Epigenome-Wide DNA Methylation Profiling in Colorectal Cancer and Normal Adjacent Colon Using Infinium Human Methylation 450K

The aims were to profile the DNA methylation in colorectal cancer (CRC) and to explore cancer-specific methylation biomarkers. Fifty-four pairs of CRCs and the adjacent normal tissues were subjected to Infinium Human Methylation 450K assay and analysed using ChAMP R package. A total of 26,093 differentially methylated probes were identified, which represent 6156 genes; 650 probes were hypermethylated, and 25,443 were hypomethylated. Hypermethylated sites were common in CpG islands, while hypomethylated sites were in open sea. Most of the hypermethylated genes were associated with pathways in cancer, while the hypomethylated genes were involved in the PI3K-AKT signalling pathway. Among the identified differentially methylated probes, we found evidence of four potential probes in CRCs versus adjacent normal; HOXA2 cg06786372, OPLAH cg17301223, cg15638338, and TRIM31 cg02583465 that could serve as a new biomarker in CRC since these probes were aberrantly methylated in CRC as well as involved in the progression of CRC. Furthermore, we revealed the potential of promoter methylation ADHFE1 cg18065361 in differentiating the CRC from normal colonic tissue from the integrated analysis. In conclusion, aberrant DNA methylation is significantly involved in CRC pathogenesis and is associated with gene silencing. This study reports several potential important methylated genes in CRC and, therefore, merit further validation as novel candidate biomarker genes in CRC.

Loss of MBD2 attenuates MLL-AF9-driven leukemogenesis by suppressing the leukemic cell cycle via CDKN1C

Acute myeloid leukemia (AML) is a deadly cancer characterized by an expanded self-renewal capacity that is associated with the accumulation of immature myeloid cells. Emerging evidence shows that methyl-CpG-binding domain protein 2 (MBD2), a DNA methylation reader, often participates in the transcriptional silencing of hypermethylated genes in cancer cells. Nevertheless, the role of MBD2 in AML remains unclear. Herein, by using an MLL-AF9 murine model and a human AML cell line, we observed that loss of MBD2 could delay the initiation and progression of leukemia. MBD2 depletion significantly reduced the leukemia burden by decreasing the proportion of leukemic stem cells (LSCs) and inhibiting leukemia cell proliferation in serial transplantation experiments, thereby allowing leukemic blasts to transition to a more mature state reflecting normal myelopoiesis. Both gene expression analyses and bioinformatic studies revealed that MBD2 negatively modulated genes related to myeloid differentiation, and was necessary to sustain the MLL-AF9 oncogene-induced gene program. We further demonstrated that MBD2 could promote LSC cell cycle progression through epigenetic regulation of CDKN1C transcription probably by binding to its promoter region. Taken together, our data suggest that MBD2 promotes AML development and could be a therapeutic target for myeloid malignancies.

Hyperglycemia Altered DNA Methylation Status and Impaired Pancreatic Differentiation from Embryonic Stem Cells

The prevalence of type 2 diabetes (T2D) is rapidly increasing across the globe. Fetal exposure to maternal diabetes was correlated with higher prevalence of impaired glucose tolerance and T2D later in life. Previous studies showed aberrant DNA methylation patterns in pancreas of T2D patients. However, the underlying mechanisms remained largely unknown. We utilized human embryonic stem cells (hESC) as the in vitro model for studying the effects of hyperglycemia on DNA methylome and early pancreatic differentiation. Culture in hyperglycemic conditions disturbed the pancreatic lineage potential of hESC, leading to the downregulation of expression of pancreatic markers PDX1, NKX6−1 and NKX6−2 after in vitro differentiation. Genome-wide DNA methylome profiling revealed over 2000 differentially methylated CpG sites in hESC cultured in hyperglycemic condition when compared with those in control glucose condition. Gene ontology analysis also revealed that the hypermethylated genes were enriched in cell fate commitment. Among them, NKX6−2 was validated and its hypermethylation status was maintained upon differentiation into pancreatic progenitor cells. We also established mouse ESC lines at both physiological glucose level (PG-mESC) and conventional hyperglycemia glucose level (HG-mESC). Concordantly, DNA methylome analysis revealed the enrichment of hypermethylated genes related to cell differentiation in HG-mESC, including Nkx6−1. Our results suggested that hyperglycemia dysregulated the epigenome at early fetal development, possibly leading to impaired pancreatic development.

