Vol. 6, 1 549- 1558, December 1995 cell Growth & Differentiation 1549

3 The abbreviations used are: AFP, a-fetoprotein; HNF, hepatocyte nuclearfactor; C/EBP, CCAAT/enhancer binding protein.

Sequence Requirements for a-Fetoprotein Gene Expression. S #{149}

during Liver Regeneration

Yin tin, David K. un, Jean Vacher, andMiriam H. Feuerman2

Department of Biochemistry, State University of New York, Health

Science Center at Brooklyn, Brooklyn, New York 1 1 203 [Y. L., D. K. J.,M. H. Fl, and Institut de recherches cliniques de Montr#{233}al, 1 10 avenuedes Pins Ouest, Montreal, Quebec H2W1 R7, Canada lJ. V.1

Abstract

The a-fetoprotein (AFP) gene is expressed in fetalliver and in adult liver undergoing regeneration ortumorigenesis. It has been shown previously that threedistal enhancers, a proximal promoter, and a dominantnegative postnatal repressor element are required for thetissue-specific and developmental regulation of AFP geneexpression. Using transgenic mice, we have determinedthe sequence requirements for AFP gene inductionduring liver regeneration. Two DNA sequences werefound in all transgenes appropriately regulated inresponse to liver regeneration: a distal sequencebetween 1 01 0 and 838 bp upstream of the structuralgene and a proximal sequence within 1 1 8 bp of thetranscriptional initiation site. In situ hybridizationanalysis showed that transgene expression during liverregeneration was first found in all hepatocytes and thenlocalized to perinecrotic hepatocytes surrounding thecentral vein. This pattern of expression is reminiscent ofthat observed after birth for the transgenes, suggestingthat repression of AFP gene expression after birth andliver injury may be regulated by similar mechanisms.

Introduction

The liver is one ofthe few organs capable of regeneration inthe adult. After chemical damage on 70% surgical resection,the liven will undergo regeneration, ending when it hasreached its original mass (1-4). Compensatory liver growthis associated with expression of so-called oncofetal antigens(5), proto-oncogenes, as well as the anti-oncogene p53(6-9).

Biological function varies between hepatocytes, depend-ing on the location within the liver lobule, the basic struc-tunal unit of the liver. Zone i hepatocytes, proximal to theportal tniads, have a greater proliferative potential (1 0, 1 i).Zone 3 hepatocytes, adjacent to the central vein, are in-volved in detoxification of chemicals in the blood stream(10, 12). Carbon tetrachlonide (CCI4) poisoning leads todeath of zone 3 hepatocytes accompanied by an inflam-matory response (1 3, i 4).

Received 8/22/95; revised 10/10/95; accepted 10/11/95.I This work was supported by USPHS Grant CA55739 from the National

Cancer Institute.

2 To whom requests for reprints should be addressed, at Department of

Biochemistry, Box 8, SUNY Health Science Center at Brooklyn, 450 Clark-son Avenue, Brooklyn, NY 11203. Phone:(718) 270-1258; Fax:(718) 270-

331 6; E-mail: mfeuerman@netmail.hscbklyn.edu.

AFP3 is a classic oncofetal antigen. The AFP gene isexpressed in the fetal yolk sac, liver, and gut but not innormal adult tissues (5, i 5). Immediately after birth, the AFPgene is expressed in all hepatocytes. As levels ofAFP mRNAdecrease, transcripts are host first in peniportal hepatocytes(zone i ). After 1 week, AFP mRNA is localized to zone 3hepatocytes (proximal to the central vein) and is host entirelywithin 1 month after birth (1 6, i 7). High level AFP geneexpression is induced in adult liver upon regeneration ortumonigenesis (5, 14, 18, i9). Localization of AFP geneexpression in adult regenerating liver is dependent on theprotocol used to damage the liver (1 3, 1 4, 18, 20). Two to3 days after treatment with a single dose of CCI4, AFP geneexpression was detected in peninecrotic hepatocytes(13, 18).

In recent years, a great deal has been learned about themolecular control mechanisms regulating AFPgene expres-sion. Transfection assays using primary hepatocytes andhepatoma cell lines have shown that three distal enhancersand a complex promoter regulate AFP gene expression invitro (21-23). Experiments in transgenic mice have shownthat developmental regulation of AFP gene expression wasdependent on the dominant effects of the proximal 1 kb ofAFP 5’-flanking sequence (24, 25). Transgene expressionwhen portions of this sequence have been deleted suggeststhat multiple elements within i kb ofthe transcriptional startsite are required for full postnatal repression of AFP geneexpression. Deletion of the sequence from -838 to -604in a transgene carrying the remaining 7.6 kb of AFP 5’-flanking sequence led to persistent AFP transgene expres-sion in the adult liver, specifically in zone 3 hepatocytes,residing close to the central vein. Extending the deletion to-250 resulted in even higher transgene expression sun-rounding the central vein, leading to the identification of adominant negative element required for postnatal repres-sion of AFP gene expression (26, 27).

