Unraveling Epigenetic Regulation in Embryonic Stem Cells

Embryonic stem (ES) cells can replicate indefinitely while retaining the capacity to differentiate into functionally distinct cell types. ES cells proliferate and differentiate without detectable genetic changes, indicating that these processes are controlled by epigenetic factors. Here we describe what is known about the epigenetics of ES cells and speculate that a dynamic balance among at least three epigenetic elements (chromatin structure, DNA methylation, and microRNAs), in conjunction with transcription factors, contributes to the maintenance of pluripotence. Understanding the interactions among these factors will be critical to the development of improved strategies to reprogram differentiated cells or direct differentiation of pluripotent cells

Mre11 Protein Complex Prevents Double-Strand Break Accumulation during Chromosomal DNA Replication

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A comprehensive microarray-based DNA methylation study of 367 hematological neoplasms

Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs

Epigenomic profiling of prostate cancer identifies differentially methylated genes in TMPRSS2:ERG fusion-positive versus fusion-negative tumors

Background: About half of all prostate cancers harbor the TMPRSS2:ERG (T2E) gene fusion. While T2E-positive and T2E-negative tumors represent specific molecular subtypes of prostate cancer (PCa), previous studies have not yet comprehensively investigated how these tumor subtypes differ at the epigenetic level. We therefore investigated epigenome-wide DNA methylation profiles of PCa stratified by T2E status. Results: The study included 496 patients with clinically localized PCa who had a radical prostatectomy as primary treatment for PCa. Fluorescence in situ hybridization (FISH) "break-apart" assays were used to determine tumor T2E- fusion status, which showed that 266 patients (53.6 %) had T2E-positive PCa. The study showed global DNA methylation differences between tumor subtypes. A large number of differentially methylated CpG sites were identified (false-discovery rate [FDR] Q-value Conclusions: This study identified substantial differences in DNA methylation profiles of T2E-positive and T2E-negative tumors, thereby providing further evidence that different underlying oncogenic pathways characterize these molecular subtypes

Application of a low cost array-based technique — TAB-Array — for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells

Abstract5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at base resolution. Implementing this approach, termed “TAB-array”, we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular progenitors and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we identified a large number of novel dynamically methylated genomic regions that are implicated in the development of these lineages. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease

Array-based DNA methylation profiling of primary lymphomas of the central nervous system

<p>Abstract</p> <p>Background</p> <p>Although primary lymphomas of the central nervous system (PCNSL) and extracerebral diffuse large B-cell lymphoma (DLBCL) cannot be distinguished histologically, it is still a matter of debate whether PCNSL differ from systemic DLBCL with respect to their molecular features and pathogenesis. Analysis of the DNA methylation pattern might provide further data distinguishing these entities at a molecular level.</p> <p>Methods</p> <p>Using an array-based technology we have assessed the DNA methylation status of 1,505 individual CpG loci in five PCNSL and compared the results to DNA methylation profiles of 49 DLBCL and ten hematopoietic controls.</p> <p>Results</p> <p>We identified 194 genes differentially methylated between PCNSL and normal controls. Interestingly, Polycomb target genes and genes with promoters showing a high CpG content were significantly enriched in the group of genes hypermethylated in PCNSL. However, PCNSL and systemic DLBCL did not differ in their methylation pattern.</p> <p>Conclusions</p> <p>Based on the data presented here, PCNSL and DLBCL do not differ in their DNA methylation pattern. Thus, DNA methylation analysis does not support a separation of PCNSL and DLBCL into individual entities. However, PCNSL and DLBCL differ in their DNA methylation pattern from non- malignant controls.</p

Methylation Markers of Early-Stage Non-Small Cell Lung Cancer

Despite of intense research in early cancer detection, there is a lack of biomarkers for the reliable detection of malignant tumors, including non-small cell lung cancer (NSCLC). DNA methylation changes are common and relatively stable in various types of cancers, and may be used as diagnostic or prognostic biomarkers.We performed DNA methylation profiling of samples from 48 patients with stage I NSCLC and 18 matching cancer-free lung samples using microarrays that cover the promoter regions of more than 14,500 genes. We correlated DNA methylation changes with gene expression levels and performed survival analysis.We observed hypermethylation of 496 CpGs in 379 genes and hypomethylation of 373 CpGs in 335 genes in NSCLC. Compared to adenocarcinoma samples, squamous cell carcinoma samples had 263 CpGs in 223 hypermethylated genes and 513 CpGs in 436 hypomethylated genes. 378 of 869 (43.5%) CpG sites discriminating the NSCLC and control samples showed an inverse correlation between CpG site methylation and gene expression levels. As a result of a survival analysis, we found 10 CpGs in 10 genes, in which the methylation level differs in different survival groups.We have identified a set of genes with altered methylation in NSCLC and found that a minority of them showed an inverse correlation with gene expression levels. We also found a set of genes that associated with the survival of the patients. These newly-identified marker candidates for the molecular screening of NSCLC will need further analysis in order to determine their clinical utility

A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1 that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs

Differential Allelic Expression in the Human Genome: A Robust Approach To Identify Genetic and Epigenetic Cis-Acting Mechanisms Regulating Gene Expression

The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60∶40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms

Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology