Mechanisms of cell fate acquisition in the differentiation of pluripotent stem cells

Mammalian development requires the creation of hundreds of cell types, each with distinct patterns of gene expression, all sharing the same genetic sequence. Epigenetic mechanisms, such as histone tail modification, are proposed to be critical for this process. The embryonic stem cell's pluripotency, the ability to differentiate into all cell types within the body, makes these cells an excellent model for studying epigenetic change during development. Understanding the histone tail modifications used to maintain and establish cell fate is critical for improving differentiation protocols, evaluating the potency of adult cells, and reprogramming adult cells to an embryonic stem cell-like state. Ultrastructurally, we noted that pluripotent human and mouse embryonic stem cells had scant cytoplasm and a large euchromatin-rich nucleus. Both acetylation and trimethylation of lysine 9 on histone H3 increased genome-wide upon the induction of differentiation. The brachyury T locus, a key transcription factor of mesendoderm, had bivalent chromatin modification in pluripotency, with trimethylation on lysine 4 and lysine 27 on histone H3. This bivalency resolved first to only lysine 4 trimethylation when the locus was actively producing transcript, and lysine 9 and lysine 27 trimethylation when the locus was silenced and never to be active again. In pluripotency or mesendoderm, approximately 20% of protein-coding loci are non-transcribed, 40% are transcriptionally initiating but not elongating and 40% are productively transcribing with initiation and elongation by RNA polymerase. Loci transitioning between transcriptional states are nearly exclusively (98-99% of loci changing state) transitioning to or from initiation without elongation, indicating that elongation and initiation are distinctly regulated steps during differentiation. Nuclear effectors of the canonical Wnt or TGF-beta signaling cascades are binding approximately 3000-6000 loci during this step of differentiation, with the co-binding of Groucho ( Wnt signaling) and coSmad4 (TGF-beta signaling) promoting the gain of initiation at a ten-times higher rate than the loss of initiation. Together these findings indicate a rich rote for transcriptional regulation, interactions with chromatin, extracellular signaling and a potential feed-forward mechanism are all involved in cell fate acquisition during differentiation of pluripotent cells