Project Details
Projekt
Molecular mechanisms of Tet1 and Tet2 mediated DNA demethylation
Applicant
Dr. Tomasz Jurkowski
Subject Area
Biochemistry
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277430654
Almost all living organisms contain modified nucleotides in their genomes. These DNA modifications are part of epigenetic regulatory mechanisms that in mammals are crucial for proper embryonic development, regulation of gene expression, genomic imprinting, genome stability and maintenance of cellular differentiation state. Whereas the processes of genomic DNA methylation and the responsible enzymes are fairly well investigated, the processes of active DNA demethylation are not. In 2009, a family of tumor suppressor genes (comprising TET1, TET2 and TET3 in mammals) has been identified that oxidizes the methyl groups of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products, the 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), which are involved in active DNA demethylation. The discovery of TET enzymes has started a revolution in the DNA modification field, as it uncovered an active enzymatic DNA demethylation pathway and a set of novel modified DNA bases carrying new epigenetic information. Tet1 and Tet2 are the main enzymes responsible for the introduction of 5hmC and higher 5mC oxidation products in embryonic stem cells (ES cells) and primordial germ cells (PGCs), where they are essential for erasure of parental imprinting.Despite significant progress in uncovering the distribution and biological function of the novel DNA modifications, the molecular and mechanistic understanding of the biochemical properties of TET enzymes underlying their genome targeting, sequence specificity and regulation of activity are poorly investigated.Therefore, the general aim of this project is to elucidate the molecular principles governing the genomic targeting of Tet1 and Tet2, as well as the regulation of their activity, and to relate these biochemical properties to the different biological functions of these enzymes in cells. In particular, we plan to use a mixture of in vitro assays (activity assays and TAB sequencing with recombinant Tet enzymes) and cell based methods (ChIP-seq and live cell imaging) to investigate the sequence and modification state preference of Tet1 and Tet2 catalytic domains, as well as the CXXC domain of Tet1 and the IDAX protein. Furthermore, to understand the interplay and contribution of selected post-translational modifications and protein partners on Tet regulation, we will examine their effects on Tet1 activity and genomic location.Successful completion of this project will provide important insights on the biochemical properties of Tet enzymes and shed light on the molecular mechanisms of active DNA demethylation in ES and PGC cells.
DFG Programme
Priority Programmes
