Abstract
The formation of differentiated cell types from pluripotent progenitors involves epigenetic regulation of gene expression. DNA hydroxymethylation results from the enzymatic oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) 5-mC dioxygenase enzymes. Previous work has mapped changes in 5-mC during differentiation of intestinal stem cells. However, whether or not 5-hmC regulates colonocyte differentiation is unknown. Here we show that 5-hmC regulates gene expression during colonocyte differentiation and controls gene expression in human colon cancers. Genome-wide profiling of 5-hmC during in vitro colonic differentiation demonstrated that 5-hmC is gained at highly expressed and induced genes and is associated with intestinal transcription factor binding sites, including those for HNF4A and CDX2. TET1 induction occurred during differentiation, and TET1 knockdown altered gene expression and inhibited barrier formation of colonocytes. We find that the 5-hmC distribution in primary human colonocytes parallels the distribution found in differentiated cells in vitro, and that gene-specific 5-hmC changes in human colon cancers are directly correlated with changes in gene expression. Our results support a model in which 5-hmC regulates differentiation of adult human intestine and 5-hmC alterations contribute to the disrupted gene expression in colon cancer.
Highlights
Differentiation of the intestinal epithelium along the spatially distinct crypt-luminal axis requires gene expression changes mediated in part by epigenetic pathways[1]
To determine if 5-hmC has a functional role in regulating colonocyte differentiation, we mapped 5-hmC changes during cell-cell adhesion-initiated differentiation of T84 colon adenocarcinoma cells since a similar system had previously been used to map chromatin regulatory regions of small intestinal differentiation[14]
We examined HNF4A binding sites by Tet-assisted bisulfite sequencing (TAB-seq)[22], which allows for quantification of cytosine, 5-mC, and 5-hmC at single base resolution
Summary
Our results identify a previously unappreciated role for 5-hmC in regulating epithelial differentiation. Previous reports have identified 5-hmC loss as a hallmark of cancer, including colon cancer[23,29,30,31,32,33,34] Many of these studies measured only global 5-hmC content without genome-wide mapping. Our studies reveal both locus-specific losses and gains of 5-hmC in cancer These 5-hmC changes occur at genes coding for proteins involved in regulating intestinal development and cancer cell proliferation. Correlation of 5-hmC alterations with tumor-associated changes in gene expression suggests that 5-hmC changes can contribute to both gene activation and repression in cancer These epigenetic lesions are of particular interest since they are potentially reversible with epigenetic targeting agents, and future work should determine whether or not any of these locus-specific 5-hmC changes predict response to these drugs. By defining regions with altered 5-hmC in colon cancer, our work provides a starting point for identifying key genomic regions that could be epigenetically altered in other epithelial malignancies and advances our understanding of the molecular mechanisms underlying tumorigenesis
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