Bacterial Infection Promotes DNA Hypermethylation
Y.A. Bobetsis1,
S.P. Barros1,
D.M. Lin1,
J.R. Weidman2,
D.C. Dolinoy2,
R.L. Jirtle2,
K.A. Boggess3,
J.D. Beck4, and
S. Offenbacher1,*
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;
2 Duke University Medical Center, Department of Radiation Oncology Environmental Safety, Durham, NC;
3 University of North Carolina at Chapel Hill, Center for Oral and Systemic Diseases, UNC School of Dentistry, and Department of Obstetrics and Gynecology, UNC School of Medicine, Chapel Hill, NC, USA 27599; and
4 University of North Carolina at Chapel Hill, Center for Oral and Systemic Diseases, Department of Dental Ecology, UNC School of Dentistry, Chapel Hill, NC, USA 27599
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Figure 1. Translocation of C. rectus to the placenta at E16.5. Nested PCR for detection of C. rectus in placenta at E16.5. Dams were infected by an intrachamber injection with C. rectus. Seven out of 15 placentas from fetuses challenged with intra-uterine growth restriction had a positive signal. PC, positive control for C. rectus-spiked placenta.
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Figure 3. Quantitative RT-PCR analysis for Igf2. The relative expression of Igf2 from unchallenged placenta (n = 6) and from growth-restricted challenged placenta (n = 6) was compared. Reactions were performed 2 independent times in triplicate. Mean values and error bars are presented. Statistical analysis was performed with one-way ANOVA. There was a significant (p = 0.0005) 2.3-fold decrease in the relative expression of Igf2 in the challenged placenta at E16.5.
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Figure 2. C. rectus infection induced structural abnormalities in the placenta. H&E staining from unchallenged (a,b) and growth-restricted challenged (c,d) placentas. In the intra-uterine growth-restricted challenged samples (n = 12), there is a significant relative decrease in the labyrinth area, with a concomitant increase in the decidua when compared with the control samples (n = 8). The spongiotrophoblast layer (S) is not altered. (e) Statistical analysis was performed with one-way ANOVA. *p < 0.001, **p = 0.04. (b,d) Histological sections indicated by rectangles in (a) and (c) with high-power magnification. Scale bar in (a) and (c), 500 µm, and in (b) and (d), 125 µm. (L) labyrinth, (S) spongiotrophoblast layer, (Sp) spongiotrophoblast cells, (D) decidua.
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Figure 4. C. rectus infection induces hypermethylation of the placental-specific P0 promoter region of Igf2 in the placenta. DNA from unchallenged and from intra-uterine growth-restricted placentas were bisulfite-treated and sequenced (details in MATERIALS & METHODS). (a) The location of the CpG islands and the differentially methylated regions (DMR) of the Igf2 gene, as well as the location of the 4 promoters (P0, P1, P2, P3). (b) The 16 CpG dinucleotides (vertical gray lines) within the P0, which are also part of the DMR0. This DNA fragment extends from sites 7782 to 8512 (GenBank Accession #U71085). To facilitate the sequencing process, we divided this region into 3 areas (amplicons 1–3). (c) For each CpG locus, from 50 to 90 clones were sequenced per experimental group, giving a total of 390 clones. The 6 red vertical lines represent the CpG sites that were hypermethylated by more than 10% after C. rectus infection. *The CpG sites where hypermethylation was statistically significant (p < 0.05). (d) Depicts the percentage of methylation at each of the CpG sites from the unchallenged (red) and challenged with Intra-uterine Growth Restriction (blue) placentas. Mean % methylation and error bars are presented. Statistical analysis can be found in MATERIAlS & METHODS. *CpG sites where hypermethylation was statistically significant (p < 0.05). OR, odds ratio; parentheses include the confidence interval. Four CpG sites were significantly hypermethylated (7845, 7869, 8461, and 8472), and these sites appear to be clustered.
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