Regulation of pluripotency and lineage decisions by histone methylation

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Embryonic stem cells (ESCs) provide an unrivalled venue to study the basic processes defining pluripotency and lineage specification, as well as the potential to harness this knowledge for emerging therapies in regenerative medicine. Recent progress with ESCs has identified three stages within the pluripotent window; namely Nanog positive ESCs (Nanog on), Nanog negative (Nanog off) and Epiblast stem cells (EpiSCs). Work by the applicants, supported during the first funding period, has identified linkages between these ESC transitions and histone 3 trimethylation at lysines 4 (H3K4me3) and 27 (H3K27me3), which are also hallmarks of the opposing regulatory systems based on gene activation/maintenance by the trithorax-Group (trx-G) and silencing by the Polycomb-Group (PcG) respectively. Unexpectedly, we recently found that H3K27 trimethylation of lineage specific genes is acquired during transition from Nanog on to Nanog off states. Furthermore many of the ectodermal lineage specific gene promoters appear to be specifically regulated by the trx-G factor, Mll2. In the proposed research, we will systematically examine and compare selected trx-G, PcG and certain other proteins for protein-protein interactions, genome binding sites and expression profiles after conditional mutagenesis, in the three pluripotent stages. These analyses, which have already begun, are focused on the two histone methyltransferases already known to be involved in H3K4 and H3K27 methylation (Mll2 and Ezh2 respectively). Using robust protein tagging, gene targeting and conditional mutagenesis strategies, together with mass spectrometry and deep sequencing methods, we aim to identify key aspects of the regulatory hierarchy of pluripotency.