REVIEW

Epigenetic control of transcriptional regulation in pluripotencyand early differentiationDeniz Gokbuget1,2 and Robert Blelloch1,2,*

ABSTRACTPluripotent stem cells give rise to all cells of the adult organism,making them an invaluable tool in regenerative medicine. In responseto differentiation cues, they can activate markedly distinct lineage-specific gene networks while turning off or rewiring pluripotencynetworks. Recent innovations in chromatin and nuclear structureanalyses combined with classical genetics have led to novel insightsinto the transcriptional and epigenetic mechanisms underlying thesenetworks. Here, we review these findings in relation to their impact onthe maintenance of and exit from pluripotency and highlight the manyfactors that drive these processes, including histone modifyingenzymes, DNA methylation and demethylation, nucleosomeremodeling complexes and transcription factor-mediated enhancerswitching.

KEY WORDS: Epigenetics, Pluripotency, Transcriptional regulation,Embryonic stem cells, Epiblast-like cells, Mouse

IntroductionCell-fate transitions are ultimately driven by changes in geneexpression. Gene expression is regulated by the binding oftranscription factors (TFs) to specific and conserved DNA motifswithin enhancers and promoters, which then, in collaboration withchromatin-modifying enzymes, leads to target gene activation orrepression (Long et al., 2016). Although promoters are directlyadjacent to the genes they regulate, enhancers can be thousands tomillions of bases away.Enhancers and promoters show a range of activities. Inactive

enhancers and promoters show minimal expression of their targetgenes and tend to have a high degree of nucleosome occupancy andthe presence of repressive histone marks such as histone H3 lysine27 trimethylation (H3K27me3) and H3K9 di- and trimethylation(H3K9me2/3). A subset of TFs can reverse this inactive state byrecruiting nucleosome remodeling complexes and histonemodifying enzymes, resulting in an accumulation of activatingmarks and ultimately increased transcription. These activatingmarks include histone H3K4 trimethylation (H3K4me3) andmonomethylation (H3K4me1), which are found at activepromoters and enhancers, respectively. Active enhancers andpromoters also correlate with actively transcribing RNApolymerase II (Pol II) and histone H3K27 acetylation (H3K27ac).In addition, Pol II transcribes a class of non-coding RNAs termedenhancer RNAs (eRNAs) at active enhancers, which are proposed to

participate in enhancer-promoter communication (Li et al., 2016).Intermediate enhancer states such the ‘primed’ and ‘poised’ statesare also observed, and are associated with subsets of marks that arethought to allow for rapid activation or repression in response to cellsignaling cues. Although the different marks correlate well with theactivity of enhancers and the expression of their cognate genes, thesequence of events required to interconvert between activity states isonly partially understood. Furthermore, it remains to be clarifiedmechanistically how enhancers and promoters work in concert tomodulate gene expression and to what degree physical contactsbetween these elements matter.

Pluripotent stem cells of the mammalian epiblast, which give riseto all cells in the embryo, have proven to be valuable models forunderstanding transcriptional regulation. This transient in vivopopulation of cells can be cultured indefinitely in vitro underdefined conditions. These cells, termed embryonic stem cells(ESCs), enable the study of mechanisms involved in the earliest fatedecisions in embryonic development. During early differentiationboth in vivo and in vitro, ESCs undergo molecular andmorphogenetic changes, while retaining their ability to contributeto all cells of the adult animal (Smith, 2017). These changes occurover a span of several days and have led to the definition of distinctpluripotent states. However, there is much confusion in the literatureregarding the terminology used to describe these states. In a recentreview, Austin Smith aimed to clarify this issue by specificallydefining distinct pluripotent states (Smith, 2017). In agreement withthis Review, we use the following terminology. ESCs cultured in thepresence of leukemia inhibitory factor (LIF) and 2i (glycogensynthase kinase-3 beta and mitogen-activated protein kinase kinaseinhibitors) (Ying et al., 2008) will be considered as naïvepluripotent cells. They are similar to early pre-implantationepiblast cells (Hayashi et al., 2011; Nakamura et al., 2016).Epiblast-like cells (EpiLCs) differentiated in either serum alone orFGF/activin will be defined as exhibiting the formative pluripotentstate; these cells are similar to late epiblast cells (Krishnakumaret al., 2016; Hayashi et al., 2011). We also discuss results fromstudies of mouse ESCs cultured in serum plus LIF (S/L; alsodescribed as metastable ESCs) and mouse epiblast stem cells(EpiSCs) cultured in FGF/activin, both of which are moreheterogeneous cell populations. S/L-cultured ESCs converge on abulk transcriptome comprising a mixture of naïve and formativemarkers (Guo et al., 2016), whereas EpiSCs converge on a bulktranscriptome similar to late gastrula-stage ectoderm and are distinctfrom EpiLCs (Kojima et al., 2014; Tsakiridis et al., 2014).

A remarkable feature of transitions between naïve and otherpluripotency states is the large degree of epigenetic reprogrammingthat occurs in the absence of major changes in the expression ofmany of core pluripotency genes (Buecker et al., 2014; Ficz et al.,2013; Galonska et al., 2015; Habibi et al., 2013; Krishnakumaret al., 2016; Marks et al., 2012). This epigenetic reorganization isthought to rewire the ESC genome in preparation for rapid lineage

1The Eli and Edythe Broad Center of Regeneration Medicine and Stem CellResearch, Center for Reproductive Sciences, University of CaliforniaSan Francisco, San Francisco, CA 94143, USA. 2Department of Urology, Universityof California San Francisco, San Francisco, CA 94143, USA.