Genome-wide DNA methylation pattern in systemic sclerosis microvascular endothelial cells: Identification of epigenetically affected key genes and pathways

Background: The etiology of systemic sclerosis is not clear, but there is evidence suggesting a critical role for epigenetic alterations in disease pathogenesis and clinical expression. We sought, in this study, to characterize the genome-wide DNA methylation signature in systemic sclerosis microvascular endothelial cells. Methods: We performed a genome-wide DNA methylation study in microvascular endothelial cells derived from seven diffuse cutaneous systemic sclerosis patients compared to seven age-, sex-, and ethnicity-matched healthy controls. We paired matched samples on Illumina HumanMethylation450 (three diffuse cutaneous systemic sclerosis microvascular endothelial cells and three controls), and reproduced the results in an independent set of matched patient and controls using Illumina Infinium MethylationEPIC (four diffuse cutaneous systemic sclerosis patients and four controls) to identify differentially methylated genes. Results: We identified 71,353 differentially methylated CpG sites in systemic sclerosis microvascular endothelial cells using Infinium MethylationEPIC microarray in the first group (0.081% of representative probes) and 33,170 CpG sites in the second group using HumanMethylation450 microarray (0.073% of representative probes) in diffuse cutaneous systemic sclerosis microvascular endothelial cells. Among the two groups of subjects, we identified differential methylation of 2455 CpG sites, representing 1301 genes. Most of the differentially methylated CpG sites were hypermethylated (1625 CpG), corresponding to 910 genes. Common hypermethylated genes in systemic sclerosis microvascular endothelial cells include NOS1, DNMT3A, DNMT3B, HDAC4, and ANGPT2. We also identified hypomethylation of IL17RA, CTNNA3, ICAM2, and SDK1 in systemic sclerosis microvascular endothelial cells. Furthermore, we demonstrate significant inverse correlation between DNA methylation status and gene expression in the majority of genes evaluated. Gene ontology analysis of hypermethylated genes demonstrated enrichment of genes involved in angiogenesis ( p = 0.0006). Pathway analysis of hypomethylated genes includes genes involved in vascular smooth muscle contraction ( p = 0.014) and adherens junctions ( p = 0.013). Conclusion: Our data suggest the presence of significant genome-wide DNA methylation aberrancies in systemic sclerosis microvascular endothelial cells, and identify novel affected genes and pathways in systemic sclerosis microvascular endothelial cells.

Alteration of mRNA 5-Methylcytosine Modification in Neurons After OGD/R and Potential Roles in Cell Stress Response and Apoptosis

Epigenetic modifications play an important role in central nervous system disorders. As a widespread posttranscriptional RNA modification, the role of the m5C modification in cerebral ischemia-reperfusion injury (IRI) remains poorly defined. Here, we successfully constructed a neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) model and obtained an overview of the transcriptome-wide m5C profiles using RNA-BS-seq. We discovered that the distribution of neuronal m5C modifications was highly conserved, significantly enriched in CG-rich regions and concentrated in the mRNA translation initiation regions. After OGD/R, modification level of m5C increased, whereas the number of methylated mRNA genes decreased. The amount of overlap of m5C sites with the binding sites of most RNA-binding proteins increased significantly, except for that of the RBM3-binding protein. Moreover, hypermethylated genes in neurons were significantly enriched in pathological processes, and the hub hypermethylated genes RPL8 and RPS9 identified by the protein-protein interaction network were significantly related to cerebral injury. Furthermore, the upregulated transcripts with hypermethylated modification were enriched in the processes involved in response to stress and regulation of apoptosis, and these processes were not identified in hypomethylated transcripts. In final, we verified that OGD/R induced neuronal apoptosis in vitro using TUNEL and western blot assays. Our study identified novel m5C mRNAs associated with ischemia-reperfusion in neurons, providing valuable perspectives for future studies on the role of the RNA methylation in cerebral IRI.

Increased methylation of gene promoters in the trophoblast of spontaneous miscarriages with trisomy 16

Обнаружен повышенный уровень нарушений эпигенетической регуляции экспрессии генов в трофобласте хориона спонтанных абортусов с трисомией 16. Среди гиперметилированных генов была значимо обогащена группа генов секретируемых белков, значительное количество генов кодировали рецепторы и транскрипционные факторы. Гиперметилирование выявленных генов является потенциальной причиной гибели эмбрионов с трисомией 16 на ранних стадиях развития. An increased level of abnormal epigenetic regulation of gene expression in the trophoblast of spontaneous miscarriages with trisomy 16 was found. Among the hypermethylated genes, the group of genes of secreted proteins was significantly enriched, and a significant number of genes encoded receptors and transcription factors. Hypermethylation of the identified genes is a potential cause of death of embryos with trisomy 16 in the early stages of development.

