Year 2

The developmental progression of embryonic stem cells into specialist cell types involves the activation of lineage-specific programs of gene expression and the silencing of genes involved in maintaining pluripotency. These changes are known to include epigenetic modifications such as DNA methylation and deposition of distinct histone marks across the genome. However, much remains to be learned as to how ES cells differ from differentiated progeny. In particular, it has remained unclear as to whether and how the 3D-structures of the ES cells differ from that of differentiated progeny and how such differences relate to function. The development of novel approaches has made it possible to address these fundamental questions. Specifically formaldehyde cross-linking approaches in conjunction with chromosome-capture studies, named HiC, have made it possible to identify the entire spectrum of interacting genomic elements. This analysis showed that the ES and pro-B cell genomes are organized as topological domains, consisting of clusters of loops, organized like beads-on-a-string. Here, we extended the approach to describe the genome topologies of differentiated human lymphoid and myeloid cells derived from human induced pluripotent stem cells. Specifically, we have generated differentiated human myeloid and DP thymocytes from induced pluripotent stem cells (iPS). Both myeloid cells as well as thymocytes were fixed and examined using in situ HiC analysis. We found that a large fraction of genes were differentially localized in human thymocytes derived from embryonic stem cells versus human myeloid cells. Prominent among the loci that showed differential localization was the Bcl11b gene. We plan to scale up the in situ HiC described here to chart the 3D-genomes of human lymphoid and myeloid cells in order to more to determine genomic regions that are differentially localized in the heterochromatic versus euchromatic compartments. We also would aim to describe in detail the 3D-architecture of lymphoid and myeloid genomes that are derived from human induced pluripotent stem cells.