Reversing DNA methylation by heterokaryon-mediated reprogramming

• Main Author: Piccolo, Francesco M.
• Other Authors: Fisher, Amanda
• Published: Imperial College London 2013
• Subjects: 610
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616802

Genomic imprinting directs the allele‐specific expression of a subset of loci according to their parental origin. Monoallelic expression of these genes is regulated by imprinting control regions (ICRs) and is established in the embryonic germ line through differential DNA methylation. Differentiated cells can be reprogrammed to pluripotency by several strategies including the ectopic expression of specific ‘inducers’ and by transfer of nuclei into enucleated eggs. Cellular fusion of somatic cells with a pluripotent stem cell partner can also lead to dominant pluripotent reprogramming. Although ES cells (derived from the inner cell mass) and embryonic germ cells (EG, derived from primordial germ cells) can both reprogram, EG cells are unique in being able to erase genomic imprints from the somatic partner. In order to characterize the earliest events in successful reprogramming, as well as EG‐specific DNA demethylation, I generated experimental heterokaryons between B‐lymphocytes and mouse stem cell lines. I showed that ES cells that lack Polycomb Repressor Complex 2 (PRC2) failed to reprogram B cells and were unable to induce two early events that characterise successful B cell reprogramming; a global redistribution of HP1α and an increased serine 10 phosphorylation. In the second part of my study I confirmed that EG cells were able to induce DNA demethylation at several ICR in B cells following fusion. I present evidence that this reprogramming of the somatic genome requires Tet1 and Tet2 and is accomplished through a two‐step process involving both DNA synthesis and conversion of 5methylcytosine into 5hydroxymethylcytosine.