Computational and experimental methods for deciphering the epigenetic code in hematopoietic stem cell lineage commitment

Abstract: The emergence of post-genomic approaches that couple chromatin immunoprecipitation (ChIP) assays to high-throughput technique has greatly advanced our ability to investigate epigenetic features associated with normal cell development and disease. Nevertheless, these techniques exhibit some limitations. Firstly, standard protocols usually require millions of cells, thus limiting their application to the study of abundant cell populations. Second, high-throughput techniques generate vast amounts of data, the handling and analysis of which pose several bioinformatic challenges to biomedical researchers. In this thesis we present a miniaturized ChIP-chip assay (miniChIP-chip) for the genome-wide analysis of chromatin modifications from 10,000 cells as well as software (ChIPeasy) for the visualization and analysis of ChIP-chip datasets. Employing the experimental and computational approaches described in these articles, we investigated the signatures of selected histone modifications in rare murine hematopoietic stem cells (HSCs) and progeny, the analysis of which had previously been limited due to low frequencies of purified HSCs in adult animals. The distinct histone modification maps generated in this study indicated that promoters of mature blood cell regulators were primed for lineage-specific expression by bivalent histone methylation marks (co-modification of H3K4me3 and H3K27me3) in HSCs and progenitors prior to lineage commitment. In HSCs, bivalent signatures at promoters associated differently with combinations of other histone modifications depending on the downstream resolution of the bivalent marks, suggesting that the fate of lineage-specific genes is partly established already at the stem cell level. During T cell maturation, gene expression programs that are important for other lineages acquired increased H3K27me3 and H3K9me3 silencing, but a large majority also retained the activating and bivalent chromatin states. This result suggested that a residual epigenetic plasticity or perhaps priming capacity might be retained in these mature cells. In summary, this study has provided insight into some of the unique epigenetic features of hematopoietic stem cell lineage commitment and differentiation.