©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Human C-reactive Protein Gene in Transgenic Mice (*)

(Received for publication, August 10, 1994; and in revised form, October 4, 1994)

Carol Murphy (1)(§) Johannes Beckers (1)(¶) Ulrich Rüther (1) (2)(**)

From the  (1)European Molecular Biology Laboratory, 69012 Heidelberg, Federal Republic of Germany and the (2)Institut für Molekularbiologie, Medizinische Hochschule Hannover, OE 5250, 30623 Hannover, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human C-reactive protein (hCRP) is a major acute-phase reactant in man. The regulation of the hCRP gene in transgenic mice is similar to that in humans. To map DNA regions required for the correct regulation of the hCRP gene, several constructs have been generated, and their expression in transgenic mice has been analyzed. Constructs lacking DNA regions surrounding the poly(A) site of the gene are not expressed either before or after induction in transgenic mice. Minimal regions 540 base pairs upstream and 1.2 kilobases downstream of the hCRP gene are sufficient for liver-specific expression. Extended 5`- and 3`-flanking regions are required to silence the expression prior to induction. Our findings demonstrate that regulatory sequences shown to confer inducible expression of the hCRP gene in hepatoma cell lines are insufficient in transgenic mice.


INTRODUCTION

The acute phase response is characterized by dramatic alterations in the expression pattern of several liver proteins, hCRP (^1)being the most abundant in humans (see Pepys and Baltz(1983)). The level of expression of hCRP increases from a basal serum level of less than 1 µg/ml to more than 300 µg/ml during the acute phase response. The proteins induced during the acute phase response are for the most part species-specific. In mouse, for instance, the expression of serum amyloid proteins (SAA) is greatly enhanced (Pepys and Baltz, 1983) while the expression of CRP is hardly induced (Whitehead et al., 1990). Nevertheless, the expression of human CRP was found to be regulated in transgenic mice as well as in man (Ciliberto et al., 1987a). A very low basal level of hCRP is strongly increased after induction. Therefore, all the sequences required for the correct regulation of the hCRP gene were contained within the 31-kb genomic fragment injected. The search for a suitable experimental system to further investigate the hCRP gene regulation led to the use of human hepatoma cell lines. However, even though these cells are known to lose liver-specific properties (discussed in Baumann(1989)), they were successfully used to demonstrate that both interleukin 1 and 6 acute phase response elements (Arcone et al., 1988; Ganter et al., 1989; Majello et al., 1990) and binding sites for hepatocyte nuclear factor 1 (Toniatti et al., 1990a) were present in the hCRP promoter. Although these in vitro studies were a first step toward the characterization of the hCRP promoter, it was not known whether the identified elements were sufficient for the correct in vivo regulation of the hCRP gene.

In the present study, we have attempted to delineate the DNA flanking regions of the hCRP gene that are responsible for its in vivo regulation.


EXPERIMENTAL PROCEDURES

Generation of Constructs

Construct 42 contained 17 kb 5` of the cap site and 11.3 kb 3` of the poly(A) sequence (corresponding to -17 kb to +13.6 kb in Fig. 1). The 31-kb ClaI fragment was isolated for microinjection from a previously described human cosmid, pCOS-CRP1 (Ciliberto et al., 1987a).


Figure 1: Organization of the human CRP gene. Restriction enzyme sites are positioned relative to the cap site of the hCRP gene. The coding part of the two exons (I, II), the 3`-untranslated region (3` UTR), and the CRP pseudogene () are indicated.



Construct 101 was generated by isolation of an AccI fragment containing 541 bp 5` of the cap and 1.2 kb 3` of the poly(A) corresponding to position -0.541 to 3.5 kb in Fig. 1. This construct and the following construct 4 contained the minimal elements for the in vitro regulation of the hCRP gene (Arcone et al., 1988).

Construct 4 contained 1.7 kb 5` of the cap site and 1.6 kb 3` of the poly(A) site, corresponding to -1.745 to +3.91 kb (Fig. 1). This construct was microinjected alone and also co-microinjected with the matrix attachment region (McKnight et al., 1992).

Construct 97 was generated by subcloning the BamHI-EcoRI fragment (position -1.7 to +1.8 kb) with the polyadenylation signal of SV40. This construct lacked the polyadenylation site and the 3` surrounding sequences of construct 4.

Construct 61 contained 12.7 kb 5` of the cap and 1.6 kb 3` of the poly(A) site, from position -12.7 to +3.91 kb, and it represented construct 4 with extended 5`-flanking sequences.

Construct 47 contained 1.7 kb 5` of the cap and 8.2 kb 3` of the poly(A) site, from position -1.745 to +10.5 kb (Fig. 1). In this construct and the following ones various lengths of 3`-flanking sequences were added to construct 4.

