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Bacterial Infection Promotes DNA Hypermethylation
1 University of North Carolina at Chapel Hill, Center for Oral and Systemic Diseases, Department of Periodontology, UNC School of Dentistry, CB #7455, DRC Rm 222, Chapel Hill, NC, USA 27599-7455; Correspondence: * corresponding author, steve_offenbacher{at}dentistry.unc.edu
Maternal oral infection, caused by bacteria such as C. rectus or P. gingivalis, has been implicated as a potential source of placental and fetal infection and inflammatory challenge, which increases the relative risk for pre-term delivery and growth restriction. Intra-uterine growth restriction has also been reported in various animal models infected with oral organisms. Analyzing placental tissues of infected growth-restricted mice, we found down-regulation of the imprinted Igf2 gene. Epigenetic modification of imprinted genes via changes in DNA methylation plays a critical role in fetal growth and development programming. Here, we assessed whether C. rectus infection mediates changes in the murine placenta Igf2 methylation patterns. We found that infection induced hypermethylation in the promoter region-P0 of the Igf2 gene. This novel finding, correlating infection with epigenetic alterations, provides a mechanism linking environmental signals to placental phenotype, with consequences for development.
Key Words: epigenetics • chronic inflammation • oral pathogen • fetal growth
DNA methylation is an ancestral mechanism for neutralizing potentially damaging DNA elements in the genome (Hendrich and Tweedie, 2003). One fundamental level of epigenetic genome modification involves methylation of cytosine at position C5 in CpG dinucleotides, the most common DNA modification used in eukaryotes. Functionally, DNA methylation regulates many cellular processes, such as embryonic development, transcription, and genomic imprinting. Genomic imprinting is defined as an epigenetic modification of a specific parental chromosome in the gamete or zygote that leads to differential expression of the two alleles of a gene in the somatic cells of the offspring (Robertson, 2005). A substantial proportion of imprinted genes are also involved in the control of fetal growth and placental development (Isles and Holland, 2005). Properly established and maintained, DNA methylation patterns are essential for mammalian development and for the normal functioning of the adult organism. Growing numbers of human diseases, syndromes, and disorders are correlated with a gain or loss of DNA methylation (Robertson, 2005). In development, Igf2 can affect the growth of the fetus, and has a particularly unique and important action in placental growth (Constancia et al., 2002). The paternally expressed Igf2 gene (chromosome 7 in the mouse) is part of a cluster of imprinted genes with a homologous cluster on the syntenic region on chromosome 11p15.5 in the human (DeChiara et al., 1991; Paulsen et al., 1998). Deletion of the murine Igf2 promoter region 0 blocks placental IGF2 expression and leads to reduced placental growth, followed by fetal growth restriction (Constancia et al., 2002). Moreover, Igf2 knockout mice are 40% smaller at birth (DeChiara et al., 1990). Pre-term births and low birthweight have been associated with an increased risk for life-threatening disabilities, including diabetes, neurodevelopmental anomalies, and coronary heart disease (Christianson et al., 1981; Hattersley and Tooke, 1999). Analysis of emerging data indicates that maternal infections during pregnancy and attendant inflammatory responses may play a role in pre-term births and growth restriction (Goldenberg et al., 2000). Periodontal infections have been associated with an increased risk for pre-term births (Offenbacher et al., 2001), and increasing evidence points to C. rectus as a key pathogen (Buduneli et al., 2005). In this study, we analyzed the placental Igf2 expression in mice and investigated the methylation status of the placental Igf2 promoter (P0) region following C. rectus challenge in pregnant mice.
Mouse Infection Model All mouse experiments were carried out under the approval of the UNC Institutional Animal Care and Use Committee and have been described in detail previously (Lin et al., 2003; Yeo et al., 2005). A stainless steel coil (1 x 0.4 cm) was surgically implanted subcutaneously into female adult BALB/C mice. After healing, mice were mated overnight, and pregnant dams received an intrachamber injection of 100 µL of 109 CFU/mL live C. rectus strain 314 (log growth) or PBS at E7.5. Mice were killed at E16.5, and fetuses (n = 144 from 27 unchallenged dams and n = 193 from 32 challenged dams) and placental tissues (n = 106 from 19 unchallenged dams and n = 105 from 19 challenged dams) were collected. We measured the weight and crown-rump length, and performed statistical analysis using a mixed-model method (Little et al., 1996), to account for the fact that effects of challenge on littermates are clustered within dams.