High levels of AFPgene expression in the adult liver is anindication of growth. Two models can be proposed forre-activation of AFP gene expression in the adult: (a) ex-pression is the reversal of the normal postnatal repression;and (b) expression is the result of a novel induction process.As a first step to understanding the molecular mechanismsinvolved, we have identified cis-acting DNA sequencesrequired for AFPgene expression during liver regeneration.Since there are no suitable in vitro models of liver regen-eration, expression of various transgenes was studied inmice. It has been shown previously that expression of trans-genes with 7.6 kb ofAFP 5’-flanking sequence responded toliver regeneration (28). To more closely define the requiredsequences, a series of transgenes using different portions ofthe AFP 5’-flanking sequence to drive expression of areporter gene were tested for expression during liver

A A

AIbEAFP

o�JAFPEA1b

AFP -0

Fig. 1. Transgenes used in the study. A, albumin/AFP gene locus. AFP

sequences are shaded. Circles, enhancers; ovals, promoters; rectangles, genebodies. The three AFP enhancers are centered 2.25, 5.0, and 6.5 kb upstream

of the transcriptional start site. B, transgenes used. The AIb�AFP transgeneincludes the albumin enhancer between -9 and -12 kb upstream of the

albumin coding sequence and the proximal 1 kb of AFP 5-flanking se-quence. The AFP�AIh transgene contains the AFP enhancers, the DNAsequence between 1 and 7.6 kb upstream of the AFPgene, and an albumin

Promoter sequence within 600 bp upstream of the albumin coding sequence(25). The �1 , � and �7 transgenes each include 7.6 kb of AFP 5-flanking

sequence with portions of proximal the 1 .0 kb deleted. A nested set ofdeletions was used for the construction of the ,�1 ,.�3, and �7 transgenes. The

5-boundary was -838 hp. with deletions ending at -604, -250, and - 1 18hp for M, .�3, and �7, respectively (27).

B

Fig. 2. Expression of the AIb, AFP and AFP,AIb transgenes during liver

regeneration. Transgenic mice were injected with either a 10% solution ofCd4 in mineral oil (CCI,) or mineral oil alone (Mm Oil) and sacrificed 72 hlater. AFP, endogenous AFP gene; AFPMG, AFP minigene; AIb, endogenousalbumin gene; AIb MG, albumin minigene. A, Northern blot of poIylA)� liverRNA from AIb1AFP transgenic mice 72 h after injection with CCI4 or mineraloil. Transgene and endogenous RNA was detected by hybridization to theAFP exon 1 probe (toppanel). A duplicate blotwas hybridized to a ribosomal

protein probe, rpL32, as an indication of amount and quality of RNA loadedon the gel (bottom panel). B. RNase protection assay of total liver RNA from

AFP[AIb transgenic mice 72 h after injection with Cd4 or mineral oil.Transgenes and endogenous albumin RNA were detected by RNase protec-

tion assay using a probe consisting of exons 1-4 of the albumin gene (toppanel). AFP mRNA, an indication of liver regeneration, was also detected byRNase protection assay using an AFP probe spanning exons 3 and 4 (bottom

panel).

1550 AFP Gene Expression during Liver Regeneration

. S S : AFP AIbEAFP

Mm Oil CCL

B 51 -838604

-838 -250

-838 -118

regeneration. We found that appropriately regulated trans-genes had approximately 300 bp AFP 5’-flanking sequencein common: a distal sequence located between 1010 and838 bp upstream of the AFP coding sequence; and 1 1 8 bpadjacent to the transcriptional start site. AFP tnansgene ex-pnession was found in surviving hepatocytes in all regions ofthe liver lobule early after CCI4 injection, then becamelocalized to peninecrotic hepatocytes. This changing zonalexpression after CCI4 treatment (19) is similar to what isobserved for both transgenes (26) and the endogenous geneafter birth (16, 17).

Results

Identification of the Sequences Required for Induction ofAFP Gene Expression during Liver Regeneration. To deter-mine if AFP gene expression during liver regeneration ne-quired the three AFP enhancers or the proximal 1 kb of5-flanking sequence, two lines of tnansgenic mice werescreened for tnansgene expression during liven regeneration.As shown in Fig. 1, AIb(AFP has the albumin enhancer(lying between 9 and 1 2 kb upstream of the albumin tran-scriptional start site) and the 1 kb of 5-flanking sequenceimmediately adjacent to the AFP transcriptional start sitedriving expression of the AFP minigene. The AFP minigeneencoded exons i , 2, 3, 1 4, and i 5, producing an mRNA of500 nucleotides. The reciprocal tnansgene, the AFPEAIbtnansgene, consisted of the three AFP enhancers (locatedbetween 7.6 and 1 .0 kb upstream of the AFP transcriptionalstart site) and the albumin promoter (to -600 bp), drivingexpression of an albumin minigene. The albumin minigeneencoded exons 1 , 2, and 1 5, producing an mRNA of 400nucleotides (25). Liven regeneration was induced in trans-genic mice, between 6 and 1 2 weeks of age by i.p. injectionof a 1 Dub solution of CCI4 in mineral oil. Mice were injected

