Epigenetic mechanisms that enforce pluripotency in embryonic stem cells

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07025
Investigator: 
Award Value: 
$1,161,000
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Status: 
Active
Public Abstract: 

Embryonic stem (ES) cells have the unique ability to self-renew while maintaining a pluripotent state. They can readily be differentiated into all cell types upon exposure to the appropriate stimuli. The differentiation of ES cells into specialist cell types involves the activation of lineage-specific programs of gene expression and the silencing of genes that promote pluripotency. These changes are now well 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 how the 3D-structures of the ES cell genome change upon developmental progression into a fully committed cell type and during reprogramming. Thus, we are now faced with the fundamental question as to how the 3D-structures of human ES cell genomes differ from that of differentiated progeny and how such differences relate to the establishment and maintenance of pluripotency versus differentiation. This is the focus of the studies proposed in this application.

Statement of Benefit to California: 

Our studies would provide insights into the mechanisms that underpin the abilities of human embryonic stem cells to self renew and to differentiate into specific cell lineages. This research will serve as a foundation for understanding the basic properties of human embryonic stem cells and differentiated progeny. Understanding and modifying the properties of stem cells would directly impact novel approaches that are being developed to study stem cell models of many types of diseases and regenerative medicine. It would also permit the development of new avenues for the diagnosis and treatment of human disease and help to maintain the position of California as a leader in basic and applied biomedical research.

Progress Report: 

The development of novel experimental and computational approaches has made it possible to identify the spectrum of interacting genomic elements across the entire genome. Hence now it is feasible to assign specific enhancers to distinct promoters and to identify the ensemble of anchors associated with the folding pattern of the genome. During the past year we have identified the folding patterns of human embryonic stem cells to iPS cells derived from human B cells. Briefly, iPS cells derived from human B cells were generated as follows. Human B cells were isolated from human cord blood and transduced with viral vectors expressing Oct4, Sox2, KLF4 and c-Myc. From this population iPS cells were derived and expanded. Next the interactome of the iPS cells was determined and compared to that of human embryonic stem cells. Using an ensemble of computational strategies we found that the majority of genomic regions in iPS cells derived from human B cells was indistinguishable from that of human embryonic stem cells. However, we also found exceptions. Specifically, we found that approximately 700 genomic regions located throughout the genome showed differential nuclear positioning upon comparing iPS cells derived from human lymphoid cells to that of human embryonic stem cells. These data indicate that the majority of the genome derived from that of human embryonic stem cells shows a similar pattern in chromatin folding as compared to that of iPS cells derived from human B cells but that they differ from each other in a subset of genomic regions.

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.