*Author for correspondence (robert.blelloch@ucsf.edu)

D.G., 0000-0002-3038-4071; R.B., 0000-0002-1975-0798

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diversification with the onset of gastrulation (Fig. 1) (Smith, 2017).Global levels of DNA methylation markedly increase when ESCsexit from naïve pluripotency, accompanied by increased expressionof DNA methyltransferases (DNMTs) 1, 3A and 3B (Von Meyennet al., 2016a; Ficz et al., 2013; Habibi et al., 2013; Hackett et al.,2013; Leitch et al., 2013). Furthermore, bivalent H3K27me3 andH3K4me3 marks are established at CpG-rich island (CGI)-positivepromoters (Marks et al., 2012). In contrast, the expression of mostpluripotency genes, including octamer-binding transcription factor4 (OCT4, also known as POU5F1) and SRY-Box 2 (SOX2),remains unchanged, although the enhancers driving expression ofthese genes do change (Buecker et al., 2014; Chen et al., 2018;Galonska et al., 2015). Understanding how these various epigeneticand chromatin-based changes regulate the transition betweenpluripotent states, and the various molecular factors that mediatethem, will contribute to our understanding of cell-fate decisions indevelopment and disease, and could help us to direct cell-fatedecisions in the context of regenerative medicine.In this Review, we focus on studies that have used state-of-the-art

technologies combined with classical genetics to tease out themechanistic relationships underlying the regulation of transcriptionin pluripotent states, as well as during the exit from pluripotency.Using the terminologies defined above, we present emergingconcepts of transcriptional regulation in pluripotency at the levels ofhistone modifications, DNAmethylation, nucleosome accessibility,chromatin looping and enhancer switching. We also highlight howcrosstalk between these chromatin modifications and TFs caninfluence pluripotency.

Histone modifying enzymesHistone modifying enzymes are a diverse group of proteinscatalyzing the covalent post-translational modification of histones,most prominently at their N-terminal ‘tail’ regions. Most ofthese enzymes are ubiquitously expressed and have no directDNA-binding domains, and thus need to be recruited in a celltype-specific and dynamic manner. Although the list of histone

modifications and their associated enzymes continues to grow, wefocus here on a subset of these enzymes that catalyze the addition ofmarks with well-known functions in transcriptional activation andrepression.

MLL proteinsIn mammals, mixed lineage leukemia (MLL)/Set1A/B proteinsare responsible for H3K4 methylation, which is a marker of poised,primed and active enhancers and promoters. The H3K4 mono- anddimethyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) areenriched at enhancers (Hu et al., 2013). In ESCs, loss of bothMLL3/4 or MLL4 alone results in comparably reduced H3K4me1levels, suggesting that MLL4 is the major driver of H3K4me1(Fig. 2). Indeed, MLL4 knockout (KO) mice show early embryoniclethality at around embryonic day (E)9.5, whereas MLL3 KO micesurvive until birth (Lee et al., 2013). Surprisingly, even in theabsence of MLL3/4, expression of core pluripotency genesincluding Sox2, Nanog and Oct4 remains unchanged when ESCsare cultured in naïve or S/L conditions. In addition, colonyformation assays suggest no major defects in self-renewal.However, levels of H3K27ac at MLL3/4-bound sites aredecreased, as is eRNA transcription, Pol II density and expressionof nearby genes (Dorighi et al., 2017; Wang et al., 2016).

Upon removal of 2i, MLL4-deficient ESCs fail to adopt atranscriptome characteristic of S/L-cultured ESCs but maintain anaïve-like ESC state (Fig. 2) (Cao et al., 2018). Molecularly, this isconfirmed by decreased levels of H3K4me1 and H3K27ac levels atS/L-induced genes. Furthermore, MLL3/4-deficient ESCs show animpaired capacity to give rise to somatic lineages during embryoidbody differentiation, as evidenced by persistence of manypluripotency markers and a failure to induce expression ofdifferentiation markers (Wang et al., 2016). Interestingly, thefailure in enhancer activation and exit of naïve pluripotency inMLL4-deficient ESCs can be reversed by depletion of the H3K4demethylase LSD1 (KDM1A), which co-occupies MLL4-boundsites (Cao et al., 2018). This finding suggests that MLL4 counteractsthe repressive role of LSD1 at bound enhancers and that itsdeparture from specific enhancers during differentiation isassociated with their decommissioning via LSD1. In line with thisinterpretation, impairing MLL4 recruitment to chromatin bydeletion of its N-terminal PHD finger cluster mimics the MLL4KO phenotype (Cao et al., 2018).

The effect of MLL3/4 on H3K27ac levels and gene expressionappears to be independent of their methyltransferase activity, aspoint mutations or complete deletion of their catalytic SET domainresults in few changes (Cao et al., 2018; Dorighi et al., 2017). Thissurprising finding questions the importance of H3K4me1 inenhancer activity. However, given H3K4me1 is reduced but notabolished inMLL3/4 mutant cell lines, compensation by other MLLproteins might explain this finding. H3K4me1 does appear to playan important role in chromatin looping. Loss of MLL3/4 protein orof just its methyltransferase activity results in a decrease in cohesinoccupancy and long-distance enhancer-promoter interactions,particularly at large clusters of enhancers termed super-enhancersand enhancers that gain MLL3/4-dependent H3K4me1 during earlyESC differentiation (Yan et al., 2018). Another study identified aninteraction between H3K4me1-containing nucleosomes andchromatin remodeling complexes, which suggests H3K4me1might have a role in recruiting these factors (Local et al., 2018).These data suggest that H3K4me1 might confer robustness toenhancer activation by creating a local environment that favorscohesin recruitment and H3K27 acetylation.