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with mineral oil alone as control. Tnansgene expression wasassayed 72 h after CCI4 injection, when endogenous AFPgene expression is at its highest level (29). Northern blotanalysis in Fig. 2A shows that neither endogenous AFP nortnansgene mRNA (AFP MG) were detected in five AIbEAFPtnansgenic mice injected with mineral oil alone. However,both mRNAs were detected in five transgenic mice injectedwith CCI4. Alb�AFP transgene expression closely parallelsendogenous AFP gene expression. When the endogenousgene was expressed at higher levels, the minigene was alsoexpressed at higher levels. The variability in minigene and

Cell Growth & Differentiation 1551

AFP message was most likely due to differences in responseto Cd4 injection as a duplicate blot hybridized to the rpL32nibosomal protein probe showed comparable RNA loadingof each sample.

This was in contrast to the results for the AFPEAIb trans-gene in Fig. 28. RNase protection assay was used to deter-mine tnansgene expression 72 h after CCI4 injection. Hy-bnidization of endogenous albumin mRNA to a probeconsisting of the first four exons protected a 275-nucheotidefragment from digestion with RNase One. Albumin mini-gene mRNA protected a 1 05-nucleotide fragment fromRNase digestion. There was no difference in transgenemRNA levels between six mice injected with mineral oilalone and seven mice injected with CCI4. The Cd4 injec-tion was sufficient to induce expression of the endogenousAFP gene, as shown by RNase protection assay using anAFP gene probe (Fig. 2B, bottom). Hybridization of endo-genous AFP mRNA with probe protected a i 90-nucleotidefragment from digestion with RNase. Comparison of theexpression of these two transgenes, AFPEAIb and AIbEAFP,suggests that the sequence within 1 kb upstream of the AFP

transcriptional start site was necessary for appropriate in-duction of expression during liver regeneration induced by

CCI4 injection.To more closely define the AFP 5’-flanking sequences

required for tnansgene expression during liver regeneration,the expression of three additional transgenes were ana-lyzed. Each consists of portions of i .0 kb of the proximal5’-flanking sequence combined with all three AFP enhanc-ens (- 1 .0 to - 7.6 kb upstream of the transcriptional startsite) linked to the AFP minigene. Ideally, these sequenceswould be tested in the absence of all other transcriptional

control elements; however, the proximal i kb of AFP 5’-flanking sequence alone was not sufficient for transgeneexpression (28). As before, tnansgenic mice were injectedwith either the CCI4 solution or mineral oil as control. Micewere sacrificed 72 h later, and tnansgene expression wasdetermined by RNase protection assay. Hybridization of theAFP probe with AFP minigene mRNA yielded a 1 21 -nude-otide fragment upon digestion with RNase. As shown in Fig.3A, the �i tnansgene, with the deletion from -604 to-838, was highly expressed in fetal liver and significantlyrepressed in three control transgenic adult mice injectedwith mineral oil alone. However, after injection with CCI4,minigene expression was increased 30-fold.

The same analysis was performed on �3 transgenic mice.Like the M transgene, the z�3 transgene contained all threeenhancers. However, the sequences between 838 and 250bp upstream of the AFP transcriptional start site were de-leted, reducing the AFP sequences that may respond to liver

regeneration. As shown in Fig. 3B, this transgene was highlyexpressed in the four adult mice injected with mineral oil,although no endogenous AFP gene expression was de-tected. Twenty-four h after injection with CCI4, no signifi-cant increase in either endogenous or transgene mRNA wasdetected. By 48 h after injection, endogenous AFP mRNAwas detected. Seventy-two h after injection with CCI4,when endogenous AFP mRNA is at its highest level, mini-gene expression was increased over normal adult levels(Fig. 38, midd!e panel). Endogenous AFP mRNA was de-tected in �3 tnansgenic mice injected with CCI4, indicatingthat sufficient liven regeneration had been induced for ex-pression of AFP (Fig. 313, top pane!). This analysis showsthat �3 transgene expression is increased in response toliver regeneration.

