in the future, ligands are identified for these proteins, then these ligands could influence liver-specific gene expression.
What controls the controllers? As a cascade of sequential transcriptional controls has been shown to determine early Drosophila development, it is reasonable to assume that mammalian development
might require similar cascades. Recently, the first evidence of a cascade has been found with the mRNA levels of HNF-I~, C/EBP0t, HNF-30c and HNF-4 being shown to be at least partially controlled at the level of transcription [15%16]. The subsequent cloning of the genomic sequences encoding these transcription factors has allowed the analysis of their upstream regions for critical regulatory elements that might act earlier in development. C/EBP0t was the first of these genes cloned and in addition to some novel regulatory sites, has been shown to contain a site for binding by C/EBP-family members [17.]. A second example of such autoregulation comes from HNF-3]3, which contains an HNF-3-binding site in addition to one for a novel cell-specific factor [18.]. Neither C/EBP0c nor HNF-313 contain sites for the binding of other known hepatocyte transcription factors. Analysis of the two remaining factors, HNF-I~ and HNF-4, has revealed a different pattern of regulation. The examination of a series of differentiated and dedifferentiated hepatoma cell lines has shown that HNF-1 and HNF.4 DNA-binding activi W disappears upon dedifferentiation and reappears upon differentiation, while the DNA-binding activity of C/EBP and HNF-3 remains constant [16,19"']. In vivodata linking HNF-1 and HNF-4 has been derived from studies on mice homozygous for the albino locus. The animals die shortly after birth because of the inability of their livers to regulate gluconeogenesis. The analysis of the transcription rates and mRNA levels in the livers of these mice showed not only that several liver-specific mRNAs such as albumin are greatly decreased but that HNF-1 and HNF-4 mRNAs are also missing and that their rate of transcription is severely limited. In contrast, the level of HNF-3 and C/EBP mRNAs is hardly affected [20.]. Kuo et al. [19"] have analyzed the HNF-lc~ promoter and found it to contain binding sites for both HNF-3 and HNF-4. While deletion of the HNF-3 site causes a slight drop in the expression of a receptorencoding gene that is driven by the HNF-I= promoter, deletion of the binding site for HNF-4 causes a profound
257
258 Geneexpressionand differentiation drop. Furthermore, dedifferentiatecl hepatomas that lack both HNF-I= and HNF-4 can be stimulated to transcribe HNF-I~ mRNA from the endogenous HNF-lrx gene after being stably transfected with an HNF-4 expression construct. Thus, it appears that the HNF-4 protein plays a regulatory role in the production of HNF-I~, establishing for the first time the dependence of one hepatocyte factor on another. The next obvious question to be asked is that of what regulates HNF-4 gene expression? Preliminary findings from our laboratory suggest that neither HNF-1 nor HNF-4 is involved (W Zhong, FM Sladek, JE Damell, unpublished data). While control at the level of transcription is an important method for regulating hepatocyte transcription factors, other models of control surely contribute to final protein levels (for a general review see [21]). There is evidence of HNF-1 [3o-,15.,16], C/EBP-[3 [6"'] and D/EBP (another C/EBP family member [22]) being controlled at levels other than transcription.
HNF-3 and HNF-4 as tools to study endodermal development The possible involvement of HNF-1,-3,-4 and C/EBP in early liver development has recently been summarized [2]. Here, we mention only some of the recent, provocative information concerning Drosophila homologs of HNF-3 and HNF-4 that are expressed in early embryonic stages in the precursors to gut cells. In the DNA-binding domain of HNF-30g 86/110 amino acids were found to be identical to forkhead, a product of a homeotic gene of Drosophila that is known to lie midway in the pathway of differentiation of the cells that form the anterior and posterior gut in flies [7",23]. In fly embryos, the forkheadencoded protein occurs in the hindgut, salivary placode and foregut [23], while in the adult mouse, HNF-3 family members are present in the large intestine, salivary gland and possibly the esophagus (E Lai, V Prezioso, JE Darnell, unpublished data).