Naïve Formative Lineagecommitment

Gastrulation

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Fig. 1. States of pluripotency. Embryonic stem cells transition throughdistinct states of pluripotency (naïve, formative) before they undergo lineagecommitment (and then gastrulation). This transition is accompanied by variouschanges in chromatin, including gain of bivalent domains and global DNAmethylation, global gain of H3K9 dimethylation (H3K9me2) and loss ofchromatin accessibility. In addition, many genes are regulated by changingsets of enhancers.

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MLL3/4 are not the only methyltransferases found at enhancers(Dorighi et al., 2017). A recent study demonstrated that MLL1 isrequired for EpiSC maintenance and is also predominantly boundat enhancers (Zhang et al., 2016). Genetic loss or pharmacologicalinhibition of MLL1 results in reprogramming to a naïve-like ESCstate along with a reduction of H3K4me1 at MLL1-bound sites inEpiSCs. Thus, MLL1-dependent H3K4 methylation appears to berequired for maintenance of the EpiSC state. Another recent studyfound that MLL2 and MLL4 deletion in ESCs results in loss ofH3K4me1 at partially overlapping sets of distal regulatoryelements, suggesting redundant and non-redundant functions ofthese MLL proteins (Morgan et al., 2017). A careful dissection ofthe relative contributions of MLL proteins to H3K4me1 and targetgene transcription at different classes of enhancers and promotersin well-defined pluripotency conditions is necessary to clarify therole of MLL proteins and H3K4me1 in ESC transcriptionalregulation. Of particular interest would be to determine the extentto which MLL function is required for maintenance of enhanceractivity as opposed to de novo activation during cell statetransitions.MLL proteins also act in concert with other activating chromatin-

modifying enzymes to shape enhancer activity in ESCs. Forexample, the H3K27 demethylase UTX (KDM6A) cooperates withMLL4 to recruit P300 and enhance H3K27 acetylation (Fig. 2)(Wang et al., 2017), which in turn further promotes H3K4methylation. The cooperativity of MLL4, UTX and P300ultimately leads to greatly enhanced transcriptional activation. Thecatalytic activity of UTX is dispensable for this mechanism.Furthermore, during ESC differentiation, UTX suppresses enhancerof zeste 2 (EZH2)-dependent H3K27 methyltransferase activityfollowing DNA replication, providing a transient state of openchromatin allowing binding of fate-specifying TFs to the nascentDNA strand (Petruk et al., 2017). It will be interesting to seewhetherthis function of UTX occurs in cooperation with P300 andMLL3/4,and to assess the impact of this mechanism on target genetranscription.

Histone deacetylasesAs ESCs differentiate, global levels of H3K27ac decrease,correlating with a globally more closed chromatin state anddecreased gene expression (Efroni et al., 2008; Lee et al., 2004;Meshorer et al., 2006). This deacetylation of histones, including thatof H3K27ac, is catalyzed by class I histone deacetylases (HDAC1-3), which assemble into large multi-subunit complexes includingother chromatin-modifying enzymes. Sin3-Hdac, NuRD andCoREST are examples of such complexes that are essential duringearly embryonic development (Kelly and Cowley, 2013).

To investigate the redundancy between ESC-expressed class IHDACs, a recent study generated inducible HDAC1/2 double KOsin S/L-cultured ESCs and demonstrated that HDAC1/2 loss resultsin defects in chromosomal segregation, pronounced cell death anddecreased core pluripotency factor expression, which isaccompanied by globally increased levels of histone acetylation(Jamaladdin et al., 2014). Although these data highlight HDAC1/2as crucial regulators of pluripotency, many of the observed defectscould be indirect. It remains unclear whether the observedphenotype is due to increased histone acetylation levels or linkedto catalytic-independent functions similar to transcriptionalrepression by class IIa HDACs, which involves inhibition of TFactivity through direct interaction and recruitment of co-repressors(Dressel et al., 2001; Zhang et al., 2001, 2002). This could beaddressed by generating catalytically dead HDAC mutants. Inaddition, the role and impact of the opposing activities of the histoneacetyltransferases P300/CBP should be investigated at HDAC1/2-dependent loci.

Using a proteomics approach, a recent study identified SIN3-HDAC Complex Associated Factor (FAM60A, also known asSINHCAF) as a new ESC-specific subunit of the Sin3a-HDACcomplex (Streubel et al., 2017). FAM60A colocalizes genome-widewith other complex members at promoters and is required forSIN3A localization. SIN3A and FAM60A depletion results in acommon set of deregulated target genes, including genes associatedwith transforming growth factor (TGF) beta signaling and G1 cell

MLL3/4

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ac

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Fig. 2. MLL3/4 complex functions in ESCs. The MLL3/4 histone methyltransferases function as part of the COMPASS complex to promote H3K4mono- and dimethylation (me1/2), using S-Adenosyl methionine (SAM) as a cofactor. In addition, MLL4 counteracts LSD1 to preserve H3K4me1. MLL3/4also co-operate with P300 and UTX to promote H3K27 demethylation and acetylation (ac). Biologically, MLL3/4 proteins are required for exiting naïvepluripotency and proper differentiation into somatic lineages.

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cycle phase length. As with the HDAC1/2 double KO cells, it wouldbe important to address the role of H3K27 deacetylation in theregulation of SIN3A-HDAC target genes and how it could regulatethe short and unique G1 phase of pluripotent cells.