The last transgene tested for induction of gene expressionduring liver regeneration was �7, where the sequence be-tween -838 and -ii8 bp has been deleted from theproximal i kb of 5’-fhanking sequence. As shown in Fig. 3C,

minigene mRNA was detected in all mice tested. No endo-genous AFP mRNA was detected in mice injected withmineral oil or mice sacrificed 1 6 h after CCI4 injection. By24 h after treatment, there was a significant increase in bothendogenous AFP and tnansgene mRNA. An additional in-

crease in both minigene and endogenous gene expressionwas found 72 h after injection. This clearly shows that z�7transgene expression was regulated in response to liverregeneration.

Localization of Gene Expression. Although M , z�3, andL�7 transgene expression was detected in normal adult liver,not all postnatal repression of AFP gene expression hadbeen lost. In the fetus, all transgenes are expressed through-out the liver lobule. However, in the adult, expression wasrestricted to zone 3 hepatocytes, proximal to the centralvein of the liver lobule (26). To determine if this limitationin transgene expression affected expression during liverregeneration, in situ hybridization experiments were per-

formed. The first exon of the AFP gene, which recognizesequal lengths of both endogenous gene and minigenetranscripts, was used as probe. Thus, hybridization is acombination of both endogenous and transgene expression.Since M , z�3, and i�7 transgenes were expressed at signif-icantly higher levels than the endogenous gene, it wasunlikely that the endogenous gene contributes significantlyto the detected hybridization. However, based on theNorthern blot analysis in Fig. 2A, the endogenous gene andminigene transcripts were expected to contribute equally tothe detected signal in the AIbEAFP tnansgenic mice.

Following the same protocol, AIbEAFP, M , z�3, and �7transgenic mice were injected with either CCI4 or mineraloil as control and sacrificed 72 h hater; then liver tissue wasprepared for in situ hybridization. In AIbEAFP transgenicmice, there was no detectable hybridization in control miceas shown in Fig. 4A (light field) and Fig. 4B (dark field).However, 72 h after injection with CCI4, hybridization wasobserved in the peninecrotic hepatocytes surrounding thecentral vein (shown in Fig. 4, C and 0). No hybridizationwas detected around the portal tniads. The backgroundhybridization throughout the section was also observedwhen a sense RNA was used as probe (data not shown),suggesting that it does not represent gene expression. Asshown at higher magnification (Fig. 4, E and F) AIbEAFPtransgenic mice developed the typical histopathology ofCCI4 intoxication with inflammation (1 3, 14). Only a frac-tion of peninecrotic hepatocytes expressed these transcripts,similar to what was observed for the endogenous gene (datanot shown; Refs. 13, 14, and 18).

M transgene expression in control mice was observed inhepatocytes within one to two cell layers of the central vein,as shown in Fig. 5, A and 8. However, after CCI4 injection,peninecrotic hepatocytes expressed AFP minigene mRNA(Fig. 5, C and D). Therefore, liver regeneration led to achange in the pattern of transgene expression.

/n situ hybridization showed that �3 transgenic miceinjected with mineral oil alone expressed high levels of AFPminigene mRNA localized to four to six hepatocyte layerssurrounding the central vein (Fig. 6, A and B). Seventy-twoh after injection with Cd4, hybridization was found inpeninecrotic hepatocytes (Fig. 6, C and D). Higher magni-

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1552 AFP Gene Expression during Liver Regeneration

AM

p M F F MinOiI Cd4

Fig. .3. Expression of the �1 ,

and �7 transgenes after CCI4 in-

jection. RNA was prepared andanalyzed for the endogenous AFP

gene and �1 transgene expressionby RNase protection assay. A, �1,Lanes: P, undigested probe; M,

marker-pBR322 digested with

Mspl; F, fetal; Cd4, mice injectedwith Cd4 and sacrificed 72 h at-

er; Mm Oil, mice injected withmineral oil and sacrificed 72 h

later. Top panel, AFP probe; bot-torn panel, albumin probe. B, �3.Lanes: P. undigested probe; M,

marker; 24, adult mouse sacri-ficed 24 h after CCI., injection; 48,

adult mouse sacrificed 48 h after

CCI., injection; CCI.,, mice 72 hafter injection; Mm Oil. mice 72 h

after injection with mineral oil.Top panel, AFP probe using 20 �.tg

of RNA for detection of endoge-nous AFP gene expression. Middle

panel, AFP probe using 1 �g ofRNA insuring probe excess to de-tect the changes in �3 transgenemRNA abundance. Bottom panel,

albumin probe. C, � Lanes: M,

marker; P, undigested probe; 0,mineral oil injected and sacrificed72 h later. Mice injected with

Cd4 and sacrificed 16, 24, 40,

48, and 72 h later as indicated.

Top panel, AFP probe; bottompanel, albumin probe. AFP, endo-genous AFP mRNA; AFPMG. AFPminigene; AIb, albumin mRNA.

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fication (Fig. 6, E and F) showed that hybridization to theAFP gene probe was limited to peninecrotic hepatocytes.

