Polycomb Determines Responses to Smad2/3 Signaling in Embryonic Stem Cell Differentiation and in Reprogramming

Abstract

Integration of extrinsic signals, epigenetic regulators, and intrinsic transcription factors establishes pluripotent stem cell identity. Interplay between these components also underlies the capacity of stem cells to undergo differentiation, and of differentiated cells to re-establish the pluripotent state in direct reprogramming. Polycomb repressive complexes are epigenetic regulators that play key roles in stem cell identity and in differentiated cell fates. Smad2 and Smad3 (Smad2/3), the intracellular mediators of the Nodal/ Activin/transforming growth factor (TGF) b cell-cell signaling pathway also are implicated in stem cell pluripotency and in differentiation. Here, we show that Polycomb imposes responses to Smad2/3-mediated signaling to selectively regulate expression of the master pluripotency factor Oct4 during initiation of differentiation, but not in the selfrenewing pluripotent ground state. During reprogramming back to the ground state, we find that the enhancement of reprogramming efficiency stemming from blocking Nodal/ Activin/TGFb signaling also depends on Polycomb. These context-dependent responses to Smad2/3 imposed by Polycomb action provide a mechanism for selective gene regulation that can reconcile the apparently conflicting roles of this signaling pathway in pluripotency, differentiation, and reprogramming.

Inhibiting Smad2/3 signaling in pluripotent mouse embryonic stem cells enhances endoderm formation by increasing transcriptional priming of lineage‐specifying target genes

“…Although the degree of Prdm14 overexpression in that study was not specified, it is likely to be far higher than that induced by blocking Smad2/3 signaling. Indeed, we had not previously noticed any change in the ability of ALKi treated ESCs to differentiate (Dahle & Kuehn 2013). However, to confirm this under the specific conditions used here, and to examine the capacity for ESCs treated with ALKi in the pluripotent state to undergo endodermal differentiation, we removed LIF and discontinued ALKi treatment at the same time.…”

“…We have recently defined a novel paradigm for this pathway: activated Smad2/3 recruits the Jumonji C domain-containing histone demethylase Jmjd3 to remove the repressive H3K27me3 modification deposited by PRC2, thereby allowing target gene expression. We have found this mechanism in the differentiation of mouse ESCs and in reprogramming of mouse fibroblasts (Dahle et al 2010; Dahle & Kuehn 2013), while it was reported independently in the differentiation of human ESCs (hESCs) into endoderm (Kim et al 2011). In undifferentiated ESCs, blocking Smad2/3 signaling leads to an increase in H3K27me3 levels at the Oct4 locus, encoding one of the key pluripotency transcription factors.…”

“…1B, P ¼ 0.04). We next checked expression, expecting either no change as we previously found for Oct4 (Dahle and Kuehn, 2013), or a decrease given that Polycomb deposition of H3K27me3 is normally associated with repression. Surprisingly, we found Prdm14 expression increased approximately 2.5-fold with ALKi treatment (Fig.…”

“…In undifferentiated ESCs, blocking Smad2/3 signaling leads to an increase in H3K27me3 levels at the Oct4 locus, encoding one of the key pluripotency transcription factors. However, a decrease in Oct4 gene expression is only found once cells begin to differentiate, suggesting that the full elaboration of this signaling paradigm occurs only after cells exit the pluripotent state (Dahle & Kuehn 2013). This lack of effect on target gene expression prior to differentiation could be related to distinct regulatory functions for PRC2 in pluripotent cells (Aloia et al 2013).…”

Dynamic Protein Interactions of the Polycomb Repressive Complex 2 during Differentiation of Pluripotent Cells

“…2B, 3). Intracellular signaling via the SMAD pathway has been linked in several studies to Polycomb regulation of differentiation, but to our knowledge this is the first report of a physical interaction of an Smad protein with the PRC2 complex (29,30).…”