Polycomb repressive complexes 1 and 2Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) areepigenetic repressors (Di Croce and Helin, 2013) that are essentialfor early embryonic development, in which they function to

maintain proper gene expression patterns (Boyer et al., 2006;O’Carroll et al., 2001). Both complexes comprise a catalytic subunitas well as multiple regulatory subunits. The core of canonical PRC1is formed by chromobox-domain (CBX) proteins, PCGF familymembers 2 or 4 (PCGF2/4) and the ubiquitin E3 ligase subunitsRING1A/B, which catalyze the ubiquitylation of H2AK119(H2AK119ub) (Fig. 3A). Non-canonical PRC1 complexes in turnare devoid of CBX proteins, but instead contain RING1 and YY1Binding Protein (RYBP) or YY1 Associated Factor 2 (YAF2)

RING1ARING1B

CBXPCGF

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Repression of developmental genes in ESCsPrevention of premature ESC differentiation

Correctly timed ESC differentiation

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Long-term suppression of pluripotency genes during ESC differentiationEstablishment of bivalency at developmental genes

Pol II

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me

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GC GCme me

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Fig. 3. PRC1 and PRC2 functions in ESCs. (A) The PRC1 complex, consisting of core members RING1A/B, CBX and PCGF proteins, is responsible forubiquitylation (ub) of H2AK119. Additional regulatory proteins such as RYBP can tune its ubiquitylation activity and PCGF6 can promote its recruitment byMAX/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 andactive Pol II oppose PRC2 recruitment, whereas MTF2 recruits PRC2 to unmethylated CG-rich regions. PRC2 activity in turn opposes DNA methylation byDNMT1 at non-CGI regions. Furthermore, NSD1-dependent H3K36 trimethylated domains can restrict PRC2-dependent H2K27me3 domains. In S/L-culturedESCs, PRC2 is responsible for the establishment of bivalent domains at developmental genes before lineage commitment and long-term suppressionof pluripotency genes upon differentiation.

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(Di Croce and Helin, 2013; Rose et al., 2016). They also containPCGF1, PCGF3, PCGF5 or PCGF6. By contrast, PRC2 consists ofthe core subunits EZH1/2, suppressor of zeste 12 (SUZ12) andembryonic ectoderm development (EED), with EZH1/2 catalyzingH3K27 methylation (Fig. 3B). Non-core subunits segregate PRC2into two major subtypes, PRC2.1 [containing PCL homolog (PCL1-3)alongside EPOP or LCOR] and PRC2.2 (containing JARID2 andAEBP2). Both PRC1 and PRC2 have the capacity to bindH3K27me3 and H2AK119ub, respectively, reinforcing eachother’s binding to chromatin (Blackledge et al., 2014; Cooperet al., 2014; Min et al., 2003; Wang et al., 2004).PRC2 has been extensively studied in ESCs and is responsible for

genome-wide H3K27me2 and H3K27me3 (Chamberlain et al.,2008; Ferrari et al., 2014; Margueron et al., 2008; Montgomeryet al., 2005; Pasini et al., 2004, 2007; Schoeftner et al., 2006; Shenet al., 2008). Accordingly, reintroduction of the PRC2 subunitEZH2 in EZH1/2 double KO ESCs is sufficient to accurately re-establish wild-type H3K27me2/3 patterns, even after those markshave all been lost (Højfeldt et al., 2018). This suggests that PRC2binding and de novo methylation occur independent of previousH3K27 methylation. In line with this, it has been shown that H3K27methylation is a stepwise process involving transient mono- anddimethylated states (Højfeldt et al., 2018). Recent evidence suggestsEZH1 preferentially deposits H3K27 monomethyl marks, whereasEZH2 shows a preference for depositing K27 di- and trimethylmarks (Lee et al., 2018). Interestingly, histone linker lengthenhances methyltransferase activity of EZH2. Future studiesshould address the extent to which differential substratepreference of EZH1/2 and histone linker length relates to localK27 methylation patterns.PRC2 preferably binds CGIs, however DNA methylation and

active transcription oppose PRC2 binding at these sites (Di Croceand Helin, 2013) (Fig. 3B). Remarkably, inhibition of Pol II bindingor elongation in naïve ESCs alone rapidly induces global ectopicPRC2 recruitment to nucleosome-depleted CGIs associated withPRC2 binding in differentiated tissues (Riising et al., 2014). Thiseffect is reversible following Pol II inhibitor washout, suggestingPRC2 can be displaced by Pol II. Similar to Pol II recruitment,dCas9-mediated recruitment of the SWI/SNF nucleosomeremodeling complex in S/L-cultured ESCs is sufficient to opposelocal PRC1/2 function (Braun et al., 2017). These results suggestthat factors promoting a local environment permissive fortranscription are sufficient to oppose PRC2, and that the absenceof such factors is a prerequisite for local PRC2 activity.A recent study profiled an array of epigenetic modifications upon

transitioning ESCs from naïve to S/L-cultured conditions andreported a global enrichment of PRC2-dependent H3K27me3 innaïve ESCs (van Mierlo et al., 2019). This enrichment is mainlyattributed to extended eu- and heterochromatic regions outside ofCGIs, which were previously shown to gain H3K27me3 in S/L-cultured compared with naïve ESCs (Marks et al., 2012).Interestingly, deletion of the PRC2 component EED in naïveESCs results in a gain of DNA methylation over these non-CGIregions, suggesting a requirement of PRC2 in naïve ESCs foropposing widespread DNA methylation. Overall, these studieshighlight extensive competitive crosstalk between PRC2 activity,transcription and DNA methylation, which is essential for theepigenetic landscape of naïve and S/L-cultured ESCs. Futurestudies are needed to better characterize the epistasis of thiscrosstalk, the nature of the factors involved and how these activitieslink to pluripotency gene expression and activation of fate-specifying gene programs.