RNase protection assays cleanly showed an increase in �7minigene mRNA after CCI4 injection. As shown in Fig. 7, in

situ hybridization experiments detected �7 transgene mRNAtightly localized around the central vein in the liven lobules ofmineral oil-injected mice. Expression was found in virtuallyallhepatocytes in these cell layers (Fig. 7, A and B). Seventy-twoh after injection with Cd4, transgene mRNA was only foundin peninecrotic hepatocytes (Fig. 7, C and D).

Pattern of Transgene Expression after CCI4 Intoxication.It has been shown that AFPgene expression after damageby Cd4 can be found in both peripontal and peninecnoticzones 48 h after treatment. Expression then becomesrestricted to peninecrotic hepatocytes by 72 h after injec-tion (19). To determine if transgene expression was sub-ject to the same regulation as the endogenous gene, in

situ hybridization experiments were performed on liven

sections of mice sacrificed at various times after CCI4injection. As shown in Fig. 8, 24 h after injection withCCI4, z�3 tnansgene expression was found in zone 1 andzone 2 hepatocytes. It is unclear if the absence of ex-pression in zone 3 hepatocytes was due to cell deathalone on to regulation of gene expression. After 48 h (Fig.8, C and D), there was significant disruption of tissuemorphology, and tnansgene expression was distributedthroughout the liven lobule. Expression was higheraround the portal tniads than the central veins. As thetissue regained its normal appearance oven most of theliven lobules, z�3 transcripts were localized to a fewpeninecrotic hepatocyte layers (Fig. 8, Eand F). z�3 trans-gene expression not only increased quantitatively in re-sponse to liven regeneration, but the location of the hepa-tocytes expressing the tnansgene also changed. The samepattern of expression was observed for the �i and z�7tnansgenes (data not shown).

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Cell Growth & Ditferentiation 1553

Fig. 4. In situ hybridization ofliver tissues from Alb,AFP trans-

genk mice. Liver section from amouse injected with mineral oil

after in situ hybridization withthe AFP exon 1 probe, light field

(A( and clark field (B). Liver 5cc-

tion from a mouse injected with

CCI4 in mineral oil, light field (C

and Fl and clark field ( 0 and F).Hybridization appears white in

dark field. A-D, bar, 100 �.tm. Eand F. har, 50 f.LM. pt, portal tn-

ads; cv, central veins..pt�

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Fig. 5. In situ hyhridization of liver tissues from .�1 transgenic mice. In situhybridization of a liver section from a mouse injected with mineral oil, light

field (A( and dark field (B(. Liver section from a mouse injected with CCL, inmineral oil, light field (C( and dark field (0). Bar, 100 �m. pt. portal tniads;

cv, central veins.

Discussion

Since many ofthe factors regulating AFPgene expression inmice cannot be duplicated in vitro, transgenic mice havebeen generated to study AFPgene expression during devel-opment and pathology (25, 27, 28). We have used some ofthese lines of transgenic mice to identify AFP 5’-flankingsequences required for gene expression during liven regen-eration. Hammer et al. (28) have already shown that atransgene using the entire 7.6 of AFP 5’-flanking sequencewas appropriately regulated in response to liven negenena-tion. We have more precisely identified the required se-quence by using transgenes with smaller regions of AFP

‘

sequence. Analysis of the expression of two lines of trans-genic mice, AFPEAIb and AIb1AFP, showed that the imme-

diate 1 kb of AFP 5’-flanking sequence was necessary forappropriate AFP expression during liver regeneration, evenwhen combined with a hetenologous enhancer. In agree-ment with previous reports, when at least 234 hp of this

sequence, between -838 and -604 (the M, �3, and �7tnansgenes), were removed, tnansgene expression wasreadily detected in normal adult liver (26, 27). Cd,4 admin-istration led to an increase in tnansgene expression. In situ

hybridization experiments showed that expression of theAIbEAFP, �i , z�3, and z�7 transgenes was limited to penine-crotic hepatocytes 72 h after CCI4 administration, as ob-served for the endogenous gene. Finally, it was shown thatthe pattern of tnansgene expression after Cd4 injectionfollowed the same course as the endogenous gene. At earlytimes, it was found in zone 1 and zone 2 hepatocytes andlater localized to peninecrotic hepatocytes. The change inzonal tnansgene expression was a second indication of reg-ulation of transgene expression in response to liver regen-

enation. Therefore, all transgenes with expression respon-sive to liven regeneration, AIbEAFP, �i , �3, and �7

contained approximately 300 bp of AFP 5-flanking Se-quence in common: 172 bp between -1010 and -838;and 1 1 8 bp adjacent to the transcriptional start site. Thissuggested that either one on both of these sequences wererequired for AFPgene expression during liven regeneration.