The Drosophila homolog of HNF-4 is also remarkably conserved in the DNA-binding and dimerization/ligandbinding domains (91% and 68% amino acids identical, respectively). The Drosophila transcripts are deposited in eggs as maternal mRNA, they then disappear and finally reappear during organogenesis in the midgut. In Drosophila, HNF-4 is present in gut cells, fat bodies and in Malpigian tubules, which are the equivalents of the murine intestine, liver and kidney, respectively, where HNF-4 occurs in adult mice (W Zhong, JE Damell, unpublished data).
Hepatocyte-specific expression: general conclusions Taken together, the work published during the past year allows some general statements to be made regarding liver-specific gene expression. First, as with other transcription factors (e.g. Jun, Fos and ATF/CREB), each
liver-enriched factor belongs to a sub-family of related factors with similar but not necessarily identical DNAbinding specificities. Therefore, each liver-specific regulatory site is recognized by more than one protein and each of the proteins will recognize more than one sequence. Second, heterodimers form between various members of certain families and although their biological function is not yet clear, they will almost certainly be shown to play some role in liver-specific gene expression (see [14] for a general review on heterodimers). Third, it appears that factors with limited tissue distribution are not the only ones involved in tissue-specific gene expression: ubiquitous factors also appear to play some role. Fourth, while most of the transcription factors appear to be controlled at the level of transcription, examples of post-transcriptional regulation of the factors are also beginning to appear and need to be investigated further. Finally, certain aspects of gut development appear to be conserved between flies and rodents allowing the speculation that at least some of the factors involved in hepatocyte-specific expression in adult cells may also play an important role in differentiation and development.
Conclusion While our knowledge of liver-specific gene expression has increased tremendously, we have still not determined how a group of transcription factors, none of which is strictly liver-specific, manages to direct liver-specific transcription. While the restricted expression could be due to a unique combination of the current factors and their various isoforms, it seems equally likely that there are other as yet unidentified parameters involved. For example, a co-factor of HNF-10~ has recently been identified that does not bind DNA but that selectively stabilizes HNF-10t homodimers and enhances HNF-la transcriptional activity [24".]. Other largely unexplored possibilities include negative-acting factors and chromatin structure. Whatever other events are involved, the description of the present group of hepatocyte transcription factors represents an important first step to understanding liverspecific gene expression.
Acknowledgements We thank R Costa and D Mendel for providing copies of their manuscripts prior to publication, and A English for assistance in preparing the manuscript.
References and recommended reading Papers of particular interest, publishedwithin the annual period of review, have been highlighted as: ,, of special interest •o of outstanding interest 1.
DERMANE, KRAUTER K, WAIJJNG L, WEINBERGER C, RAY M, DARNELLJE JR: Transcriptional Control in the Production
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LAI E, DARNELLJE JR: Transcriptional Control in Hepatocytes: a Window on Development. Trends BiocloemSci 1991,
16:427-430.
Mechanisms of liver-specific gene expression Sladek, Darnell 3. ..
MENDELDB, HANSEN LP, GRAVES MK, CONLEY PB, CRABTREE GR: HNF-Ict and HNF-]~ (vHNF-I) Share Dimerization and Hornet Domains, but not Activation Domains, and Form Heterodimers In Vitro. Genes Dev 1991, 5:1042-1056. The cloning of HNF-11], a variant or isoform of HNF-lct, and evidence of heterodimer formation between HNF-I~] and HNF-lct opens a Pandora's box of new possibilities for liver-specific gene expression (see [4"']).
XAm'HOPOULOSKG, PREZJOSO VR, CHEN XX"S, SLADEK FM, CORTESER, DARNELLJE JR: The Different Tissue Transcription Patterns of Genes for HNF-1, C/EBP, HNF-3, and HNF4, Protein Factors that Govern Liver-specific Transcription. Proc Natl Acad Sci USA 1991, 88:3807-3811. The authors establish that the expression of C/EBP, HNF-3, HNF-4 and to a lesser degree HNF-1 is controlled at the level of transcription, suggesting the possibility of a transcriptional cascade.
4.
16.
REY-CAMPOSJ, CHOUARD T, YANrV M, CEREGHINI S: vHNF-1 is a Homeoprotein that Activates Transcription and Forms Heterodimers with HNF-1. F_MBOJ 1991, 10:1445-1457. Similar to [3*'], the main difference being that these authors detect transactivation by HNF-113 (here called vHNF-1 ) and the formation of HNF-10t-HNF-113 heterodimers in the kidney.