The nature of the factors that recruit PRC2 in ESCs is beginningto come to light. Recent studies have identified a crucial role for thepolycomb-like protein MTF2 in the recruitment of PRC2 tounmethylated CGIs (Li et al., 2017; Perino et al., 2018) (Fig. 3B).Crystal structures of MTF2 suggest that it specifically bindsunmethylated CpG-containing DNA via its winged-helix domain(Li et al., 2017). In support of this finding, a proteomic screen usingan unmethylated CpG-containing PRC2 motif as bait enriched forMTF2 at stoichiometric levels compared with core PRC2components (Perino et al., 2018). Interestingly, in addition to aCpG-rich context, MTF2 binding preferably occurs at sites with aspecific DNA helical shape involving an increased minor groovewidth and a decreased propeller twist. PRC2 activity is furtherrestricted by the H3K36 methyltransferase NSD1, as indicated bythe finding that NSD1 depletion results in genome-wideaccumulation of H3K27me3 domains (Streubel et al., 2018)(Fig. 3B). Thus, sequence-specific DNA binding proteins andepigenetic context are crucial factors for defining PRC2-bindingdomains.

The core PRC1 subunits RING1A and RING1B also promote therecruitment of PRC2. Loss of RING1B catalytic activity results in adepletion of H2AK119ub and negatively affects PRC2 recruitmentand activity in S/L-cultured ESCs, albeit to a lesser extent than doesloss of RING1B altogether (Illingworth et al., 2015). Similarly,mutation of RING1A/B ubiquitylation sites in the histone variantH2A.Z.1 decreases PRC1/2 recruitment to bivalent promoters inESCs (Surface et al., 2016). Therefore, RING1B has both catalytic-dependent and catalytic-independent roles in PRC2 recruitment andgene silencing, the relative importance of which varies in a locus-specific manner (Rose et al., 2016). Notably, whereas EZH2,SUZ12 and EED are dispensable for ESC self-renewal in naïve andS/L conditions, RING1A/B are required to prevent prematuredifferentiation in S/L-cultured ESCs (Chamberlain et al., 2008;Endoh et al., 2008; Pasini et al., 2004; Shen et al., 2008; van Mierloet al., 2019). This suggests that RING1A/B have essential functionsin ESCs that extend beyond cooperative binding with PRC2. Suchfunctions could depend on their roles in protein complexes that aredistinct from PRC1, such as the BCL6 co-repressor complex(Gearhart et al., 2006).

PRC2 targets in ESCs are enriched for developmental genes(Boyer et al., 2006), suggesting that PRC2 is involved in the timelyexpression of these genes upon ESC differentiation. However,transcriptomic analysis of SUZ12 KO cells shows only minordifferences during early differentiation compared with wild-typecells (Riising et al., 2014). In contrast, PRC2 is required at laterstages of differentiation for maintaining correct expression levels ofpluripotency and differentiation genes. Future studies shouldaddress which factors initiate transcriptional repression duringESC differentiation and why PRC2 is required for maintenance ofrepression.

Recent studies have also highlighted lineage-specific roles forboth PRC1 and PRC2 activity. For example, one study revealeddynamic changes in PRC1 and PRC2 interactomes duringdifferentiation of ESCs to neural precursor cells (NPCs), (Kloetet al., 2016). Core PRC2 remains intact in NPCs, with a switch ofsubstoichiometric non-core interactors such as EPOP and LCOR. Incontrast, PRC1 core subunit composition undergoes a switch inCBX and PCGF isoforms. Furthermore, H3K27me1/2/3 showdynamic redistribution during early ESC differentiation both atenhancers and at promoters (Juan et al., 2016). Interestingly,shifting the balance of H3K27me2 to H3K27me3 using ahyperactive EZH2 mutant results in ESC differentiation biased

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toward a neuronal program and renders cells refractory to 2iconditions.Non-canonical PRC1 complexes often show distinct but also

overlapping functions compared with canonical PRC1. Forexample, the PCGF6-containing non-canonical PRC1 complex(PRC1.6) is required for ESC self-renewal through suppression ofgerm cell-related genes (Endoh et al., 2017; Stielow et al., 2018;Zhao et al., 2017b) (Fig. 3A). Strikingly, the interaction of PCGF6with RING1B is required for H2AK119ub and H2K27me3marks atbound sites. The recruitment of PCGF6 is not dependent onH3K27me3 but on the TFs MGA/MAX, highlighting theimportance of TFs in the recruitment of these repressivecomplexes (Endoh et al., 2017). PCGF1, a member of anothernon-canonical PRC1 complex, is dispensable for ESC self-renewal,but required for differentiation. PCGF1 is necessary for PRC1recruitment and surprisingly results in gene activation rather thanrepression (Yan et al., 2017). Similarly, PCGF3 and 5 positivelyinfluence gene expression via P300 recruitment during mesodermspecification (Zhao et al., 2017a).Other evidence for the importance of non-canonical PRC1

configurations in ESCs was shown in a recent study addressing therole of RYBP and YAF2 (Rose et al., 2016). Loss of both RYBP andYAF2 results in a reduction of H2AK119ub at PRC1 target sites,particularly ones with low PRC1 occupancy, in the absence of anychange in global H2AK119ub levels or RING1B recruitment(Fig. 3A). Thus, RYBP/YAF2 shape PRC1 activity rather thaninfluence its recruitment, and this activity is required for normalH2AK119ub levels at sites with low PRC1 occupancy. In addition,the reduction of H2AK119ub in RYBP/YAF2 double KO cellscorrelates well with changes in H3K27me3 levels, without majorchanges in SUZ12 occupancy, supporting a model in which PRC1activity dictates local PRC2 activity. At a subset of PRC1/2 sitesexperiencing the strongest changes in H2AK119ub and H3K27me3upon RYBP/YAF2 deletion, PRC1/2 occupancy is reduced, andcognate genes are de-repressed. This suggests there is a PRC1/2activity-based threshold below which PRC1/2 occupancy cannot besustained, allowing gene-activating cues to take control. Overall,these results highlight that non-canonical PRC1 complexes areimportant regulators of ESC pluripotency and differentiation, andshow that PRC complex subunit composition dictates whether itacts as an activator or repressor within the same cell type.