Nested-PCR Analysis for the Detection of C. rectus
Quantitative RT-PCR for Igf2
DNA Methylation Analysis
Morphometric Analysis
Nested-PCR Analysis for the Detection of C. rectus Intra-uterine growth restriction was observed in 22.8% of the fetuses of challenged litters, compared with 2.7% from unchallenged dams, p < 0.01 (data not shown). We could identify the presence of C. rectus in placental tissues by nested PCR detected in 7 out of 15 samples analyzed from growth-restricted E16.5 fetuses. Nested PCR analysis of C. rectus-16S rDNA from DNA extracted from E16.5 placental samples is shown in Fig. 1  . Although in our model the infection occurred at a site distant from the uterus, analysis of these data indicates that the bacteria translocated to the placenta being detected in 46.7% of the placentas of intra-uterine growth-restricted fetuses.
Placental Morphometry and IGF2 Expression In both mice and humans, maternal blood passes through spiral arteries into a space created by the conceptus that is densely filled with placental villi, termed a ’labyrinth’ in the mouse. In the morphometric analysis, placental samples showed structural alterations within the challenged placental layers, with a relative decrease in the labyrinth area (Fig. 2 ), and a concomitant increase in the decidual layer, also enlarged due to the presence of focal areas of inflammatory infiltrate. In previous experiments using microarrays of C. rectus-challenged placentas, we found significant down-regulation of IGF2 (data not shown). Because of the central role of this gene in regulating fetal growth, real-time PCR assay was used to confirm this finding. Quantitative PCR for Igf2 expression showed a significant 2.3-fold decrease (p = 0.0005) in placental tissue from intra-uterine growth-restricted fetuses (Fig. 3).
Methylation Analysis To address whether there was an alteration in methylation patterns, we applied the bisulfite sequencing method to quantify placental DNA methylation in the Igf2 promoter region 0, which regulates murine placental expression of IGF2 and also contains the differentially methylated region 0. The location of CpG islands and the differentially methylated regions of the Igf2 gene, as well as the location of the 4 promoters, are shown in Fig. 4a . The positions of the 16 CpG dinucleotides within Igf2 promoter region 0 are illustrated (Fig. 4b). We sequenced a total of 390 clones, using 3 amplicon regions (Fig. 4b) to evaluate methylation levels for each CpG loci. Since this promoter region is maternally imprinted, there was methylation across this region in unchallenged placental tissues (Figs. 4c, 4d). However, there was an overall 10.3% increase (p < 0.0001) in CpG methylation across this entire promoter region in placental DNA from infected intra-uterine growth-restricted placentas. There was a general trend for increased methylation in 11 of the 16 CpG sites following C. rectus challenge, with 6 sites demonstrating a CpG hypermethylation greater than 10% relative to unchallenged tissues (Fig. 4c, shown in red). This hypermethylation was statistically significant (p < 0.05) at 4 CpG sites (Figs. 4c, 4d, indicated by asterisks). At CpG locus 7845, there was a 1.89-fold increase in methylation relative to control [OR = 1.89 (95% CI, 1.03–3.45)]; at site 7869, a 2.51-fold increase (95% CI, 1.04–6.07); at site 8461, a 1.79-fold increase (95%CI, 1.07–2.98); and at site 8472, a 2.24-fold increase (95% CI, 1.07–4.71). It is interesting to note that the hypermethylated regions are clustered within the Igf2 promoter region-P0. The concordance of the hypermethylation patterns in each region was very high. For example, analysis of amplicon 1 showed that the overall concordance for site methylation between CpG 7845 and 7869 was 76.7% (p < 0.0001). Similarly, analysis of amplicon 3 revealed that the concordance for methylation at CpG sites 8461 and 8472 was 89.4% (p < 0.0001). Thus, hypermethylation appears to be a site-directed process, rather than a non-specific increased methylation across the entire Igf2 promoter region P0.