15.
•
..
5.
CAt Z, UMEK RM, MCKNtGHT SL: Regulated Expression of
C/EBP Isoforms During Adipose Conversion of 3T3-LI Cells. Genes Dev 1991, 5:1538-1552. The cloning of two isoforms of C/EBP (C/EBP~ and -y), which form heterodimers with each other and with C/EBPct, expands the possibilities for liver-specific gene expression (see [3"*]). Results from a time-course experiment correlating adipocyte differentiation and the appearance of different C/EBP isoforms suggest that C/EBP[3 and C/EBPy might regulate C/EBPtx. • .
6. ••
WILUAMSSC, CANTWELL CA, JOHNSON PF: A Family of C/EBP-related Proteins Capable o f Forming Covalently Linked Leucine Zipper Dimers In Vitro. Genes Dev 1991, 5:1553-1567. Similar to [5"*], with three C/EBP-related genes cloned by these authors (CRP1, CRP2 = C/EBP~; CRP3 = C/EBPy). All three of these factors activate transcription in vivo, and form heterodimers that contain disulfide bonds crosslinking paired 'zipper-helices'. 7.
LAI E, PREZIOSOVR, TAt W, CIIEN WS, DARNEH.JE JR: Hepatocyte Nuclear Factor 3ct Belongs to a Gene Family in Mammals that is Homologous to the Drosophila Homeotic Gene forkhead. Genes Dev 1991, 5:416-427. The cloning of HNF-3~I and HNF-3y establishes a new family of transcription factors that contain a previously unidentified DNA-binding domain. Homology to the Drosophila forkbead product suggests that these factors might play a role in the endodermal differentiation of the gut. • .
8.
LI C, LAI C, SIGMAN DS, GAYNOR RB: Cloning of a Cellular Factor, lnterleukin Binding Factor, that Binds to NFAT-Uke Motifs in the Human lmmunodeficiency Virus Long Terminal Repeat. Proc Natl Acad Sci USA 1991, 88:7739-7743.
SLADEKFM, ZHONG W, LM E, DARNELLJE JR: Liver-enriched Transcription Factor HNF-4 is a Novel Member of the Steroid Hormone Receptor Superfamily. Genes Dev 1990, 4:2353-2365. The fourth and final transcription-factor family that is involved in liver differentiation is identified with the cloning of the gene encoding HNF4. A member of the steroid-hormone-receptor superfamily, it raises the intriguing possibility that it, and therefore the genes it regulates, may be influenced by an as yet unidentified ligand.
17. •
CHRISTYRJ, KAESTNER Kid, GEIMAN DE, LANE MD: CC.AAT/ Enhancer Binding Protein Gene Promoter: Binding of Nuclear Factors During Differentiation of 3T3-L1 Preadipocytes. Proc Natl Acad Sci USA 1991, 88:2593-2597. The first work analyzing the promoter of a liver-enriched transcription factor. The authors find that C/EBP7 is regulated not only by a factor present in undifferentiated cells (preadipocytes) but also by a C/EBPlike factor. This resuh suggests the occurrence of autoregulation. 18. •
PANI L QIAN X, CLEVIDENCE D , COSTA RH: The Restricted Promoter Activity of the Liver Transcription Factor HNF-3~ Involves a Cell-specific Factor and Positive Autoactivation. Mol Cell Biol 1992, in press. In this work the HNF-3~ promoter is analyzed and evidence for autoregulation is described. Another novel DNA-binding activity, which is restricted to liver, is also highlighted and this appears to play a role in HNF-313 expression. 19.
Kuo CJ, CONLEY PB, CHEN L, SLADEK FM, DARNELLJE JR, CRABTREEGR: A Transcriptional Hierarchy Involved in Mammalian Cell-type Specification. Nature 1992, 355:458-460. In this work, transient and stable transfection experiments show HNF-4 to be an important regulator of HNF-1, providing the first solid evidence of a cascade of transcription factors in mammalian development. •*
20. •
TONJES RR, XANTHOPOULOS KG, DARNELL JE JR, PAUL D: Transcriptional Control in Hepatocytes of Normal and CI4 c°s Albino Deletion Mice. ~9IBOJ 1992, 11:127-132. In vivo correlation between HNF-1 and HNF-4 gene expression is provided by run-on experimental analysis of nuclei isolated from fetal albino mutants deficient in certain liver functions. 21.
