Carolina Haefliger

DNA methylation profiling of human chromosomes 6, 20 and 22

DNA methylation is the most stable type of epigenetic modification modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of six annotation categories showed that evolutionarily conserved regions are the predominant sites for differential DNA methylation and that a core region surrounding the transcriptional start site is an informative surrogate for promoter methylation. We find that 17% of the 873 analyzed genes are differentially methylated in their 5′ UTRs and that about one-third of the differentially methylated 5′ UTRs are inversely correlated with transcription. Despite the fact that our study controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.

Identifying DNA Methylation Biomarkers of Cancer Drug Response

In the last few years, DNA methylation has become one of the most studied gene regulation mechanisms in carcinogenesis as a result of the cumulative evidence produced by the scientific community. Moreover, advances in the technologies that allow detection of DNA methylation in a variety of analytes have opened the possibility of developing methylation-based tests. A number of studies have provided evidence that specific methylation changes can alter the response to different therapeutic agents in cancer and, therefore, be useful biomarkers. For example, the association of the methylation status of DNA repair genes such as MGMT and MLH1 illustrate the two main mechanisms of response to DNA damaging agents. Loss of methylation of MGMT, and the subsequent increase in gene expression, leads to a reduction in response to alkylating agents as a result of enhanced repair of drug-induced DNA damage. Conversely, the increase in methylation of MLH1 and its resulting loss of expression has been consistently observed in drug-resistant tumor cells. MLH1 encodes a mismatch repair enzyme activated in response to DNA damage; activation of MLH1 also induces apoptosis of tumor cells, and thus loss of its expression leads to resistance to DNA-damaging agents. Other methylation-regulated genes that could serve as biomarkers in cancer therapy include drug transporters, genes involved in microtubule formation and stability, and genes related to hormonal therapy response. These methylation markers have potential applications for disease prognosis, treatment response prediction, and the development of novel treatment strategies.

IL-10 is excluded from the functional cytokine memory of human CD4+ memory T lymphocytes

Epigenetic modifications, including DNA methylation, profoundly influence gene expression of CD4+ Th-specific cells thereby shaping memory Th cell function. We demonstrate here a correlation between a lacking fixed potential of human memory Th cells to re-express the immunoregulatory cytokine gene IL10 and its DNA methylation status. Memory Th cells secreting IL-10 or IFN-γ were directly isolated ex vivo from peripheral blood of healthy volunteers, and the DNA methylation status of IL10 and IFNG was assessed. Limited difference in methylation was found for the IL10 gene locus in IL-10-secreting Th cells, as compared with Th cells not secreting IL-10 isolated directly ex vivo or from in vitro-established human Th1 and Th2 clones. In contrast, in IFN-γ+ memory Th cells the promoter of the IFNG gene was hypomethylated, as compared with IFN-γ-nonsecreting memory Th cells. In accordance with the lack of epigenetic memory, almost 90% of ex vivo-isolated IL-10-secreting Th cells lacked a functional memory for IL-10 re-expression after restimulation. Our data indicate that IL10 does not become epigenetically marked in human memory Th cells unlike effector cytokine genes such as IFNG. The exclusion of IL-10, but not effector cytokines, from the functional memory of human CD4+ T lymphocytes ex vivo may reflect the need for appropriate regulation of IL-10 secretion, due to its potent immunoregulatory potential.

DNA Methylation Analysis: Providing New Insight into Human Disease

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.

DNA Methylation Analysis

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.

Chapter 6 – DNA Methylation Analysis: Providing New Insight into Human Disease

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.

Methylation Analysis in Cancer

Aberrant DNA methylation is an early and common event in human cancers. Methylation acts as an epigenetic regulator of gene expression and is involved in cancer development as well as resistance to drug treatments. Specific methylation patterns have been shown for different cancer types and there is evidence that methylation can be used as a diagnostic tool. Several methods have been developed to study methylation on a genome wide basis. However they are labor intensive and can assess only a limited number of tissues at a time preventing the assessment of these genes in larger populations. Methylation microarrays now offer the possibility to validate these candidate genes statistically filling the gap between genome wide discovery methods and single gene assays which could be adjusted to routine clinical use. Here we show how all these methods can be combined to broaden our knowledge regarding DNA methylation and transform some of this information into powerful diagnostic tests.