These transgenic mice were studied previously to identifyelements mediating postnatal repression of AFP gene ex-pression. Camper and Tilghman (25) showed that 1 kb ofAFP 5’-flanking sequence was required for appropriate de-velopmental regulation of the AFP gene. Albumin gene

components were used to replace AFP elements in thatstudy because albumin and AFP are evolutionanily related,are expressed in the same tissues, and are induced at thesame time in development (1 5, 25). The same substitution isuseful in this work because albumin gene expression eitherremains constant (29, 30) on is repressed (31, 32) during

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1554 AFP Gene Expression (luring Liver Regeneration

Fig. 6. In situ hybridization of liver

tissues from .�3 transgenic mice.Section from a liver of a mouse in-jectecl with mineral oil after hybrid-ization with the AFP exon 1 probe,light field (A) and dark field (B).Liver section from a mouse injectedwith CCI4 in mineral oil, light field

(Cand E( and dank field (Dand F).A-V. bar, 100 pm; Eand F, bar, 50I.L5l. pt, portal tniads; cv, central

veins.

liver regeneration. It is also unlikely that the small amountof AFP or albumin mRNA sequence in these transgenescontributed to control of gene expression because it hasbeen shown that AFP 5-flanking sequence conferredappropriate gene regulation on unrelated reporter genes(33, 34)

Two models can be proposed for the induction of AFPgene expression during liver regeneration: (a) AFP geneexpression during liver regeneration is the reversal of post-natal repression of gene expression; and (b) induction ofAFP gene expression is an independent event, requiringdifferent regulatory activities. The LXi, �3, and �X7 trans-genes were persistently expressed only in the zone 3 hepa-tocytes of the adult liver, where repression of AFP geneexpression occurs last during normal development. Yet,transgene expression in all three cases responded to liverregeneration, suggesting that a reversal of the mechanism ofzone 3 repression does not entirely account for induction ofgene expression during liver regeneration. The transgeneswere repressed in the adult zone 1 and 2 hepatocytes,suggesting that reversal of postnatal repression in periportaland mid-lobular hepatocytes can contribute to AFP geneexpression during liver regeneration. Multiple mechanismscould be required for the final level of AFPgene expressionbecause it has been shown that hepatocytes in differentregions of the liver lobule express different genes and ne-spond differently to hepatotoxins (10, 12, 13).

There were two regions of the AFP 5-flanking sequencefound in all transgenes regulated in response to liver regen-eration: a distal 172 bp located between 1010 and 838 bpupstream of the mRNA cap site; and a proximal 1 1 8-bpsequence adjacent to the mRNA cap site. Within 200 bp ofthe transcriptional start site lay recognition sites for a num-ben of transcription factors that have been shown to play akey role in liver-specific gene expression. It has been shownpreviously that HNF-1, recognizing two sites in the AFPpromoter (at -60 and - 1 20 bp), increases gene transcnip-tion in hepatoma cell lines (35-38). At each site, there are

also potential binding sites for the C/EBP transcription factorfamily (38, 39). In addition, at the distal HNF-i site (at - 1 20bp), there is an overlapping NF-i binding site (36, 38-40).It has been suggested that postnatal repression and subse-quent activation of AFPgene expression during liver negen-enation may be modulated through competition for tran-scniption factor binding at these sites (40, 41 ). It has alreadybeen shown that the concentrations of some of these factorschange during liver regeneration. The concentration ofC/EBPa, high in quiescent liven, decreases in relation to theconcentrations of C/EBPI3 and � after liven injury (42-45).Similarly, HNF-i binding activity to the albumin promoteris reduced during liven regeneration (31, 46).

Additional protein factor binding sites have been ne-ported within 200 bp of the AFP transcriptional start site.Wen et a!. (47) and Wen and Locker (48) have suggestedthat a protein recognizing the sequence between - 1 78 and- 1 55 bp may “couple” the activity of the AFP promoter tothe distal enhancers. Zhang et a!. (49) have also reported aDNA-binding protein recognizing the same region, called

NPIV. Because the �7 tnansgene with these regions deletedwas regulated in response to liven regeneration, it is unlikelythat either of these activities is absolutely required for ex-pression. Other factors that regulate AFPgene expression intissue culture cells include FTF in HepG2 cells (44) andsteroid and netinoid receptors in F9 embryonal carcinomacells (50). The relationship among these factors and theirrole in gene regulation during liven regeneration is stillunclean.