Construct 57 contained 1.7 kb 5` of the cap and 5.2 kb 3` of the poly(A) site, corresponding to -1.745 to +7.51 kb.

Construct 79 contained 1.7 kb of 5` and 3.8 kb of 3` sequences. The construct was prepared by combining the BamHI-HindIII fragment at -1.7 kb to +3.14 kb and KpnI-BamHI fragment from +7.5 kb to +10.5 kb. All constructs are illustrated in Fig. 2.


Figure 2: Schematic presentation of the hCRP constructs and their expression in transgenic mice. The expression data are summarized adjacent to each construct. #, construct number; a, mean value of basal level of hCRP protein expression in µg/ml of serum and standard deviation; b, mean value of factor of induction upon LPS injection; c, number of independent integration sites; d, expression not detectable.



Generation and Identification of Transgenic Mice

Transgenic mice were generated according to standard procedures (Hogan et al., 1986) by microinjection into the fertilized eggs of a cross between F1 (C57BL/6 times SJL) animals. Tail DNA was isolated at 14 days after birth and analyzed by Southern blotting.

LPS Induction of the Transgenic Mice and Estimation of hCRP Concentration in Serum

The level of hCRP protein expression in the serum was analyzed in all mice before and 15 h after induction with an intraperitoneal injection of 100 µg of LPS (Sigma, L-3254). The animals were analyzed at 4 weeks after birth to avoid fluctuations in the gene expression that may occur at different ages. The 15-h time point was chosen as it has been previously described (Ciliberto et al., 1987a) that the peak hCRP expression occurs after 15 h in transgenic mice. The serum samples were stored at -20 °C until analyzed. hCRP concentration was estimated in the serum by an enzyme-linked immunosorbent assay method using rabbit anti-hCRP immunoglobulin fraction (Dako Corp., AO73) and conjugated anti-rabbit immunoglobulin fraction peroxidase (Dako, P227). (^2)hCRP standard X923 was obtained from Dako Corp. The lower limit of detection of hCRP in mouse serum ranged from 500 pg to 1 ng/ml.

Isolation and Northern Blotting of Total RNA

At 8 weeks postpartum the mice were subjected to partial hepatectomies before and 9 h after induction with an intraperitoneal injection of LPS. Liver samples were stored at -80 °C until processed. The 9-h time point was chosen as it has been previously shown that the peak of hCRP mRNA expression in transgenic mice occurs at this time (Ciliberto et al., 1987a). To determine whether the transgenes were liver-specific in their expression pattern, total RNA was prepared from spleen, liver, heart, thymus, muscle, bone, kidney, pancreas, gonads, and brain of two F(o) mice harboring each construct.

Total RNA was isolated from mouse organs as described previously (Chomczynski and Sacchi, 1987). 20 µg of total RNA were separated on a formaldehyde gel, blotted, and probed with DNA fragments specific for the hCRP gene, mouse serum amyloid A2, and glyceraldehyde-3-phosphate dehydrogenase. Data from mice expressing mouse SAA in liver in the preinduction state were not considered for interpretation as expression of this endogenous murine gene indicates that the animal is inflamed, and therefore the preinduction level of expression of the transgene could be unreliably high. Glyceraldehyde-3-phosphate dehydrogenase was used to control the amount of RNA loaded.


RESULTS

To define the location of regulatory regions required for the expression of the hCRP gene in transgenic mice, we have undertaken a detailed analysis of the 31-kb genomic hCRP fragment (Ciliberto et al., 1987a). For most constructs, at least 10 independent integration sites were generated to analyze the inherent variation in such an in vivo approach.

The Minimal Elements Required for Inducible Expression of the hCRP Gene in Hepatoma Cells Are Insufficient in Transgenic Mice

The expression profile of construct 42, encompassing the entire 31-kb human genomic CRP fragment (Fig. 1), showed a low constitutive level of CRP expression that was induced 30-fold after LPS induction (Fig. 2). The protein values in serum were reflected in the RNA expression patterns. Five random examples are shown in Fig. 3. Furthermore, the hCRP mRNA was restricted to liver, and no correlation was found between the expression levels and the transgene copy number (data not shown). All these findings were in agreement with previous results (Ciliberto et al., 1987a) and represented the starting point for our comparisons.


Figure 3: hCRP mRNA expression pattern. Northern blot analyses for four different constructs are presented. The construct number is indicated above the corresponding panel. The numbers 1-5 indicate animals of independent integration sites. The analysis of liver RNA isolated prior to(-) and after induction (+) is shown. The filters were sequentially hybridized with probes for hCRP, SAA2, and glyceraldehyde-3-phosphate dehydrogenase. Animal 5 of construct no. 101 is the only CRP nonexpressing line as documented by the signal of glyceraldehyde-3-phosphate dehydrogenase probe.



