Epigenetic control of transcriptional regulation in pluripotency and early differentiation

Abstract

Pluripotent stem cells give rise to all cells of the adult organism, making them an invaluable tool in regenerative medicine. In response to differentiation cues, they can activate markedly distinct lineage-specific gene networks while turning off or rewiring pluripotency networks. Recent innovations in chromatin and nuclear structure analyses combined with classical genetics have led to novel insights into the transcriptional and epigenetic mechanisms underlying these networks. Here, we review these findings in relation to their impact on the maintenance of and exit from pluripotency and highlight the many factors that drive these processes, including histone modifying enzymes, DNA methylation and demethylation, nucleosome remodeling complexes and transcription factor-mediated enhancer switching.

Keywords: Embryonic stem cells; Epiblast-like cells; Epigenetics; Mouse; Pluripotency; Transcriptional regulation.

Fig. 1.

States of pluripotency. Embryonic stem cells transition through distinct states of pluripotency (naïve, formative) before they undergo lineage commitment (and then gastrulation). This transition is accompanied by various changes in chromatin, including gain of bivalent domains and global DNA methylation, global gain of H3K9 dimethylation (H3K9me2) and loss of chromatin accessibility. In addition, many genes are regulated by changing sets of enhancers.

Fig. 2.

MLL3/4 complex functions in ESCs. The MLL3/4 histone methyltransferases function as part of the COMPASS complex to promote H3K4 mono- and dimethylation (me1/2), using S-Adenosyl methionine (SAM) as a cofactor. In addition, MLL4 counteracts LSD1 to preserve H3K4me1. MLL3/4 also co-operate with P300 and UTX to promote H3K27 demethylation and acetylation (ac). Biologically, MLL3/4 proteins are required for exiting naïve pluripotency and proper differentiation into somatic lineages.

Fig. 3.

PRC1 and PRC2 functions in ESCs. (A) The PRC1 complex, consisting of core members RING1A/B, CBX and PCGF proteins, is responsible for ubiquitylation (ub) of H2AK119. Additional regulatory proteins such as RYBP can tune its ubiquitylation activity and PCGF6 can promote its recruitment by MAX/MGA. PRC1 is required for proper repression of developmental genes and premature differentiation in ESCs, and for correctly timed ESC differentiation. (B) The PRC2 complex, consisting of core members EZH1/2, EED and SUZ12, is responsible for trimethylation (me3) of H3K27. DNA methylation at CGIs and active Pol II oppose PRC2 recruitment, whereas MTF2 recruits PRC2 to unmethylated CG-rich regions. PRC2 activity in turn opposes DNA methylation by DNMT1 at non-CGI regions. Furthermore, NSD1-dependent H3K36 trimethylated domains can restrict PRC2-dependent H2K27me3 domains. In S/L-cultured ESCs, PRC2 is responsible for the establishment of bivalent domains at developmental genes before lineage commitment and long-term suppression of pluripotency genes upon differentiation.

Fig. 4.

JmjC-containing histone demethylase functions in ESCs. JMJD2A/C predominantly demethylate H3K9me3/2, whereas JMJD1A/B demethylate H3K9me2/1 at H3K4me3-positive domains, which are enriched at promoters. Both JMJD clusters are required for pluripotency gene expression in ESCs.

Fig. 5.

DNMT and TET functions in ESCs. (A) DNMT proteins catalyze the methylation of cytosine (5mC) at CG-rich regions. DNMT1 converts hemimethylated DNA to fully methylated DNA at replication forks during DNA replication, whereas DNMT3A/B catalyze de novo methylation. TET proteins, in turn, are responsible for the stepwise oxidation of 5mC via the intermediates 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC) and 5-carboxyl-cytosine (5caC). 5fC and 5caC are depurinated by TDG, which is followed by restoration of unmethylated cytosine by the base excision pathway. (B) Maintenance of DNA methylation via DNMT1 is a major driver of global DNA methylation in S/L-cultured ESCs. DNMT1 was shown to be recruited by UHRF1, which in turn localizes to G9A-dependent H3K9me2-positive domains. (C) DNMT1 is required for global methylation of the EpiLC/EpiSC genome. TET1 and TET2 have antagonistic roles in promoting formative and naïve pluripotency, respectively.

Fig. 6.

Nucleosome remodeling complexes in ESCs. Three major families of nucleosome remodeling complexes function in ESCs. The SWI/SNF complex member BRG1 promotes the recruitment of pluripotency TFs and is therefore required for naïve pluripotency and ESC survival. SMARCAD1 links SWI/SNF complexes to deimination of H3R26 and repression of H3K9me3. SMARCAD1 prevents acquisition of EpiLC features. The TIP60-P400 complex counteracts TF recruitment by decreasing chromatin accessibility, and thereby is required for ESC self-renewal and differentiation. Finally, the NuRD complex can transiently decrease chromatin accessibility at transcriptionally active promoters, which allows for reestablishment of altered TF binding patterns. NuRD modulates pluripotency gene expression under naïve conditions and during exit from naïve pluripotency.

Fig. 7.

Chromatin looping factors in ESCs. Three factors were recently shown to be responsible, together with cohesin, for the formation of distinct chromatin loops in ESCs. (A,B) CTCF and YY1 both function together with cohesin in the formation of largely distinct loops, TAD boundaries (A) and enhancer-promoter loops (B), respectively. TAD boundaries are required for insulation of TADs and activation of ubiquitously expressed genes. Enhancer-promoter loops regulate gene transcription more directly. (C) RING1B plays a role in controlling contacts between repressed promoters, thereby contributing to repression of cognate genes.

Fig. 8.

Enhancer switching between ESC pluripotency states. The transition from naïve to formative pluripotency is associated with numerous changes in enhancer activity, TF and cohesin occupancy. Naïve-expressed TFs collaborate to drive naïve-specific gene expression. Upon exit from naïve pluripotency they can be redirected through collaboration with formative-specific TFs to activate other enhancers and induce formative-specific genes or contribute to maintenance of expression of certain pluripotency genes.

Fig. 9.

Regulation of enhancer and promoter activity through TF-mediated recruitment of chromatin-modifying enzymes. Collaborative binding of multiple TFs creates an environment favoring the recruitment of chromatin-modifying enzymes. Enzymes catalyzing modifications that correlate with enhancer and gene activity include nucleosome remodeling complexes (NRCs), MLLs, P300/CBP and JMJDs. Enzymes catalyzing modifications that correlate with enhancer and gene repression include PRC1/2, HDACs and H3K9 methyltransferases (H3K9MTs). Active enhancers and promoters typically correlate with local chromatin accessibility and H3K27ac signal. In addition, active enhancers and promoters carry H3K4me1 and H3K4me3, respectively. Inactive enhancers and promoters typically lose chromatin accessibility and gain H3K9 and H3K27 methylation.