(Received for publication, August 10, 1994; and in revised form, October 4, 1994)
From the
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.
The acute phase response is characterized by dramatic
alterations in the expression pattern of several liver proteins, hCRP ()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.
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.
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.
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.
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.
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).
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.
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 -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
-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.