DNA Demethylation, DNA Repair and Reprogramming

Figure 1. Model for repair-mediated DNA demethylation

Epigenetic gene regulation is of central importance for development and disease. In the DNA of metazoans, 5-methylcytosine (m5C) is a common epigenetic mark associated with gene silencing. DNA methylation plays important roles in gene expression, genomic imprinting, X-chromosome inactivation, genomic instability, embryonic development and cancer. The pattern of DNA methylation varies in different tissues and during embryonic development and little is known how this methylation pattern is regulated. DNA methylation patterns are mitotically and meiotically heritable due to maintenance methylation, which methylates the nascent strands of hemimethylated replicating DNA. Because of this heritability, DNA methylation is commonly perceived as a very stable modification. However, it is becoming increasingly clear that DNA methylation is a dynamic process, which can be reversed by an active (i.e. enzymatic) demethylation process.

Despite dramatic progress in epigenetics during the past decade, DNA demethylation remains one of the last big frontiers and very little is known about it. DNA demethylation is a widespread phenomenon and occurs in plants as well as in animals, during development, in the adult, and during somatic cell reprogramming of pluripotency genes. For example, cell reprogramming proceeds by reactivation of pluripotency genes. Part of this reactivation is DNA demethylation. The molecular identity of the DNA “demethylase” in animal cells remains poorly understood. We showed that Growth Arrest and DNA Damage 45a (Gadd45a) is a key player in active DNA demethylation, which opened new avenues in the study of this elusive process.

Gadd45 acts by recruiting DNA repair to sites of demethylation. Methylated cytosines are excised and replaced by unmethylated nucleotide, thereby leading to demethylation (Figure 1). This is a novel epigenetic mechanism of gene activation.

The goal of our research is to further analyse the mechanism of DNA demethylation as well as the role played by Gadd45 in development. In particular, our recent discovery of the interaction between TET DNA demethylases and Gadd45a in demethylation raises new questions. Which target genes are regulated by Gadd45a? What are the sequence determinants that recruit Gadd45a to specific genes during demethylation? What is the role of Gadd45a-mediated demethylation in mammals? 

To investigate these questions, we will analyse mouse embryonic stem cells (mESC) that are mutant for Gadd45a, b and g. We plan to monitor gene expression and DNA methylation in mESCs and analyse the differentiation capacity of Gadd45-mutant cells, as well as identifying Gadd45 target genes. To address Gadd45 function, we use biochemical, molecular biological and cell biological approaches, employing embryonic stem cells, the mouse and frog model systems. Given the many unresolved questions in this burgeoning field, our work promises to be ground-breaking and therefore have a profound impact in unravelling one of the least understood processes in epigenetic gene regulation.