Pluripotency is the remarkable ability of a single cell to give rise to every cell type of the mammalian body plan. Pluripotent cells exist in epiblast of early implantation embryos. There are two well-described pluripotent cell types, those of the early versus late epiblast that can be modeled in vitro as nave embryonic stem cells versus primed epiblast cells respectively. These cells differ minimally in terms of their expression profiles, yet vastly in terms of their epigenomes. In particularly, largely distinct enhancers drive expression of the same genes in the two states. The reason for the extensive enhancer rewiring in the absence of gene expression changes is unknown, but appears to be a critical aspect of early mammalian development. Preliminary results begin to address this problem by following the function of a single transcription factor Grhl2. Grhl2 is upregulated during embryonic stem to epiblast cell transition and is able to induce previously latent enhancers to a fully active state driving expression of proximal genes. Yet, these genes do not change expression during the transition. Evaluation of potential enhancers regulating the same genes in the embryonic stem cells uncovered the Klf2/4/5- related transcription factors as likely regulators of the genes in the nave state. Indeed Grhl2 is upregulated just as Klf2/4/5 is downregulated. However, Klf2/4/5 regulates a much larger network of genes in the naive state than Grhl2 does in the primed state. Therefore, it appears that Grhl2 assumes control of a subset of Klf2/4/5 targets during the transition and that other transcription factors must assume control of other parts of the very large Klf2/4/5 network. These findings led to the hypothesis that during the early to late epiblast transition, large nave regulatory networks are broken down into much smaller primed regulatory networks, providing the late epiblast cells the flexibility to differentiate down the divergent somatic lineages that form at gastrulation, immediately following the late epiblast stage. Then each of the smaller networks can be selectively maintained among the different lineages. Indeed, the Grhl2 network is excluded from the primitive streak while remaining expressed in the surrounding epiblast cells. To test the hypothesis, there are three specific aims.
In aim 1, cutting edge technologies are used to identify all Klf2/4/5 and Grhl2 driven enhancer promoter interactions in the embryonic stem and epiblast cell states respectively in order to directly determine whether Grhl2 results in the rewiring of enhancer-promoter interactions among a subset of Klf2/4/5 targets.
In aim 2, bioinformatics and novel biochemical methods are used to uncover additional epiblast cell transcription factors that rewire other subsets of the Klf2/4/5 driven network.
In aim 3, single cell sequencing of wild-type and knockout embryos is used to explore the biological role for enhancer rewiring in vivo. Successful completion of this project will be highly significant as it will uncover novel paradigms of gene control that regulate cell fate and a cell?s unique development potential.
The proposed research is relevant to public health as it addresses the fundamental question of how enhancers and rewired during cellular differentiation to enable to changing developmental potential of stem cells. New knowledge gained through this application will advance the fields of therapeutic manipulation of transcription factors and their target enhancers in treatment of disease and cell reprogramming. Therefore, the research is relevant to NIH?s mission to foster fundamental creative discoveries that increase the nation's capacity to protect and improve human health.
|Freimer, Jacob W; Hu, T J; Blelloch, Robert (2018) Decoupling the impact of microRNAs on translational repression versus RNA degradation in embryonic stem cells. Elife 7:|