5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

Maja Klug,Michael Rehli,Sandra Schmidhofer,Reinhard Andreesen,Claudia Gebhard

doi:10.1186/gb-2013-14-5-r46

Maja Klug, Michael Rehli + Show 3 more

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https://doi.org/10.1186/gb-2013-14-5-r46

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Abstract

BackgroundCytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. However, the exact mechanisms facilitating the erasure of DNA methylation remain unclear in many cases.ResultsWe previously established human post-proliferative monocytes as a model to study active DNA demethylation. We now show, for several previously identified genomic sites, that the loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hydroxymethylcytosine. Monocytes were found to express the methylcytosine dioxygenase Ten-Eleven Translocation (TET) 2, which is frequently mutated in myeloid malignancies. The siRNA-mediated knockdown of this enzyme in primary monocytes prevented active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes.ConclusionsThe work described here provides definite evidence that TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine initiates targeted, active DNA demethylation in a mature postmitotic myeloid cell type.

Highlights

Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements
Recent pioneering work has identified the family of TenEleven-Translocation proteins (TET1-3) that catalyze the conversion of 5- methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) in mammalian cells [4], and has prompted speculations that these enzymes are involved in DNA demethylation processes [5,6]
Using methyl-CpG-immunoprecipitation (MCIp) and a global microarray-based approach to detect differences in DNA methylation [21,22], we recently identified a number of actively demethylated regions in a natural setting of postmitotic cells: the differentiation of human peripheral blood monocytes into monocyte-derived macrophages or dendritic cells (Figure 1)

Summary

Introduction

Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. DNA methylation is a frequent epigenetic modification that restricts the activity of regulatory elements, including cell type-specific gene promoters and enhancers. The initial massive erasure of 5mC in primordial germ cells, appears to be a TET-independent, passive process that is likely controlled by the downregulation of UHRF1, which facilitates the recruitment of the maintenance DNA-methyltransferase DNMT1 to nascent hemimethylated DNA at the replication fork [10]. TET3 mediated conversion of 5mC to 5hmC is essential for the reprogramming of the zygotic paternal DNA after fertilization [11,12,13]. 5hmC is gradually replaced by unmethylated cytosines during preimplantation development, suggesting that the erasure of 5hmC in zygotes is a DNA replication-dependent passive process [12]

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