FALVEYE, SCH|BLER U: How are the Regulators Regulated? FASEB J 1991, 5:309-314.
22.
MUELLERCR, MAIRE P, SCHIBLER U: DBP, a Liver-enriched Transcriptional Activator, is Expressed Late in Ontogeny and its Tissue Specificity is Determined Posttranscriptionally. Cell 1990, 61:279-291.
23.
WEIGELD, JURGENS G, KUTI'NER F, SEIFERT E, JACKEL H: The Homeotic Gene forkhead Encodes a Nuclear Protein and is Expressed in the Terminal Regions of the Drosophila Embryo. Cell 1989, 57:645-658.
9. •.
10.
FORMANBM, SAMUEt.SHH: Dimerization Among Nuclear Hormone Receptors. N Biol 1990, 2:587-594.
11.
LADIASJAA, KARATHANASlSSK: Regulation of the Apolipoprotein AI Gene by ARP-I, a Novel Member of the Steroid Receptor Superfamlly. Science 1991, 251:561-565.
12.
ZHOU D-X, YE TSB: The Ubiquitous Transcription Factor Oct-1 and the Liver-specific Factor HNF-1 are Both Required to Activate Transcription of a Hepatitis B Virus Promoter. Mol Cell Biol 1991, 11:1353-1359.
13.
SEGRmESWA: Something Old, Some Things new: the Steroid Receptor Superfamily in Drosophila. Cell 1991, 67:225-228.
14.
LAMBP, MCKNIGHTSL: Diversity and Specificity in Transcriptional Regulation: the Benefits of Heterotypic Dimerization. Trends Biochem Sci 1991, 16:417-422.
HERBST RS, NIELSCH U, SLADEK F, LAI E, BABISS LE, DARNELL JE JR: Differential Regulation of Hepatocyte-enriched Transcription Factors Explains Changes in Albumin and Transthyretin Gene Expression Among Hepatoma Cells. N Biol 1991, 3:289-296.
24. •.
MENDELDB, KHAVAm PA, CONLEY PB, GRAVES MK, HANSEN LP, ADMON A, CRABTREE GR: Characterization of a Cofactor that Regulates Dimerization of a Mammalian Homeodomaln Protein. Science 1991, 254:1762-1767. Presents the first evidence indicating that a non-DNA-binding protein (DCoH) might play a role in tissue-restricted gene expression. Unlike other such factors (also termed adapters, co-activators or bridging proteins) that have recently been described, this co-factor does not appear to interact with the basal transcription machinery. Rather, it appears to function by stabilizing HNF-lct (and HNF-113) homodimers.
FM Sladek, Environmental Toxicology Program, University of California, Riverside, California 92521, USA. JE Damell, Department of Molecular Biology, Rockefeller University, 1230 York Avenue, New York, New York 10021, USA,
259
Introduction The unique phenotype of each differentiated cell in an animal arises from the selective expression of genes in a cell- or tissue-specific fashion. Since it was discovered that the expression of the majority of liver-specific proteins is controlled at the level of transcription [1], researchers have avidly sought the regulatory transcription factors that control liver-specific gene expression. There has been considerable success in this field of study and most of the current information on liver-specific gene expression comes from the analysis of these factors. In recent years the genes encoding three positively-acting transcription factors have been cloned. These factors, which have limited tissue distribution and appear to regulate many important liver-specific genes, are all members of gene families and include: C/EBP, the original leucinezipper protein; HNF-1cx, a pou-homeo domain protein; and HNF-3cx, one of a new transcription factor family (reviewed in [2] ). During the past year, isoforms or variants from all three of these families as well as a novel hepatocyte factor belonging to the steroid-hormone-receptor superfamily have been cloned. Studies have also been initiated to elucidate the regulation of these regulators. Homologs of two of these regulatory transcription factors (namely HNF-3 and HNF-4) have been found in Drosophila.