PATH-29. COMPARATIVE ANALYSIS OF DNA METHYLATION PROFILES ASSOCIATED WITH IDH-WILDTYPE GLIOMA AND GLIONEURONAL TUMOR SUBTYPES

DNA Methylation profiles are highly robust and reproducible as a classification tool. Less is understood regarding the methylation differences that exist among gliomas and glioneuronal tumors. To address this, we analyzed differentially methylated probes (DMPs) of gliomas and glioneuronal tumors compared to normal brain white matter controls. After filtering (Δbeta >0.3, logFC >±1) of significant probes we observed that low grade glioma/glioneuronal tumors (LGGs) had significantly fewer DMPs (Hyper/Hypo) as compared to GBMs. For example, posterior fossa pilocytic astrocytomas (PA’s) showed 2861 DMPs (1916 hypo/945 hyper) versus 9653 for GBM-RTKI ((6563/3090) respectively, while tumors such as PXA and anaplastic PA showed intermediate changes between LGG’s and GBMs. Hypomethylated and hypermethylated probes were analyzed for gene ontology and KEGG pathway enrichment, with LGG subtypes showing hypomethylated probes/genes associated with cell adhesion, blood vessel development and viral infection (P-value = 10-7). In contrast, hypomethylated probes in GBM subtypes were enriched for plasma membrane and cell periphery gene ontologies (P-value = 10-52). With respect to hypermethylated probes, LGG subtypes showed enrichment for myelination, glial cell differentiation and sphingolipid metabolism (P-value = 10-5) while DNA-binding transcription factor activity was seen in GBM subtypes (P-value=10-35). Examples of the most significantly hypermethylated genes in GBM included the transcription factors, GATA3 and PAX9. Intermediate-grade gliomas such as anaplastic PA and PXA showed enrichment of hypermethylated genes similar to GBM, but of lower significance (P-values = 10-6 and 10-4). Overall, understanding of cancer-associated DNA methylation changes in glioma subtypes suggests a hierarchy of biological changes that may underlie the pathogenesis of these tumors and interestingly, highlight tumor types such as PXA and anaplastic PA as having intermediate methylation changes, between benign LGG and GBM. Hypermethylation of transcription-factor genes will be investigated in GBM and compared with changes in gene expression to understand possible roles in the pathogenesis.

DNA methylation in cockroaches is essential in early embryo development and reduces gene expression noise

The influence of DNA methylation on gene behavior, and its consequent phenotypic effects appear to be very important, but the details are not well understood. Insects offer a diversity of DNA methylation modes, making them an excellent lineage for comparative analyses. However, functional studies have tended to focus on quite specialized holometabolan species, such as wasps, bees, beetles, and flies. Here we have studied DNA methylation in a hemimetabolan insect, the cockroach Blattella germanica, a model of early-branching insects. In this cockroach, one of the main genes responsible for DNA methylation, DNA methyltransferase 1 (DNMT1), is expressed in early embryogenesis. In our experiments, DNMT1 interference by RNAi reduces DNA methylation and impairs blastoderm formation. Using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic analyses, we observed that hypermethylated genes are associated with metabolism and are highly expressed, whereas hypomethylated genes are related to signaling and have low expression levels. Moreover, the expression change in hypermethylated genes is greter than that in hypomethylated genes, whereas hypermethylated genes have less expression variability than hypomethylated genes. The latter observation has also been reported for humans and in Arabidopsis plants. A reduction in expression noise may therefore be one of the few universal effects of DNA methylation.

P828 DNA methylation profile defines epigenetic characteristics and molecular targets of Crohn’s disease

Background Inflammatory bowel disease(IBD) is known to be caused by a genetic predisposition involving multiple genes; however, there is growing evidence that abnormal interaction with environmental, particularly epigenetic, factors can have a significant contribution during the development of IBD. Although many studies, particularly genome-wide association studies (GWAS), have been performed to identify the genetic changes underlying the pathogenesis of Crohn’s disease (CD), the role of epigenetic changes in the development of complications arising from CD is poorly understood. Methods Here, we employed an unbiased approach to define DNA methylation alteration in CD patients using the Human Methylation 450K Bead Chip platform. Compared to normal controls, the majority of differential DNA methylation in CD patient samples was in the promoter, intergenic, and gene body regions. Results The DNA methylation profile in CD revealed 134 probes (23 hypermethylated and 111 hypomethylated probes) that were differentially methylated. We validated the methylation levels of 19 genes that showed hypermethylation in CD patients compared with normal control. Technical validation was performed using quantitative MSP analysis and we finally identified that the Fragile Histidine Triad (FHIT) genes were hypermethylated in a disease-specific manner. Using a large cohort for CD patients samples (n = 207), we found that FHIT is frequently methylated in CD patients (71%) by MSP and significantly increasing methylation level in CD patient samples. In addition, we confirmed the methylation level of FHIT gene between normal colon and CD patients. Due to hypermethylation of FHIT gene promoter in CD patients, we observed that the level of FHIT protein is downregulated in CD patient samples compared with normal by IHC analysis. Gene network analysis by GO and metascape for hypermethylated genes in CD patients suggested putative cellular and molecular interactions relevant to IBD pathology. Conclusion Overall, our DNA methylation profile identifies newly hypermethylated genes in CD, as well as paves the way to a better understanding of the role of epigenetics in the pathogenesis of CD, and provides direction for future research in the diagnosis/prognosis or therapeutic treatments for CD.