Mapping of histone modifications in totipotent and pluripotent cells in vivo and characterization of a novel epigenetic mark

This project focuses on revealing the epigenetic basis of in vivo pluripotency and totipotency, characterizing a putative novel epigenetic mark with relevance to pluripotency and the characterization of candidate enzymes for the regulation of this mark. Fertilization produces a totipotent zygote which can give rise to all cells in an organism, embryonic and extraembryonic. Resulting pluripotent cells can be found in the inner cell mass (ICM). On the path from fertilization to an ICM, massive changes in DNA methylation and histone modifications have been uncovered by immunofluorescence based studies, revealing global patterns. However, the gene specific role of in particular histone modifications in this reprogramming of the genome remains nearly unexplo red due to the requirement for large cell numbers in conventional chromatin immunoprecipitation (ChIP) studies. Therefore, the increasing amount of literature on epigenetics in pluripotency relies primarily on in vitro pluripotent cell types such as embry onic stem (ES) cells, although this model cell type is only at best an approximate of its in vivo "counterpart". We have developed an improved ChIP assay to facilitate genome-wide surveys of limiting cell types. This method, together with the remarkable a dvance in ultra-deep sequencing technology, opens for extensive investigation on histone modifications in in vivo totipotency and pluripotency. Furthermore, we describe an innovative strategy for genome-wide studies of a putative, novel epigenetic mark, 5 -hydroxymethylcytosine (5hmC), identified recently. The association of 5hmC with distinct histone modifications and histone variants will also be studied. Moreover, identification of an enzymatic activity that cleaves 5hmC containing DNA and a protein tha t specifically interacts with 5hmC containing DNA has encouraged further work.