Hepatocyte transcription factors Two groups have isolated cDNA clones corresponding to the 'variant' HNF-1 electrophoretic-mobility shift activity seen in extracts from dedifferentiated hepatoma cell lines [3%4"]. The amino acid sequence of the variant HNF113 protein (also referred to as vHNF-1) encoded by the cDNA is 58% identical to the original HNF-I~ protein, while the homeodomain is 92% identical, the pou-specific box is 66% identical and the dimerization domain is 72% identical [3"]. While HNF-I~ mRNA is found in roughly equal amounts in the liver and kidney, and to a lesser extent in the intestine and spleen, the HNF-1 [3 mRNA is found predominantly in the kidney with lesser amounts occurring in the other tissues, including the liver (Table 1). HNF-la and HNF-I[3 form heterodimers
256
in vitro and in the kidney but no heteroclimers are detectable in liver extracts where HNF-Icx-HNF-10t homodimers are readily found. While the appearance of HNF-I[3 DNA-binding activity has been correlated with the repression of a subset of liver-specific genes, HNF-I~ is capable of stimulating transcription in at least one transfection system [4.*].
Two other groups [5",6"'] have isolated cDNAs encoding isoforms of the transcription factor C/EBP0t: C/EBP[3 (CRP2); C/EBP8 (CRP3); and C/EBPs (CRP1). As with HNF-I~ and HNF-I[3, the amino acid sequences of these proteins vary greatly except in the conserved regions, in this case the basic and leucine-zipper regions, where they are 66-78% and 51--63% identical, respectively. The mRNA's of the isoforms also have distinctive tissue distributions with all of them, in varying degrees, being found in tissues other than the liver (Table 1). The C/EBP isoforms formed heterodimers with each other both in vitro and also intracellularly in transient transfection systems. Finally, all three C/EBP isoforms stimulate transcription from a reporter construct in transient transfection experiments. The effect of the heterodimers on transcription rates has not yet been analyzed. HNF-3]3 and HNF-37 have also recently been cloned [7..]. The HNF-3 family resembles the HNF-1 and C/EBP families in that the DNA-binding domain, but no other lengthy domain, is highly conserved (90% identity). There are also two stretches of about 10 amino acids conserved at the carboxy-termini of the proteins. The three different family members, all of which act as positive activators in transient transfection assays, have distinct tissue distributions with no one factor being expressed solely in the liver (Table 1). In fact, other HNF-3 family members have been found in lymphocytes [8] and brain cells (E Lai, personal communication). Unlike the other two families, there is no evidence for dimerization of the HNF-3 protein, and light-scattering data suggests that HNF-3 binds DNA as a monomer (E tai, S Burley, personal communication). Finally, the purification and cloning of HNF-4 implicated a fourth transcription factor family in liver-specific gene expression - - the steroid-hormone-receptor superfamily [9"]. HNF-4 mRNA is present in roughly equal amounts
Q Current Biology Ltd ISSN 0959-437X
Mechanisms of liver-specific gene expression Sladek, Darnell Table 1. Tissue distribution of hepatocyte transcription factors. Transcription factor
Tissue d i s t r i b u t i o n
Endodermal origin Liver
Intestine
Lung
Stomach
Kidney
HNF-Iot
+ +
HNF'II3
+(--)
C/EBP(z
+ +
C/EBPI~
+ +
+ +
-
+
+ +
-
+
+
+
+ +
.
+
+
ND
+(--)
+
4-
4-
+ +
+ +
4-
+(--)
4-
+(--)
+(--)
.
-
ND
ND
+
ND
4-
+ +
ND
--
4-
+ +
+
+(--)
ND
+
ND
ND
+ +
+
+
+
-
-
-
ND
ND
ND
-
HNF-3~
+ +
+
+
+
--
-
-
ND
NO
ND
+
--
HNF-37
+
+
+
-
+
-
-
-
ND
ND
ND
+
+
HNF-4
+
+
+ +
--
--
--
--
--
NO
-
ND
ND
-,
are the relative
.
+
.
+
.
a m o u n t s of t h e m R N A levels based o n n o r t h e r n b l o t or T2
absent; ND, n o t d e t e r m i n e d . Examples of p r o t e i n levels
that differ from the
4-
Testes
ND
.
+
Ovary
HNF-30~
.
4-
-
Fat
+
.
+
+ .
Thymus
.