We next analyzed construct 101, which is only 4 kb in size (Fig. 2). Though this construct still contained all the known in vitro defined acute phase response elements, its regulation differed significantly from that of construct 42. The transgenic mice generated showed a high basal level of hCRP expression, which was induced 5-fold by LPS (Fig. 2). This finding was also reflected on the RNA level, five examples of which are shown in Fig. 3. The high basal level of CRP in serum of transgenic mice generated with construct 101 was not a result of inflammation because expression of SAA2, a gene known to be induced in mice in the inflammatory state, was absent prior to LPS induction (Fig. 3). The result suggests that construct 101 lacked regulatory regions required to silence the transgene expression prior to induction.

Effect of DNase-hypersensitive Sites on the Inducible Expression of the hCRP Gene

Three LPS-inducible liver-specific DNase I-hypersensitive sites were found in transgenic mice harboring construct 42 (Toniatti et al., 1990b). Two mapped to the 5`-region adjacent to the cap site and at approximately -250 bp. The third site mapped to approximately -600 bp. Interestingly, this third DNase I-hypersensitive site mapped to a region that is conserved in the human haptoglobin and alpha(1)-antitrypsin genes (Bensi et al., 1985; De Simone et al., 1987). The sequence, TGGACACAGG, is 100% identical at position -671 of the hCRP gene. The regulation in mice of construct 4, which, compared with construct 101, contained extra 5`- and 3`-flanking DNA sequences including the latter motif, did not differ substantially from that of construct 101 except that it exhibited an even higher basal level of expression prior to induction (Fig. 2). Therefore, this DNase I-hypersensitive site is not involved in the silencing of the gene prior to induction. Since expression of constructs 4 (Fig. 4) and 101 (data not shown) was exclusively found in the liver, tissue specificity and silencing of hCRP expression prior to induction seem to be controlled by different regulatory regions.


Figure 4: Tissue-specific mRNA expression pattern of hCRP. A Northern blot analysis of a mouse harboring construct 4 is shown. The expression of hCRP in several organs is analyzed upon induction with LPS. Expression in liver is also shown before (L) and after induction (L*). P, pancreas; L, liver; H, heart; Lg, lungs; O, ovary, uterus; K, kidney; M, skeletal muscle. The upper panel (CRP) comes from a short exposure while the lower panel (CRP*) represents a longer exposure to ensure the absence of expression in other organs.



To exclude the possibility that the high basal level of expression of constructs 101 and 4 was due to the interference of surrounding sequences, construct 4 was co-injected with the matrix attachment region sequences from the chicken lysozyme gene. The matrix attachment region sequences have the property of allowing integration site-independent expression of transgenes (McKnight et al., 1992). Three transgenic mice were analyzed. all of which showed an expression pattern prior to and after induction similar to that of construct 4 alone (data not shown).

Sequences Surrounding the Poly(A) Site of the hCRP Gene Are Necessary for Its Expression

In the uninduced liver of transgenic mice harboring construct 42, a strong DNase I-hypersensitive site was identified in the vicinity of the poly(A) site of the hCRP gene (Toniatti et al., 1990b). To investigate the requirement of this region for the proper regulation of hCRP, we generated construct 97, which contained the poly(A) signal of SV40 (Fig. 2). Five transgenic mice were analyzed, none of which expressed the transgene in any organ either before or after induction. Thus, the sequences in the vicinity of the poly(A) site are necessary for hCRP promoter activity.

Investigation of Extended 5`- and 3`-Flanking Regions

Since none of the short constructs (e.g. no. 4) analyzed so far revealed a low basal level of expression that was comparable with that of construct 42, we determined the effects of increased 5`- or 3`-flanking regions. To this end constructs 61 and 47 with either additional 5`- or 3`-flanking regions, respectively, were generated (Fig. 2). Both constructs exhibited reasonably low basal serum level, which was strongly induced after LPS stimulation, a result that was also confirmed by RNA expression analysis (Fig. 3). Thus, increasing either the 5`- or 3`-flanking DNA clearly decreased the basal level of expression prior to induction indicating the presence of regulatory regions in these sequences.

The influence of 3`-flanking regions was surprising and therefore further investigated by analyzing construct 57, which encompassed a shorter length of 3`-flanking sequences compared with construct 47 (Fig. 2). Transgenic mice harboring this construct showed high levels of CRP expression prior to induction, resembling the regulation of constructs 101 and 4 ( Fig. 2and Fig. 3). This result indicated that regions 5 kb downstream of the hCRP coding sequence (3` of the +7.51 KpnI site (Fig. 1) are important for inducible expression. These 3`-regions include the hCRP pseudogene (Ciliberto et al., 1987b). We have therefore generated construct 79, which contained the sequences deleted in construct 57 attached to the sequences of construct 4 (Fig. 2). This construct exhibited a low basal expression level followed by strong induction after LPS stimulation suggesting that the +7.5 to +10.5 flanking region of the hCRP gene is important for negative control of expression.