To address the effects of maternal infection on fetal growth restriction and possible alteration of the methylation status of placental Igf2, we utilized an infection/pregnancy mouse model (Offenbacher et al., 2005), challenging the pregnant mice with C. rectus. In our model, although the infection occurred at a distant site from the uterus, the bacteria translocated to the placenta, as reflected by the nested PCR detection in 46.7% of the placentas of intra-uterine growth- restricted fetuses. There were also structural alterations in the placental layers, with a decrease in the proportion of the labyrinth. We also observed a 27% reduction in weight at E16.5 (data not shown) that is similar to the decrease in birth weight detected in mice in which the Igf2 gene has been completely deleted (De Chiara et al., 1990; Constancia et al., 2002), and is also comparable with our previous findings (Offenbacher et al., 2005; Yeo et al., 2005). Analysis of our data from real-time PCR also showed a significant 2.3-fold decrease (p = 0.0005) in IGF2 mRNA levels in placentas from intra-uterine growth-restricted fetuses. Since IGF2 synthesis is trophoblast-dependent, a two-fold decrease at a tissue level may have significant effects on fetal growth. This is supported by McMinn and colleagues, who demonstrated that, in humans with intra-uterine growth restriction, placental tissues displayed a significant 2.1- and 2.6-fold decreased expression of IGF1 and IGF2, respectively (McMinn et al., 2006). Thus, C. rectus infection suppresses Igf2 expression, and this finding provides the first report associating bacterial infection mediating placental suppression of a growth factor known to be critical to both placental and somatic fetal growth. Since the P0 promoter region of Igf2 controls the expression of this gene in the placenta (Moore et al., 1997), we tested our hypothesis, that the observed suppression of Igf2 expression could be the result of epigenetic hypermethylation of CpG sites in the promoter region. The findings indicated that bacterial infection is associated with hypermethylation of the Igf2 promoter region-P0 at 4 loci, with a site-specific odds ratio for hypermethylation ranging from 1.79 to 2.51. Previous investigations have showed that methylation of the Igf2 promoter is associated with gene silencing, and that trans acting H19 locus (Feil et al., 1994; Yang et al., 2003) is also important in controlling IGF2 gene expression. Although promoter methylation is associated with altered gene expression, it remains to be seen whether infection-mediated methylation on Igf2 promoter region-P0 is functionally related to the observed Igf2 down-regulation. However, it is interesting to note that the region of hypermethylation at CpG sites 8461 and 8472 contains an Sp1 binding element. The inability of transcription factors to bind to their DNA binding sites when methylated has been proposed as a possible mechanism of gene silencing, and may provide one possible explanation for the transcriptional down-regulation of this gene. This investigation provides the first report that infection can lead to host epigenetic modification of an imprinted gene. It is not known whether this hypermethylation occurs due to direct bacterial interaction with the tissue, or as a consequence of a host inflammatory response. For example, H. pylori, an organism that is phylogenetically closely related to C. rectus, has been reported to be associated with altered oncogene methylation patterns in the gastric mucosa at local sites of infection, a process which is strongly linked with an increased risk for cancer (Maekita et al., 2006). The potential for oral bacteria such as C. rectus to modify DNA methylation patterns locally within the oral mucosa, and thereby alter local gene expression patterns, is currently under investigation. In our experiments, the alteration in DNA methylation patterns was evaluated in the placental tissues; however, it may also occur in other fetal tissues or organs. This raises the possibility that somatic epigenetic modification may occur in utero as a consequence of infection, and also provides a potential link for exploring DNA methylation in the offspring in response to infection as it modifies development and potentially mediates long-term disabilities in various organ systems. Infection-mediated alterations in intra-uterine imprinting may underlie the linkages seen between pre-term delivery and diseases of the offspring that include neurological impairments and adult-onset conditions, such as diabetes and cardiovascular disease (Allin et al., 2006; Mericq, 2006). In humans, abnormalities in IGF regulation have been linked to mental retardation, diabetes, and atherosclerosis (Zaina and Nilsson, 2003). We have recently reported that, in pregnant mice, C. rectus challenge may lead to white matter damage and hypomyelination (Offenbacher et al., 2005). Our data showing infection-mediated hypermethylation of the placental Igf2 promoter, coupled with the knowledge that changes in expression of imprinted genes have major implications for developmental programming, may, in part, explain the poor prognosis of the infant born small for gestational age, or with pre-term birth. The role of maternal infection as a possible source of abnormal epigenetic imprinting opens up the consideration of not only the potential somatic effects of IGF abnormalities, but also the potential effects on other key imprinted genes that control growth and development.
We thank Dr. Robert Bagnell and Victoria J. Madden for imaging assistance, and Dr. John Preisser and Chris Fan for statistical analyses. This work was supported by National Institutes of Health grants RO1-DE-12453, P-60-DE-13079, and U01 DE14577, and by the University of North Carolina General Clinical Research Center grant Mp1-RR-00046.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication May 15, 2006. Revision received October 24, 2006. Accepted for publication October 25, 2006.
Journal of Dental Research, Vol. 86, No. 2,
169-174 (2007) This article has been cited by other articles:
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