JmjC domain histone demethylasesJumonji C (JmjC) domain-containing proteins are a family of iron-and alpha-ketoglutarate-dependent histone demethylases that act onmethylated H3K4, H3K9, H3K27, H3K36, H3R2 and H4R3, whichare generally associated with transcriptionally repressed chromatin.They phylogenetically separate into distinct clusters with differentsubstrate specificities (Cloos et al., 2008). These clusters include theJMJD2 cluster proteins, which consecutively demethylateH3K9me3/me2 as well as H3K36me3/2, and the JMJD1 clusterproteins, which demethylate H3K9me2/me1 (Fig. 4).The activity of the JMJD2 proteins JMJD2A (KDM4A) and

JMJD2C (KDM4C), but not that of JMJD2B (KDM4B), is cruciallyrequired for naïve pluripotency (Pedersen et al., 2016). Specifically,JMJD2A/C act redundantly to suppress H3K9 methylation, and to alesser extent H3K36 methylation, at H3K4me3-marked promotersin naïve ESCs. These data suggest that JMJD2 proteins are activelyrecruited to promoters to oppose default repression with H3K9 andH3K36methylation. Surprisingly, JMJD2C alone may play a role indifferentiation towards somatic lineages by regulating distalregulatory elements (Tomaz et al., 2017). However, the

physiological relevance of this function is unclear given thatJMJD2C KO is compatible with mouse development (Pedersenet al., 2014). Further studies are needed to clarify this apparentcontradiction.

The JMJD1 proteins JMJD1A (KDM3A) and JMJD1B(KDM3B) play a crucial role in blocking H3K9 methylation atgene-dense chromosomal regions (Kuroki et al., 2018). Loss ofJMJD1A and B results in perturbed gene expression, includingdecreased OCT4 levels and rapid cell death. This phenotype can berescued by additional deletion of the H3K9 methyltransferase G9A(EHMT2), suggesting that JMJD1A/B counteract excessive G9Aactivity to ensure both pluripotency gene expression and ESCviability.

Collectively, these studies highlight that the active recruitment ofH3K9 demethylation machinery to regulatory elements acts as anessential mechanism for maintaining pluripotency and enablingdifferentiation of ESCs (Fig. 4).

DNA methylation and demethylationDNA methylation is a well-characterized heritable epigeneticmodification of the DNA molecule itself that shows dynamicregulation in development and disease, and that typically has generepressive functions. During early embryonic development, DNA isinitially demethylated in several waves and, in mice, reaches itslowest methylation level in the blastocyst-stage embryo, after whichpoint it sharply increases. DNA methylation is also one of the mostdistinguishing epigenetic differences between naïve and laterpluripotency stages. Below, we discuss key enzymes involved inthe deposition and removal of this modification and highlight recentinsights into their impact on the transition between pluripotentstates.

DNMT-driven DNA methylationDNA methylation at the fifth position of cytosine (5mC) is a well-characterized epigenetic mark with important functions inmammalian development. 5mC is enriched at CpG dinucleotidesbut largely absent at regions of high CpG density (CpG islands)

JMJD2A

JMJD2C

K9

K4

JMJD1A

JMJD1B

me3/2

me3/2

me3

me2/1K9

K4me3

Pol II

me2/1

Pluripotency gene expression

Fig. 4. JmjC-containing histone demethylase functions in ESCs. JMJD2A/C predominantly demethylate H3K9me3/2, whereas JMJD1A/B demethylateH3K9me2/1 at H3K4me3-positive domains, which are enriched at promoters.Both JMJD clusters are required for pluripotency gene expression in ESCs.

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(Kim and Costello, 2017). The enzymes catalyzing DNAmethylation are DNMT family members including DNMT1,DNMT3A and DNMT3B (Fig. 5A). DNMT3A and DNMT3B arethe predominant de novomethylation enzymes (Okano et al., 1999),whereas DNMT1 is responsible for the maintenance of DNAmethylation, acting via its capacity to recognize, with the help ofUHRF1, hemimethylated DNA at replication forks and methylatethe opposing strand (Bostick et al., 2007; Hermann et al., 2004;Sharif et al., 2007).Naïve ESCs are globally hypomethylated relative to most other

cell types including S/L-cultured ESCs, EpiLCs and EpiSCs (VonMeyenn et al., 2016a; Ficz et al., 2013; Habibi et al., 2013; Hackettet al., 2013; Leitch et al., 2013). The conversion of S/L-culturedESCs into naïve ESCs is associated with diminishment of 5mC,which can be accelerated by deleting UHRF1 or DNMT1 (VonMeyenn et al., 2016b). Interestingly, the loss of DNMT3A/B or thedemethylation pathway components TET1/2/3 or TDG (discussedbelow) has little effect on the kinetics of this loss of methylation.The stability of UHRF1 and its recruitment of DNMT1 to DNAreplication forks is also reduced in naïve compared with S/L ESCs.This UHRF1-dependent DNMT1 recruitment partially depends onthe binding of UHRF1 to methylated H3K9 residues (Liu et al.,2013) (Fig. 5B). Accordingly, loss of G9A leads to globally reduced5mC levels (Dong et al., 2008; Myant et al., 2011). AlthoughH3K9me3 levels are not changed in the S/L to 2i transition,H3K9me2 levels are significantly reduced, possibly underlyingimpaired recruitment of UHRF1 and subsequently DNMT1 (VonMeyenn et al., 2016b). In addition, expression of PRAMEL7, a

member of the preferentially expressed antigen in melanoma(PRAME) family, in S/L ESCs was shown to induce a naïve-likeESC expression signature, which was dependent on its induction ofproteosomal degradation of UHRF1, resulting in DNAhypomethylation (Graf et al., 2017).

