Tet2-dependent processing of 5-methylcytosine and the maintenance of active higher order structures during differentiation.

• Applicant: Dr. Achim Breiling
• Subject Area: General Genetics and Functional Genome Biology
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 233693164

Lineage specific differentiation in the developing embryo is accompanied by extensive reprogramming of the epigenome, resulting in the repression of stemness specific factors, regulators of other lineages and the transcriptional maintenance of activated lineage-specific genes. A well studied developmental gene array is the mammalian Hoxa cluster which is inactive in undifferentiated pluripotent cells but activated during development in a lineage-specific, spatiotemporally regulated way. The repressed cluster is characterised by the presence of specific bivalent chromatin structures and several methylated CpG-rich regions. Using the human HOXA cluster as a model system to follow changes in DNA modification patterns during retinoic acid induced differentiation, we have previously described that the activated anterior part of the cluster becomes increasingly enriched in 5-hydroxymethylcytosine (5hmC), which is paralleled by the reduction of 5-methylcytosine (5mC). Interestingly, this 5mC-5hmC conversion followed closely the colinear activation pattern of the cluster. 5mC can be converted to 5hmC by proteins of the Ten-Eleven-Translocation (TET) family, which either maintains activated transcription or acts as intermediate for demethylation pathways. By depleting TET2 in pluripotent embryonal carcinoma cells or by analysing tissue samples from Tet2-/- mice, we found the maintenance of activated Hoxa transcription significantly disturbed. Our data suggest that gene specific 5mC-5hmC conversion by Tet2 is crucial for the maintenance of active chromatin states at lineage-specific loci.In the experiments described in this proposal we will further study the role of 5hmC during differentiation, in particular potential processing pathways that could lead to unmethylated cytosines. A major task will also be to understand the reason for the tissue specific differences of 5hmC and Tet levels observed in mammals. Therefore, we will analyse if 5hmC is further processed during RA-induced differentiation of human embryonic carcinoma cells and in several differentiated mouse tissues. In order to better understand the mechanistic role of the 5mC-5hmC conversion for Hoxa maintenance, we will further analyse if maintained Hoxa expression during mouse haematopoiesis and differentiation specific changes of higher order structures in the cluster depend on the processing of 5hmC by Tet2. Taken together, the results of the experiments proposed will show if long term maintenance of active states of developmental selector genes depends on Tet-mediated conversion of 5mC to 5hmC, which might serve an independent epigenetic mark or represent an intermediate for further processing steps leading to demethylation.

• DFG Programme: Research Grants