In addition to the region within 1 1 8 bp of the transcnip-tional initiation site, the region between 1010 and 838 bpupstream is also conserved in all of the tnansgenes thatrespond to liver regeneration. This sequence is conserved inthe rat (51). Analysis of the DNA sequence identified ho-mology to the sites recognized by C/EBPf3 in hepatitis Bvirus (52, 53) and HiNF-D in the histone H3 promoter (54).Experiments are under way to identify DNA-binding activ-ities, regulated with respect to liven regeneration, that nec-

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Cell Growth & Differentiation 1555

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Fig. 7. In situ hybridization of liver tissues from �7 transgenic mice. Liversection from a mouse injected with mineral oil, light field (A) and dark field(B). Liver section from a mouse injected with Cd4 in mineral oil, light field(C) and clark field (0). Bar, 100 �.cm. pt. portal triads; cv, central veins.

ognize this region. Additional lines of transgenic mice willbe required to determine the biological significance of thisregion.

M , �3, and �7 transgene expression in mice injectedwith Cd4 was first found in zones 1 and 2 hepatocytes, thenthroughout the liven lobule, and finally localized to zone 3hepatocytes. These lines of mice were also used to study thedevelopmental regulation of the AFP gene. Emerson et a!.(26) showed that immediately after birth �3 tnansgene ex-pression was found in all regions ofthe liven lobule. Expres-sion was gradually lost, first from peniportal hepatocytes.

fltnansgene expression assumed the normal adult pattern 6to 8 weeks after birth (26). Thus, AFPgene expression afterbirth and after CCI4-induced injury follow the same patternof localization within the liven lobule and require the samecis-acting sequences. The similarities in these two processesmay indicate similarities in the establishment (or ne-estab-lishment) of the gradient of gene expression in the livenlobule (1 3, 14, 1 6, 1 7, 19, 55, 56). It has been suggested byGleiberman et a!. (19) that the common factor is the for-mation of extracellular contacts. Hepatocyte gene expres-sion is exquisitely sensitive to cell-cell and cell-matrix in-teractions (57-61 ). AFP gene expression may also beaffected, because Gleibenman et a!. (19) showed that AFPwas found only in hepatocytes that have lost a specific cellsurface antigen found on normal adult hepatocytes (19). Apossible mediator of this regulation may be the C/EBP fam-ily of transcription factors, because their relative levels aredependent on cellular morphology in culture (60). Thenumber and location of potential C/EBP binding sites on theAFP gene may reflect the possibility that modulation of theC/EBP isoforms may regulate AFP gene expression.

In conclusion, we have used tnansgenic mice to identifyDNA sequences required for AFP gene expression duringliver regeneration in vivo. Tnansgenes regulated in responseto CCI4 injection have approximately 300 bp of AFP 5’-flanking sequence in common: a distal 172 bp locatedbetween 1010 and 838 bp upstream of the transcriptionalstart site; and a proximal 1 1 8 bp immediately adjacent tothe mRNA cap site. Within these sequences lie binding sites

for transcription factors known to be regulated in responseto liver regeneration, and future work will lead to the den-tification of the crucial factors involved. Furthermore, thesimilarity in the progression of AFP transgene expressionafter CCI4-induced injury to the progression of expressionduring postnatal development may indicate a common un-derlying mechanism for these two processes.

Materials and Methods

Animals. C3H/He inbred mice were obtained from TheJackson Laboratory. Tnansgenic mice were the gift of Dr.Shirley M. Tilghman (Princeton University, Princeton, NJ).Generation of AFP1AIb (N33i) and A1b1AFP (962L) wasdescribed previously by Camper and Tilghman (25). Gen-enation ofM (Gi), �3 (Fl), and �7 (Pi)tnansgenic mice wasdescribed previously by Vachen and Tilghman (27). De-scendants of the transgenic founder were mated withC3H/He mice, and the tnansgenic progeny were used inthese studies. Tnansgenic progeny were identified by South-em blot (62) of DNA obtained from a tail biopsy. AIbFAFP,M , �3, and �7 tnansgenic mice were identified by hybrid-ization to a 2.4-kb HhaI-EcoRI fragment as probe that nec-ognizes AFP exons 1-3 (25, 27, 28). AFP1AIb mice werealso identified by Southern blot using a 1 .2-kb Hindlll-BamHI fragment of AFP enhancer I as probe (25). Estimatesof transgene copy number based on Southern blot are:AFPEAIb (N33i), 22 copies; AIb1AFP (962L), 4 copies (Ref.25); �i (Gi ), 20 copies; z�3 (Fl ), 30 copies; and �7 (Pi ), 40

copies (Ref. 27).Liven regeneration in 6- to 1 2-week old mice was induced

by a single p. injection (10 �h/g body weight) of a 10%solution of CCI4 in mineral oil (29). Control mice wereinjected with mineral oil alone. Mice were sacrificed bycervical dislocation at various times after injection, andlivers were frozen in liquid nitrogen for RNA preparationon embedded in OCT (Miles Laboratories) for in situ

hybridization.RNA Preparation and Analysis. RNA was prepared from

liven tissue by LiCI/urea extraction (63). Poly(A)� RNA forNorthern blots was isolated by oligo-dT cellulose chroma-tognaphy (64). Five j�g of poly(A)� RNA/sample were sub-jected to formaldehyde gel electrophoresis and transferredto nitrocellulose membrane. AFP endogenous and mini-gene mRNA was detected by hybridization to the 0.44-kbHincll fragment encoding the first exon ofthe AFPgene (24,25). Ribosomal protein mRNA was detected using the pseu-dogene probe, rpL32 (65).

