H3K27 Demethylation in Hematopoietic Stem Cells

Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called hematopoietic stem cells (HSCs). HSCs are largely residing in the long bones and are the very ancestral cells to every blood cell in adult vertebrates. The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an HSC itself (self-renews) to maintain the HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells. Most cell fate decisions are executed through the action of transcription factors (e.g. GATA-1, PU.1), and there is a comprehensive understanding of the genetic programs enforced by these factors during differentiation. However, it is still largely unexplored which upstream factors may regulate the expression of these lineage-determining transcription factors. The family of polycomb group proteins (PcG) have been implicated in the regulation of cell proliferation, genomic imprinting, X inactivation, and - most interestingly - in maintenance of stem cell identity via epigenetic suppression of differentiation promoting cues (e.g. lineage-determining transcription factors). The polycomb repressive complex 2 (PRC2) catalyzes the addition of methyl groups onto lysine 27 residues of histone H3 (H3K27). H3K27 trimethylation leads to the recruitment of polycomb repressive complex 1 (PRC1) resulting in permanent repression of the respective gene. Until recently it was believed that H3K27 trimethylation was a permanent epigenetic mark, only removable via histone eviction. However, the recent identification of the H3K27 demethylases (HDMs) Utx (Ubiquitously transcribed tetratricopeptide repeat gene, X chromosome) and Jmjd3 (Jumonji domain containing 3) introduced an unexpected flexibility in terms of H3K27 trimethylation mediated gene suppression. Given that H3K27 trimethylation can be removed in an enzymatically directed manner and the well-documented importance of epigenetic regulation of gene transcription during differentiation, it is appealing to deliberate whether H3K27 HDMs might be the very molecules regulating lineage-decisions upstream of the well-known lineage-specific transcription factors. This proposal aims to address the potential role of H3K27 histone demethylation during hematopoietic ontogeny and differentiation to identify epigenetic mechanisms operating on top of well-known lineage-determining transcription factors during hematopoietic differentiation. This will enhance our understanding of how lineage decisions during hematopoiesis are made, and - in addition - may allow the identification of yet unknown cell fate determining factors. Therefore, the applicant strives to 1.) generate conditional knockout mice of the histone demethylases Utx and Jmjd3; 2.) analyze their hematopoietic-specific deletion phenotypes during ontogeny and differentiation; and 3.) develop a method of highly sensitive chromatin immunoprecipitation from as few as 1000 cells to enable whole genome methylation mapping of HSCs and their progeny.