Together, these data show that a reduction in 5mC maintenancerather than active depletion likely underlies the global DNAhypomethylation status associated with naïve ESCs (Fig. 5C).Furthermore, although these results highlight UHRF1 and DNMT1as central players in regulating the difference in DNA methylationbetween naïve and S/L-cultured ESCs, an increase in their levels isunlikely to fully explain the gain of DNA methylation that isobserved during differentiation of naïve ESCs to EpiLCs or EpiSCs.Future studies should address the epistasis of de novo versusmaintenance methylation during ESC differentiation. In addition, itwill be important to address how these global methylation changesaffect the activity of individual regulatory elements and which otherregulatory factors cooperate with DNMTs to modulate DNAmethylation and subsequently gene expression. In this context, arecent study demonstrated that promoter, genic and intergenic CGIsretain low levels of DNAmethylation in the transition from naïve toformative pluripotency, whereas intergenic and repeat elementsfollowed the global trend of gained methylation (VonMeyenn et al.,2016a). DNA methylation is also thought to contribute to theincreased transcriptional heterogeneity characteristic of S/L-cultured ESCs, specifically by increasing the fraction of cells withreduced levels of naïve ESC-specific TFs REX1 (ZFP42) andestrogen related receptor beta (ESRRB) (Singer et al., 2014).

DNMT

DNMT1

DNMT3A/B

DNMT1

DNMT1

UHRF1G9A

A DNMT/TET functions B Maintenance of DNA methylation

C Biological function

TET1/2

TET1/2

TET1/2TET1

TET2

TDG

5mC

C

5hmC

5fC

5caC

me2K9

Methylation ofEpiLC/EpiSC genome

Formative pluripotency

Naïve pluripotencyGCGC

GC GC

GCGC

GCGC

GC GC

GC GC

GC GC

Key

Fig. 5. DNMT and TET functions in ESCs. (A) DNMT proteins catalyze the methylation of cytosine (5mC) at CG-rich regions. DNMT1 converts hemimethylatedDNA to fully methylated DNA at replication forks during DNA replication, whereas DNMT3A/B catalyze de novomethylation. TET proteins, in turn, are responsiblefor the stepwise oxidation of 5mC via the intermediates 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC) and 5-carboxyl-cytosine (5caC). 5fC and5caC are depurinated by TDG, which is followed by restoration of unmethylated cytosine by the base excision pathway. (B) Maintenance of DNA methylation viaDNMT1 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 toG9A-dependent H3K9me2-positive domains. (C) DNMT1 is required for global methylation of the EpiLC/EpiSC genome. TET1 and TET2 have antagonistic rolesin promoting formative and naïve pluripotency, respectively.

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TET-mediated DNA demethylationDNA methylation is enzymatically reversed in a stepwise oxidationprocess. The enzymes ten-eleven translocation methylcytosinedioxygenase 1 and 2 (TET1 and TET2) catalyze the iterativeoxidation of 5mC to 5-carbonylcytosine (5caC) via 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC)intermediates (Kohli and Zhang, 2013) (Fig. 5A). Subsequently,thymine DNA glycosylase (TDG) excises 5fC and 5caC, generatingan abasic site that is restored to a cytosine through the base excisionrepair pathway. Alternatively, 5hmC can be removed byproliferative dilution.TET1 and TET2 are highly expressed in S/L-cultured and naïve

ESCs (Dawlaty et al., 2011; Koh et al., 2011). Although completeloss of TET activity is compatible with self-renewal in S/L-culturedcells, TET activity is essential for proper regulation of geneexpression during ESC differentiation (Dawlaty et al., 2014). Recentevidence suggests a more complex scenario in ESCs whereby TET2promotes expression of naïve ESC genes and TET1 promotesEpiLC-enriched genes (Fig. 5C) (Fidalgo et al., 2016). This switchin TET requirement was shown to depend on the zinc-finger TFZFP281, which can recruit TET1 and repress TET2 indirectlythrough a microRNA. Another study demonstrated that TET1/2could serve as transcriptional activators alongside core pluripotencyfactors in naïve ESCs (Finley et al., 2018). Future studies are neededto address potential opposing roles of TET1/2 in the transitionbetween different pluripotency states, what factors recruit TET2 andhow this links to DNA methylation at bound regulatory elements.

Nucleosome remodelingNucleosome remodeling is the energy-dependent process by whichhistones, and hence nucleosomes, are repositioned along thechromatin fiber. Frequently, nucleosome remodeling results inincreased chromatin accessibility, a feature that facilitates thebinding of sequence-specific factors (Kadoch and Crabtree, 2015).However, a recent study challenges the view that chromatinaccessibility indicates the local depletion of nucleosomes (Voonget al., 2016). Using a chemical mapping technique, this studyrevealed that active regulatory elements in ESCs that werepreviously designated as nucleosome-depleted, including thosebound by OCT4 and CCCTC-binding factor (CTCF), retainnucleosomes that are loosely bound or fragile. Although the linkbetween nucleosome occupancy and chromatin accessibilityrequires further investigation, it is clear that nucleosomeremodeling remains an important regulatory mechanism inpluripotent and differentiating ESCs, and involves a number ofremodeling factors.