DISCUSSION

In this study we have investigated the regions of the hCRP gene that are necessary for liver-specific and inducible expression. Constructs harboring various deletions 5` and 3` of the hCRP gene have been introduced into mice and their expression analyzed on both the protein and RNA level. The results indicate that 1) constructs lacking sequences surrounding the poly(A) site of the gene were not expressed either before or after induction in transgenic mice; 2) sequences 540 bp upstream and 1.2 kb downstream of the hCRP gene are sufficient for liver-specific but constitutive expression; 3) DNA flanking regions conferring tight control of the hCRP gene prior to induction are present both in the 5`- and 3`-flanking regions; and 4) regulatory sequences shown to confer inducible expression of the hCRP gene in hepatoma cell lines are insufficient in transgenic mice.

Regulation of gene expression by sequences located 3` of the open reading frame has been well documented, and examples include the human beta-globin, CD2, and keratin 18 genes (Grosveld et al., 1987; Greaves et al., 1990; Neznanov et al., 1993). These genes are constitutively expressed in a copy number-dependent and integration site-independent manner in transgenic mice only when 3` elements are included. Similarly, in the case of the hCRP gene we have not observed any expression of the transgene in the absence of the sequences surrounding the poly(A) site. This was also the case when we attempted to express the SV40 T antigen under the control of the hCRP promoter. It was necessary to include the region surrounding the poly(A) signal for transgene expression (Rüther et al., 1993). In contrast to the copy number-dependent expression observed with the beta-globin, CD2, and keratin 18 genes, we have not found any indication for copy number-dependent expression in the case of the hCRP gene.

Further work is required to define the exact sequences surrounding the poly(A) site of the hCRP gene that are required for the transgene expression. A potential candidate is the consensus site for LR1, a lipopolysaccharide-responsive factor with binding sites in the immunoglobulin switch regions and also in the heavy-chain enhancer (Williams and Maizels, 1991). The consensus site is GNCNAGGCTGA(A/C), and that found in the human CRP gene is GACAAGGCTGAT. In the rabbit where CRP is also an acute phase gene the site is also conserved (TCCAAGGCTGAC). It is not known whether LR1 is required for the expression of acute phase response genes; so far it has only been found in cultured primary B and murine liver cells (Williams and Maizels, 1991). The presence of a potential binding site in the hCRP gene, in a DNA fragment shown here to be essential for liver-specific transgene expression, merits further investigation.

The low level of expression of the hCRP gene before induction is most likely due to negative transcriptional regulation as has been shown for some inducible genes, e.g. interferon. The promoter became constitutively active when certain elements were deleted (for review, see Clark and Docherty(1993)). Similar results have been found for the hCRP promoter in human hepatoma cells (Li et al., 1990). Two negative control elements were detected in the promoter of the gene. Removal of these elements resulted in an increase in the basal level of gene expression. The negative control regions described (Li et al., 1990) are included in all the constructs used in this report and may indicate general differences between hepatoma cell lines and the transgenic mouse system.

Our results suggest that 5`- and 3`-regions of the hCRP gene can independently contribute to the low level of expression prior to induction (constructs 61 and 47, respectively). As we have not yet mapped the DNA elements responsible, we cannot exclude that identical sequences may be present 5` and 3` of the gene. Interestingly, the 3`-region involved in the negative regulation of the hCRP gene includes the hCRP pseudogene. It is possible that the pseudogene is a product of duplication, and certain regulatory sequences have been retained. To characterize the elements required for silencing the hCRP gene prior to induction, we will screen for sequences that when deleted increase the preinduction level of expression. This approach will finally lead to a fine mapping of the elements involved in the regulation of the hCRP gene. Identification of such regulatory elements will advance the understanding of the network of cytokine action in the course of inflammation for which the hCRP is a major marker protein.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft Grant Sonderforschungsbereich 244. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a European Molecular Biology fellowship.

Present address: Dept. de Zoologie et Biol. Animale, Universite de Geneve, 1211 Geneve, Switzerland.

**
To whom correspondence should be addressed. Tel.: 49-511-532-4017; Fax: 49-511-532-4283.

(^1)
The abbreviations used are: hCRP, human C-reactive protein; SAA, serum amyloid protein; CRP, C-reactive protein; kb, kilobase(s); bp, base pair(s); LPS, lipopolysaccharide.

(^2)
R. Düssinger and B. Kyewski, personal communication.


ACKNOWLEDGEMENTS

We thank David Stoddart and Barbara Stace for excellent animal husbandry and Rocio Garcia de Veas Lovillo and Birgit Bosse for technical assistance. We thank Lothar Hennighausen for the matrix attachment region construct, and Alfred Nordheim and Michael Cahill for critical reading of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.