DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference 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 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methylation and a core region surrounding the transcriptional start site as informative surrogate for promoter methylation. We find 17% of the 873 analyzed genes differentially methylated in their 5′-untranslated regions (5′-UTR) and about one third of the differentially methylated 5′-UTRs to be inversely correlated with transcription. While our study was 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.

Deciphering the information encoded in the human genome is key for the further understanding of human biology, physiology and evolution. With the draft sequence of the human genome completed, elucidation of the epigenetic information layer of the human genome becomes accessible. Epigenetic mechanisms are mediated by either chemical modifications of the DNA itself or by modifications of proteins that are closely associated with DNA. Defects of the epigenetic regulation involved in processes such as imprinting, X chromosome inactivation, transcriptional control of genes, as well as mutations affecting DNA methylation enzymes, contribute fundamentally to the etiology of many human diseases. Headed by the Human Epigenome Consortium, the Human Epigenome Project is a joint effort by an international collaboration that aims to identify, catalog and interpret genome-wide DNA methylation patterns of all human genes in all major tissues. Methylation variable positions are thought to reflect gene activity, tissue type and disease state, and are useful epigenetic markers revealing the dynamic state of the genome. Like single nucleotide polymorphisms, methylation variable positions will greatly advance our ability to elucidate and diagnose the molecular basis of human diseases.

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.

Cytokine memory for IFN-γ production by effector/memory Th1 cells plays a key role in both protective and pathological immune responses. To understand the epigenetic mechanism determining the ontogeny of effector/memory Th1 cells characterized by stable effector functions, we identified a T-cell-specific methylation pattern at the IFNG promoter and CNS-1 in ex vivo effector/memory Th1 cells, and investigated methylation dynamics of these regions during the development of effector/memory Th1 cells. During Th1 differentiation, demethylation occurred at both the promoter and CNS-1 regions of IFNG as early as 16 h, and this process was independent of cell proliferation and DNA synthesis. Using an IFN-γ capture assay, we found early IFN-γ-producing cells from 2-day differentiating cultures acquired "permissive" levels of demethylation and developed into effector/memory Th1 cells undergoing progressive demethylation at the IFNG promoter and CNS-1 when induced by IL-12. Methylation levels of these regions in effector/memory Th1 cells of peripheral blood from rheumatoid arthritis patients correlated inversely with reduced frequencies of IFN-γ-producers, coincident with recruitment of effector/memory Th1 cells to the site of inflammation. Thus, after termination of TCR stimulation, IL-12 signaling potentiates the stable functional IFN-γ memory in effector/memory Th1 cells characterized by hypomethylation at the IFNG promoter and CNS-1.Keywords: Demethylation r Human IFNG gene r IFN-γ cytokine memory r Promoter and CNS-1 r Th1 cell differentiation Additional supporting information may be found in the online version of this article at the publisher's web-site 794Jun Dong et al. Eur. J. Immunol. 2013. 43: 793-804 Introduction A unique feature of adaptive immunity is the generation of effector/memory T cells after primary activation that control pathogens and mediate effective protection during secondary challenges [1]. Depending on the stimuli and cytokine environment experienced during activation, naive T cells make decisions to proliferate and differentiate into Th1, Th2, or Th17 lineages [2]. When challenged by intracellular pathogens, naïve CD4 + T cells can adopt a proinflammatory cell fate and differentiate into effector and memory Th1 cells, which are characterized by the production of the signature cytokine IFN-γ. Th1 cells play a key role in intracellular pathogen killing and exert proinflammatory effects in organspecific autoimmune diseases [3]. During primary activation, the expression of the Ifng/IFNG gene in naïve CD4 + T cells is initiated by TCR signaling, in conjunction with instructive IL-12 signaling through lineage-specific transcription factors (such as T-bet and the IL-12-responsive transcription factor STAT4, respectively) [4][5][6]. After termination of antigen stimulation, IL-12 also controls Ifng reactivation in effector/memory Th1 cells through STAT4 and T-bet [7]. During secondary activation, TCR signaling in the absence of the original instructive signal is sufficient to trigger rapid Ifng/I...

Cell therapeutic approaches currently lack definitive quality control measures which guarantee safety in clinical applications and create consistent standards for regulatory approval. These approaches rely on isolation, purification and possibly ex vivo manipulation of donor cells. Since such cells are exposed to artificial environments, there is potential for deviations from natural growth processes. The resulting heterogeneity of cell cultures is an inherent problem. Therefore, verification of cell identity and quantification of subpopulations is mandatory. Focusing on cultured human primary cells, we tested whether DNA methylation patterns serve as distinctive cell type markers. We identified panels of cell type specific differentially methylated gene regions (CDMs) which produce unambiguous profiles for these cell types. Applying methylation sensitive single nucleotide primer extension generated binary cell type descriptors ("barcodes") which allow quantification of cell mixtures. Thus, methylation based analytics suggest themselves as promising tools for the characterization and quality control of ex vivo manipulated cells.