DNA methylation in colonic mucosa has been extensively studied in the context of colon cancer, in some cases with implications for IBD. An age dependent increase in the methylation of CpG dense genomic regions (commonly referred to as CpG islands) has been observed at genes involved in colorectal oncogenesis. Even normal appearing colonic mucosa from colorectal cancer patients displays significant epigenetic aberrations, suggesting that those precede and potentially contribute to tumor development (52
). Similar findings have been observed in the inflamed and cancerous mucosa of IBD (54
). These appear to be non-IBD specific, however, and can even be less common than in sporadic colorectal tumors (56
In spite of the extensive epigenetic studies in colon cancer, there is limited current knowledge on normal colonic mucosal epigenetic development and how it may relate to other gastrointestinal disorders such as IBD. Moreover, the timing of epigenetic differentiation and maturation is of great relevance to the developmental origins hypothesis, which postulates that environmental influences during critical periods of development cause permanent changes in organismic structure and function that can influence adult metabolism and disease risk (9
). Mammalian epigenetic mechanisms appear to be most sensitive to environment when undergoing developmental establishment or maturation (10
). Epigenetic changes during infancy and adolescence may therefore reveal important, environmentally labile pathogenic processes and help explain the peak incidence of IBD in young adulthood. Our study is the first to address genome wide epigenetic developmental changes during normal postnatal gastrointestinal development in mammals.
Approximately 1% of the interrogated genomic regions showed DNA methylation changes from late infancy to young adulthood, indicating that epigenetic development in the colonic mucosa proceeds beyond infancy in mice. Importantly, both a germfree transfer model (38
) and our current data on DSS exposure indicate that susceptibility to colitis increases during this period. Among genes that gained methylation we found an overrepresentation of genes involved in kidney development, but the expression of those tested was already undetectable at P30. Nevertheless, these findings are consistent with increased methylation at these genomic regions serving to perpetuate the transcriptional silencing of associated genes. PAX
genes, for example, are important during embryogenesis, but their constitutive expression promotes tissue hyperplasia. In particular, reactivation of PAX8
expression has been observed in several cancers, including colon cancer (59
). Increased methylation at Pax8
may therefore contribute to its persistent transcriptional silencing, stabilize the differentiated state and protect against malignant transformation. Our recent findings in a mouse model favor this reinforcing, stabilizing role of DNA methylation changes during development (30
). In further support of this paradigm, two of the six genes involved in kidney development (Bdbf
) are associated with CTCF insulator sites, which have been shown to be important regulators of tumor development (44
). Indeed, both Bdnf
) and Gdnf
) participate in the regulation of cancer. Additionally, the genomic regions with change in methylation during pediatric development were significantly more likely to associate with CTCF insulator sites in general, compared with the control Sma
I intervals. Our data support earlier observations suggesting functional links between developmental epigenetics and cancer epigenetics (42
), and indicate that relevant developmental processes in mammalian colonic mucosa proceed well beyond infancy.
Our results, both with the targeted assessment of candidate genes and with the gene expression microarray analysis, indicated that most of the DNA methylation changes we identified were not associated with expression changes at nearby genes. One potential explanation is that DNA methylation changes may in fact regulate expression of genes located quite distally. Histone modifications at enhancer elements as far as 200 kb from TSSs correlate with cell type gene expression (43
), and as mentioned earlier, imprinting control elements can regulate allele specific gene expression over hundreds of kilobases (47
). Cooperative interactions between epigenetic modifications have been shown to influence even chromosomal/chromatin rearrangements (62
). Hence, DNA methylation changes not directly related to the expression of nearby genes may have significant, yet less easily identifiable, physiologic consequences.
We did discover coordinated developmental changes in DNA methylation and expression of galectin-1 (Lgals1
) and Smad3
, underscoring the potential pathophysiological relevance of developmental epigenetics in the colonic mucosa. Lgals1
expression has been shown to decrease following chemical induction of colitis in mice, and recombinant human GAL-1 protein protected against colitis in that model system by inducing activated T-cell apoptosis (48
). In murine models, decreased expression of Smad3
has been shown to support epithelial recovery and inhibit colorectal fibrosis following chemical induction of colitis (49
). Although the DNA methylation loss associated with Smad3
was low (~3%), it was very tightly regulated between P30 and P90 and correlated with an increased expression of the gene (~1.4-fold). Similar observations have been made in human colonic mucosal T cells, in which a ~5% decrease in DNA methylation was associated with a 3-fold increase in IFNG
). Therefore, the decreased expression of galectin-1 (decreased protection) and increased expression of Smad3
(decreased regenerative capability following mucosal injury) may contribute to the greater colitis susceptibility of P90 mice, exemplifying the potential physiologic relevance of the epigenetic changes we have identified.
Although most of the identified changes in gene expression were not correlated with methylation changes, they may nonetheless yield important insights into developmentally regulated GI pathogenesis. For example, our results show that STAT1
expression, which is elevated in the inflamed colonic mucosa of CD patients (50
), decreases during murine post-infant development.
As discussed in the introduction, IBD is likely to result from an exaggerated immune response against the intestinal microflora that is transmitted by the intestinal epithelium. This pathologic response appears to be modulated by genetic and epigenetic factors and highlights the physiologic importance of a flexible but well regulated crosstalk between the immune system and the intestinal microbiome. Therefore, it is very plausible that a dynamic and complex interaction between the immune system, the gut epithelium and the luminal flora shapes the age dependent epigenetic changes of colonocytes. Our findings herein reveal potential elements of this intricate network.
In conclusion, this work provides a compendium of genomic regions that undergo pediatric epigenetic maturation and expression changes in mammalian colonic mucosa, which may be pathophysiologically relevant in different intestinal disorders including IBD and cancer. Our findings indicate a dynamic and age dependent epigenetic programming in the genome of colonic epithelial cells. These changes proceed far beyond infancy, parallel an increased predisposition to colitis and may reinforce colonocyte differentiation. Future studies will be required to address the full degree of the physiologic correlates of this epigenetic maturation, and the potential susceptibility of these DNA methylation and gene expression changes to environmental influences such as nutrition. Such studies will advance our understanding of the epigenetic basis of gastrointestinal development and disease.