RNase protection assays were performed according to themanufacturer’s (Promega) protocol. The AFP gene probecontained exons 3 and 4 and was the gift of Dr. Shirley M.Tilghman (26). The albumin gene probe consisted of exons1 through 4 made by PCR amplification of cDNA tran-scnibed from adult mouse liven mRNA. Antisense RNAprobes were generated by Sp6 transcription of templateslinearized by Pvull incorporating Is32PJCTP (800 Ci/mmol;New England Nuclear). One to 20 j�g of RNA were mixedwith probe, denatured at 85#{176}Cfor 10 mm, and hybridizedovernight in 80% fonmamide, 40 ms� piperazine-N,N’-bis-2-ethi�nesulfonic acid (pH 6.4), 0.4 M sodium acetate, andi mM-EDTA. RNA was hybridized to the AFPgene probe at55#{176}C.60#{176}Cwas used for hybridization to the albumin geneprobe. The mixture was then digested with RNase One(Promega) for i h at 37#{176}Cin 10 msi Tnis-HCI (pH 7.5), 5 m�iEDTA, and 0.2 mM sodium acetate to remove single-

1556 AFP (;c’nt’ Expression during Liver Regeneration

s�

.....:, ‘ �

cv

�;I 4

� � ;t � ,�. � � ‘ ,.

;,5,4�i� ,‘. �

j�;; .

� �i

� . : ‘ . � . � � �. : � � ; : � :...�.. �

..�6 � I pt.�

� ,� ,‘

*8’, �“i !8, � � ‘

stranded species. The enzyme was inactivated by additionof SDS, and the RNA products were precipitated in ethanol.Ethanol precipitates were resuspended in loadingbuffer (80% formamide, 10 mtvt EDTA, 0,i% SDS, 0.1%

bromophenol blue, and 0.i% xylene cyanol) heated to85#{176}Cfor 8 mm and analyzed by denaturing gel electro-phoresis on an 8% polyacrylamide/7 NI urea gel. Probelengths protected from digestion with RNase One by hy-bnidization to mRNA were as follows: endogenous AFPmRNA, 190 nucleotides; AFP minigene rnRNA, 120 nude-otides; endogenous albumin mRNA, 275 nucleotides; andalbumin minigene mRNA, 105 nucleotides. Quantitation ofmRNA was performed by laser densitometry using a Mo-lecular Dynamics Phosphorlrnager, SF. Expression of trans-gene mRNA was normalized to endogenous albumin genelevels, and the statistical significance of the difference be-tween CCI4- and mineral oil-injected mice was analyzed byeither Student’s t test or by Mann-Whitney non-parametrictest.

In Situ Hybridization Analysis. !n situ hybridization wasdone as described elsewhere (26). Fresh tissue was embed-ded in OCT (Miles Laboratories), frozen in liquid nitrogen,and stored at -70#{176}Cbefore sectioning. Frozen tissue wassectioned onto poly-L-lysine-coated slides, fixed in 4%paraformaldehyde, and hybridized to a- 1�SlUTP-labeled

Fig. 8. Pattern of .�3 tnansgene

expression at various times afterinjection with CCI,,. Mice were in-

jected with CCI4 and sacrificed at24 h (A and B), 48 h (C and D(,

and 72 6 (Eand F) later. A, C, and

E, light field: B. 0. and F. darkfield. Bar, 100 ,zm. pt. portal tn-

ads; cv, central veins.

niboprobes overnight 55#{176}Cin a humidified container. Theantisense AFP niboprobe was generated by Sp6 polymenasetranscription ofthe a 0.44-kb Hincll exon i fragment clonedinto pGEM7Z(-) linearized with EcoRl. A sense niboprobewas generated by 17 polymenase transcription of the sametemplate linearized with Hindlll. Hybridized slides weredipped in K.2 photographic emulsion (llfond) diluted 1:1with water. Slides were exposed 2 to 5 days at 4#{176}Cand thendeveloped using D-i 9 developer and general purpose fixer(Kodak). Slides were stained with hematoxylin and Eosin Ybefore examination.

Acknowledgments

We thank Dr. Shirley M. Tilghman for her generosity in providing transgenic

mice and probes required for this work. We also thank Natalie Bailey fortechnical assistance and Drs. Peter Bergold and Mary Makowske for critically

reading the manuscript.

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