.
ND
Heart
C/EBP~:
.
+ +
Spleen
C/EBP~
Shown
+ +
Brain
.
ribonuclease analysis:
.
.
ND --
+ + , m o s t a b u n d a n t ; + , m o d e r a t e ; 4-, marginal;
RNA levels are g i v e n in parentheses.
in liver, kidney and intestine but is absent in all other tissues examined. Although HNF-4 binds to its cognate DNA sites as a dimer and although there is precedence for steroid-hormone-receptor superfamily members forming heterodimers [10], HNF-4 fails to form heterodimers with a number of other family members (F Sladek, unpublished data). Furthermore, while no isoforms or variants of HNF-4 p e r se have been found, the sequence identity between
HNF-4 and known superfamily members ranges from 30-67% amino acid identity in the zinc-finger/DNAbinding domains and up to 37% identity in the dimerization/ligand-binding domain. One of the superfamily members, ARP-1, which appears to be ubiquitous and negative acting, binds many of the same DNA sites as HNF.4 does [11]. Indeed, ARP-1 and HNF-4 homodimers have been shown to compete for binding to the same DNA site in the apoClll gene both in vitro and in in vivo transfections (M Mietus-Snyder, FM Sladek, GS Ginsburg, CF Kuo, JE Darnell, SK Karanthanasis, unpublished data). This is the second recent example of an ubiquitous transcription factor influencing liver-specific gene expression, the first example being Oct-1 (a pou-homeo protein), which aids HNF-10c activation of the hepatitis B vires promoter [12]. Finally, the cloning of HNF-4 raises the possibility of yet another dimension influencing liver-specific gene expression. HNF-4 and ARP-1 are classified as orphan receptors; as yet no ligands have been found for these 'receptors' (see discussion in [13]). ff in the future, ligands are identified for these proteins, then these ligands could influence liver-specific gene expression.
What controls the controllers? As a cascade of sequential transcriptional controls has been shown to determine early Drosophila development, it is reasonable to assume that mammalian development
might require similar cascades. Recently, the first evidence of a cascade has been found with the mRNA levels of HNF-I~, C/EBP0t, HNF-30c and HNF-4 being shown to be at least partially controlled at the level of transcription [15%16]. The subsequent cloning of the genomic sequences encoding these transcription factors has allowed the analysis of their upstream regions for critical regulatory elements that might act earlier in development. C/EBP0t was the first of these genes cloned and in addition to some novel regulatory sites, has been shown to contain a site for binding by C/EBP-family members [17.]. A second example of such autoregulation comes from HNF-3]3, which contains an HNF-3-binding site in addition to one for a novel cell-specific factor [18.]. Neither C/EBP0c nor HNF-313 contain sites for the binding of other known hepatocyte transcription factors. Analysis of the two remaining factors, HNF-I~ and HNF-4, has revealed a different pattern of regulation. The examination of a series of differentiated and dedifferentiated hepatoma cell lines has shown that HNF-1 and HNF.4 DNA-binding activi W disappears upon dedifferentiation and reappears upon differentiation, while the DNA-binding activity of C/EBP and HNF-3 remains constant [16,19"']. In vivodata linking HNF-1 and HNF-4 has been derived from studies on mice homozygous for the albino locus. The animals die shortly after birth because of the inability of their livers to regulate gluconeogenesis. The analysis of the transcription rates and mRNA levels in the livers of these mice showed not only that several liver-specific mRNAs such as albumin are greatly decreased but that HNF-1 and HNF-4 mRNAs are also missing and that their rate of transcription is severely limited. In contrast, the level of HNF-3 and C/EBP mRNAs is hardly affected [20.]. Kuo et al. [19"] have analyzed the HNF-lc~ promoter and found it to contain binding sites for both HNF-3 and HNF-4. While deletion of the HNF-3 site causes a slight drop in the expression of a receptorencoding gene that is driven by the HNF-I= promoter, deletion of the binding site for HNF-4 causes a profound
257
258 Geneexpressionand differentiation drop. Furthermore, dedifferentiatecl hepatomas that lack both HNF-I= and HNF-4 can be stimulated to transcribe HNF-I~ mRNA from the endogenous HNF-lrx gene after being stably transfected with an HNF-4 expression construct. Thus, it appears that the HNF-4 protein plays a regulatory role in the production of HNF-I~, establishing for the first time the dependence of one hepatocyte factor on another. The next obvious question to be asked is that of what regulates HNF-4 gene expression? Preliminary findings from our laboratory suggest that neither HNF-1 nor HNF-4 is involved (W Zhong, FM Sladek, JE Damell, unpublished data). While control at the level of transcription is an important method for regulating hepatocyte transcription factors, other models of control surely contribute to final protein levels (for a general review see [21]). There is evidence of HNF-1 [3o-,15.,16], C/EBP-[3 [6"'] and D/EBP (another C/EBP family member [22]) being controlled at levels other than transcription.
