Maria A. Hahn

Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis.

DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.

Methylation of Polycomb target genes in intestinal cancer is mediated by inflammation

Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

5-Hydroxymethylcytosine: a stable or transient DNA modification?

The DNA base 5-hydroxymethylcytosine (5hmC) is produced by enzymatic oxidation of 5-methylcytosine (5mC) by 5mC oxidases (the Tet proteins). Since 5hmC is recognized poorly by DNA methyltransferases, DNA methylation may be lost at 5hmC sites during DNA replication. In addition, 5hmC can be oxidized further by Tet proteins and converted to 5-formylcytosine and 5-carboxylcytosine, two bases that can be removed from DNA by base excision repair. The completed pathway represents a replication-independent DNA demethylation cycle. However, the DNA base 5hmC is also known to be rather stable and occurs at substantial levels, for example in the brain, suggesting that it represents an epigenetic mark by itself that may regulate chromatin structure and transcription. Focusing on a few well-studied tissues and developmental stages, we discuss the opposing views of 5hmC as a transient intermediate in DNA demethylation and as a modified DNA base with an instructive role.

The role of 5-hydroxymethylcytosine in human cancer

The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.

Loss of the Polycomb mark from bivalent promoters leads to activation of cancer-promoting genes in colorectal tumors

In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression.

Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study, we have combined laser-capture microdissection with reduced representation bisulfite sequencing to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2. Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. Although aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Altogether, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

Methods for genome-wide analysis of DNA methylation in intestinal tumors.

Recent studies show that colorectal cancer is strongly associated with aberrant DNA methylation, which has been linked to the origin and progression of the disease. This fact indicates a need for deep analysis of DNA methylation alterations during colorectal carcinogenesis. The knowledge obtained from such studies will elucidate the mechanisms of epigenetic changes and, through the identification and characterization of DNA methylation markers and disease-specific methylation patterns, will help improve the diagnosis and treatment options for patients. The introduction of new methods for genome-wide analysis of DNA methylation has been an important step towards achieving these goals. In this review, we discuss the role of DNA methylation in intestinal carcinogenesis as well as the different methodological approaches that are currently being used for methylation analysis on a genome-wide scale.

Expression of lactoperoxidase in differentiated mouse colon epithelial cells

Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

Abstract 2775: Characterization of transcriptional and epigenetic signatures of benign thyroid adenomas: Can we improve preoperative diagnosis of thyroid nodules

Introduction: It has been estimated that as many as 50,000 patients in the United States receive unnecessary thyroidectomies due to the inability to distinguish benign thyroid nodules from malignant ones. Up to 30% of fine needle aspirates performed for thyroid nodules diagnosis are indeterminate due to overlapping cytological features between benign and malignant nodules. At present, commercially-available diagnostic tests for thyroid nodules are based on the molecular differences observed between thyroid cancer compared to normal thyroid tissue. However, the molecular signature of benign thyroid nodules is neither well-characterized nor included in currently available diagnostic panels. The objective of this study was to determine whether thyroid adenomas (TA) are distinct from normal thyroid tissue and papillary thyroid cancer (PTC). Methods: Whole transcriptome analysis was used to assess differences in transcriptional activity in 9 TA and 12 PTC tissue pairs (with matched normal adjacent thyroid tissue from the same patients). In order to evaluate epigenetic alterations associated with TA development, we used reduced representation bisulfite sequencing (RRBS) in 114 thyroid specimens (40 PTC, 28 TA and 46 matching adjacent thyroid tissues). This approach provides single base resolution of DNA methylation genome wide. Results: According to our data, transcriptome activity divided analyzed specimens into three separate groups: PTC, TA and adjacent thyroid tissues. TA demonstrated a unique transcriptional pattern, distinct from both normal adjacent thyroid tissue and PTC. Similar results were obtained by analysis of epigenetic alterations in these tissues. According to the clustering analysis of DNA methylation patterns, the majority (18 of 28) of benign nodules forms a separate cluster, distinct from adjacent normal thyroid and PTC tissue clusters. Within this group, 14 of 28 TA demonstrated a distinct DNA methylation signature associated with TA-specific hypermethylation. In fact, only 4 of 28 TA demonstrated a DNA methylation signature similar to normal thyroid tissue, suggesting that the majority of TA is not equivalent to normal thyroid. Conclusion: According to whole transcriptome analysis and genome wide analysis of DNA methylation, TA is frequently associated with specific transcriptional and DNA methylation signatures compared to normal thyroid tissue and PTC. These data indicate that the majority of thyroid adenomas are associated with a unique molecular pathway that is distinct from PTC development. Use of the adenoma-specific molecular signature can be an essential factor in the improvement of PTC diagnostic panels, helping to reduce unnecessary thyroidectomies. Citation Format: Audrey H. Choi, Ryan Lew, Michael O’Leary, Yuman Fong, John H. Yim, Maria A. Hahn. Characterization of transcriptional and epigenetic signatures of benign thyroid adenomas: Can we improve preoperative diagnosis of thyroid nodules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2775.

Abstract B03: Loss of the Polycomb mark at bivalent promoters leads to activation of intestinal stem cell genes in colorectal cancer

During tumor formation, transcription of many genes becomes deregulated. In order to elucidate epigenetic mechanisms responsible for this phenomenon, we analyzed genome-wide patterns of several histone marks, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in human stage II colorectal cancer and matching normal tissue samples. By using liquid chromatography—mass spectrometry (LC-MS/MS), we determined that colon tumors have strongly reduced global levels of 5hmC. However, Tet-assisted bisulfite sequencing showed that site-specific loss of 5hmC in tumors is not associated with localized aberrant CpG island hypermethylation. We observed that DNA hypermethylation is generally targeted to bivalent promoters, which loose H3K27me3 and the associated genes are repressed effectively. Importantly, loss of H3K27me3 at bivalent promoters is not always coupled with gain of 5mC. Instead, H3K27me3 loss is also frequently associated with activation of intestinal stem cell markers and stem cell regulators including LGR5, SOX9, CDX1, CDX2, KLF5, and SALL4 as well as activation of many genes linked to colorectal cancer progression such as MET, CLDN1, PTGS2 (COX-2) and CCND1 (cyclin D1). Activation of these genes correlates with accumulation of the active chromatin mark H3K4me3 at their promoters. Our study demonstrates two distinct features of H3K27me3 loss in cancer. Loss of H3K27me3 at bivalent genes can be accompanied by either gain of 5mC and strong repression or by persistence or gain of H3K4me3 and gene activation. We propose that instability of H3K27me3 coupled with selection for tumor-promoting events plays a major role in colorectal cancer progression most notably by causing activation of cancer driving genes including factors involved in stem cell maintenance. Citation Format: Maria A. Hahn, Arthur X. Li, Xiwei Wu, Daniel W. Rosenberg, Gerd P. Pfeifer. Loss of the Polycomb mark at bivalent promoters leads to activation of intestinal stem cell genes in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B03.