Control of the embryonic stem cell state

Abstract

Embryonic stem cells and induced pluripotent stem cells hold great promise for regenerative medicine. These cells can be propagated in culture in an undifferentiated state but can be induced to differentiate into specialized cell types. Moreover, these cells provide a powerful model system for studies of cellular identity and early mammalian development. Recent studies have provided insights into the transcriptional control of embryonic stem cell state, including the regulatory circuitry underlying pluripotency. These studies have, as a consequence, uncovered fundamental mechanisms that control mammalian gene expression, connect gene expression to chromosome structure, and contribute to human disease.

Figure 1. Models for transcriptionally active, poised and silent genes

Transcription factors, cofactors, chromatin regulators and ncRNA regulators can be found at active, poised and silent genes. At active genes, enhancers are typically bound by multiple transcription factors (TFs), which recruit cofactors that can interact with RNA polymerase II at the core promoter. RNA polymerase II generates a short transcript and pauses until pause-release factors and elongation factors allow further transcription. Chromatin regulators, which include nucleosome remodeling complexes such as Swi/Snf complexes and histone modifying enzymes such as TrxG, Dot1 and Set2, are recruited by transcription factors or the transcription apparatus and mobilize or modify local nucleosomes. Poised genes are rapidly activated when ESCs are stimulated to differentiate. At poised genes, transcription initiation and recruitment of TrxG can occur, but pause-release, elongation and recruitment of Dot1 and Set2 do not occur. The PcG and SetDB1 chromatin regulators can contribute to this repression, and these can be recruited by some transcription factors and by ncRNAs. The RNA polymerase II “ghost” in this model of poised genes reflects the low levels of the enzyme that are detected under steady-state conditions. Silent genes show little or no evidence of transcription initiation or elongation and are often occupied by chromatin regulators that methylate histone H3K9 and other residues. Some of these silent genes are probably silenced by mechanisms that depend on transcription of at least a portion of the gene (Buhler and Moazed, 2007; Grewal and Elgin, 2007; Zaratiegui et al., 2007).

Figure 2. Core regulatory circuitry

Oct4, Sox2 and Nanog collaborate to regulate their own promoters, forming an interconnected autoregulatory loop. The Pou5f (Oct4), Sox2 and Nanog genes are represented as red boxes and proteins as blue balloons. These core transcription factors (O/S/N) function to activate expression of protein-coding and miRNA genes necessary to maintain ESC state, but they also occupy poised genes encoding lineage-specific protein and miRNA regulators whose repression is essential to maintaining that state. Additional transcription factors, such as the c-Myc/Max heterodimer (M/M), cause pause release at actively transcribed genes. A subset of the cofactors and chromatin regulators implicated in control of ES cell state (Table 1) are shown.

Figure 3. Relationships between core and other transcription factors in regulatory circuitry and gene control

A) Overlap between actively transcribed genes occupied by core transcription factors (union of Oct4, Sox2 and Nanog bound genes) and those of occupied by c-Myc. Active genes were defined as the set of genes occupied by both RNA polymerase II and nucleosomes with histone H3K79me3. B) Frequency distribution showing how c-Myc, Tcf3, Smad1, STAT3, Esrrb, Tbx3, Zfx, Ronin and Klf4 are associated with Oct4/Sox2/Nanog-occupied loci. Oct4, Sox2 and Nanog are the three transcription factors in the first bin. Binding was called at a high confidence (p<10⁻⁹) threshold within a 50bp window, so the actual number of factors bound to Oct4/Sox2/Nanog-occupied loci is somewhat higher. C) Frequency distribution showing how often c-Myc, Tcf3, Smad1, STAT3, Esrrb, Tbx3, Zfx, Ronin and Klf4 are associated with Oct4/Sox2/Nanog-occupied genes (p<10⁻⁹). D) Gene tracks showing example of an actively transcribed gene (Max) occupied by an Oct4/Sox2/Nanog enhancer and other transcription factors implicated in ESC control. ChIP-Seq data was obtained from GSE11431, GSE11724, GSE12680 and GSE22557.

Figure 4. Signaling to core regulatory circuitry

A) Model of an enhancer where transcription factors associated with Wnt, LIF and BMP4 signaling (STAT3, Tcf3 and Smad1) occupy sites near the core regulators. B) Oct4 distal enhancer provides an example of a DNA element that is bound by the core regulators and signaling transcription factors and contains sequence motifs for each of these factors. C) Frequency distribution showing how often signaling transcription factors (STAT3, Tcf3 and Smad1) are associated with Oct4/Sox2/Nanog-bound loci throughout genome. Binding was called at a high confidence (p<10⁻⁹) threshold within a 50bp window, so the actual number of signaling transcription factors bound to Oct4/Sox2/Nanog-occupied loci is somewhat higher. ChIP-Seq data was obtained from GSE11431, GSE11724, GSE12680 and GSE22557.

Figure 5. Mediator and cohesin contribute to gene control in core circuitry

A) ChIP-Seq data at the Pou5f gene for transcription factors, mediator and cohesin, and the transcription apparatus (Pol2 and TBP). Note evidence for crosslinking of most components to both enhancer elements and core promoter. The numbers on the Y-axis are reads/million. ChIP-Seq data was obtained from GSE11431, GSE11724, GSE12680 and GSE22557. B) Model for DNA looping by mediator and cohesin. Oct4, Sox2 and Nanog bind mediator, which binds RNA polymerase II at the core promoter, thus forming a loop between the enhancer and the core promoter. The transcription activator-bound form of mediator binds the cohesin loading factor Nipbl, which provides a means to load cohesin. Both mediator and cohesin are necessary for normal gene activity. This model contains a single DNA loop, but multiple enhancer may be bound simultaneously, generating multiple loops.

Figure 6. ESC core regulatory circuitry and differentiation

A) This model of core regulatory circuitry incorporates selected protein-coding and miRNA target genes. Oct4, Sox2 and Nanog directly activate transcription of genes whose products include the spectrum of transcription factors, cofactors, chromatin regulators and miRNAs that are known to contribute to ESC state. Oct4, Sox2 and Nanog are also associated with SetDB1 and PcG-repressed protein-coding and miRNA genes that are poised for differentiation. B) The loss of ESC state during differentiation involves the silencing of the Oct4 gene, the proteolytic destruction of Nanog by caspase-3, and miRNA-mediated reduction in Oct4, Nanog and Sox2 mRNA levels.