HNF-3 and HNF-4 as tools to study endodermal development The possible involvement of HNF-1,-3,-4 and C/EBP in early liver development has recently been summarized [2]. Here, we mention only some of the recent, provocative information concerning Drosophila homologs of HNF-3 and HNF-4 that are expressed in early embryonic stages in the precursors to gut cells. In the DNA-binding domain of HNF-30g 86/110 amino acids were found to be identical to forkhead, a product of a homeotic gene of Drosophila that is known to lie midway in the pathway of differentiation of the cells that form the anterior and posterior gut in flies [7",23]. In fly embryos, the forkheadencoded protein occurs in the hindgut, salivary placode and foregut [23], while in the adult mouse, HNF-3 family members are present in the large intestine, salivary gland and possibly the esophagus (E Lai, V Prezioso, JE Darnell, unpublished data).
The Drosophila homolog of HNF-4 is also remarkably conserved in the DNA-binding and dimerization/ligandbinding domains (91% and 68% amino acids identical, respectively). The Drosophila transcripts are deposited in eggs as maternal mRNA, they then disappear and finally reappear during organogenesis in the midgut. In Drosophila, HNF-4 is present in gut cells, fat bodies and in Malpigian tubules, which are the equivalents of the murine intestine, liver and kidney, respectively, where HNF-4 occurs in adult mice (W Zhong, JE Damell, unpublished data).
Hepatocyte-specific expression: general conclusions Taken together, the work published during the past year allows some general statements to be made regarding liver-specific gene expression. First, as with other transcription factors (e.g. Jun, Fos and ATF/CREB), each
liver-enriched factor belongs to a sub-family of related factors with similar but not necessarily identical DNAbinding specificities. Therefore, each liver-specific regulatory site is recognized by more than one protein and each of the proteins will recognize more than one sequence. Second, heterodimers form between various members of certain families and although their biological function is not yet clear, they will almost certainly be shown to play some role in liver-specific gene expression (see [14] for a general review on heterodimers). Third, it appears that factors with limited tissue distribution are not the only ones involved in tissue-specific gene expression: ubiquitous factors also appear to play some role. Fourth, while most of the transcription factors appear to be controlled at the level of transcription, examples of post-transcriptional regulation of the factors are also beginning to appear and need to be investigated further. Finally, certain aspects of gut development appear to be conserved between flies and rodents allowing the speculation that at least some of the factors involved in hepatocyte-specific expression in adult cells may also play an important role in differentiation and development.
Conclusion While our knowledge of liver-specific gene expression has increased tremendously, we have still not determined how a group of transcription factors, none of which is strictly liver-specific, manages to direct liver-specific transcription. While the restricted expression could be due to a unique combination of the current factors and their various isoforms, it seems equally likely that there are other as yet unidentified parameters involved. For example, a co-factor of HNF-10~ has recently been identified that does not bind DNA but that selectively stabilizes HNF-10t homodimers and enhances HNF-la transcriptional activity [24".]. Other largely unexplored possibilities include negative-acting factors and chromatin structure. Whatever other events are involved, the description of the present group of hepatocyte transcription factors represents an important first step to understanding liverspecific gene expression.
Acknowledgements We thank R Costa and D Mendel for providing copies of their manuscripts prior to publication, and A English for assistance in preparing the manuscript.
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FM Sladek, Environmental Toxicology Program, University of California, Riverside, California 92521, USA. JE Damell, Department of Molecular Biology, Rockefeller University, 1230 York Avenue, New York, New York 10021, USA,
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