The SWI/SNF familyThe ATPase-dependent SWI/SNF (or BAF) nucleosomeremodeling complex is a large multi-subunit complex that isthought to facilitate the binding of sequence-specific factors byevicting nucleosomes and thereby increasing chromatinaccessibility (Kadoch and Crabtree, 2015). The catalytic subunitof the ESC-expressed SWI/SNF complex BRG1 (SMARCA4), isessential for maintaining pluripotency (Ho et al., 2009) and isstrongly enriched at pluripotency TF binding sites, at which it wasrecently shown to be required together with topoisomerase II for therecruitment of pluripotency TFs (Miller et al., 2017) (Fig. 6). Anunexpected role of another embryonic tissue-enriched SWI/SNFfamily member SMARCAD1 was discovered in a screen for factorsbinding deiminated histone 3 arginine 26 (H3R26Cit), a markimportant for early embryonic development (Christophorou et al.,

2014). SMARCAD1 is enriched in S/L-cultured ESCs comparedwith EpiSCs and its depletion results in the appearance of someEpiSC features (Xiao et al., 2017). SMARCAD1 chromatin bindingand H3R26Cit counteract H3K9me3 deposition; however, it isunclear how this mechanism links to gene expression changesobserved upon SMARCAD1 depletion and to what degreenucleosome remodeling is involved. These results highlight SWI/SNF nucleosome remodeling complexes as integral parts ofpluripotency TF recruitment and maintenance of pluripotencystates (Fig. 6). Future studies are needed to identify the subunitcomposition of these complexes, the factors regulating their activityand localization, and their exact mechanism of action.

The TIP60-P400 complexThe TIP60-P400 complex, which is part of the INO80 family,exhibits lysine acetyltransferase (KAT) and ATPase-dependentnucleosome remodeling activities. In ESCs, it is largely dispensablefor transcriptional activation and appears rather to repressdevelopmental genes (Chen et al., 2013; Fazzio et al., 2008). Arecent paper described KAT/ATPase-dependent and -independentfunctions of TIP60-P400 in ESCs (Acharya et al., 2017).Specifically, although TIP60-P400 proteins are essential for self-renewal, their catalytic activities appear to be surprisinglydispensable. Instead, TIP60-P400 occupancy at promoterproximal sites reduces chromatin accessibility resulting in generepression (Fig. 6). TIP60 KAT activity is, however, required forproper ESC differentiation, acting to regulate the timing ofmesodermal and endodermal marker expression (Fig. 6). Futurestudies are needed to decipher the molecular mechanism of howcatalytic and non-catalytic functions of TIP60/P400 impact geneexpression and chromatin accessibility in such an opposing mannerduring ESC self-renewal and differentiation.

The NuRD complexThe nucleosome remodeling and deacetylation (NuRD) complexacts in many cellular contexts as a repressor. It is composed ofenzymatic and non-enzymatic subunits. The two enzymaticactivities are lysine deacetylation activity mediated via HDAC1/2,and SWI/SNF-type ATPase-based nucleosome remodeling activitymediated via chromodomain helicase DNA-binding protein 4(CHD4). The non-enzymatic subunits include the general histonechaperones RBBP4/7, as well as proteins that are specific to NuRD,namely GATAD2A/B, MTA proteins (MTA1,2 and/or 3),CDK2AP1 and MBD2/3. A recent study in which the NuRDsubunit MBD3 was reintroduced into MBD3-depleted ESCs (viatamoxifen-dependent rapid nuclear translocation) suggests thatNuRD has both activating and repressing gene modulatoryfunctions in ESCs (Bornelöv et al., 2018). Interestingly, thisrestoration of NuRD activity results in enriched binding at activepromoters and enhancers, where it temporarily decreases chromatinaccessibility, displaces Pol II and reduces nascent transcription. As aresult, pluripotency TFs are temporarily displaced at a subset of sitesand they then rebind to different degrees (Fig. 6). This change couldpotentially account for the active or repressive genemodulation seenin response to NuRD restoration. During exit from naïvepluripotency, NuRD-dependent changes in nucleosome and Pol IIoccupancy are associated with differential binding of thepluripotency TFs KLF4, ZFP42 and OTX2 to select enhancersand expression of their cognate genes (Bornelöv et al., 2018).Collectively, these data imply a role for NuRD-dependentnucleosome remodeling in resetting TF binding at activeregulatory elements, allowing a transcriptional state permissive for

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activation or repression to evolve (Fig. 6). Although more studiesare needed to demonstrate whether this proposed function of NuRDoccurs in other cellular contexts, it suggests that nucleosomeremodeling may be continuously involved in modulating theactivity of regulatory elements in ESCs.

Chromatin loopingChromatin exhibits a dynamic structure that enables interactionsbetween distal elements via the formation of loops. Some of theseloops occur stereotypically across development and across species,as in the case of topologically associated domains (TADs), whereasothers exhibit cell-type specificity such as super enhancers incontact with their target gene promoters. A major factor involved inthese loops is the cohesin complex, a ring-like multi-subunitcomplex that is essential for sister chromatid cohesion through itscapability to topologically enclose two chromatin fibers(Merkenschlager and Odom, 2013). A subset of cohesin sites isco-occupied by theMediator complex, which is a global regulator ofPol II-dependent transcription (Allen and Taatjes, 2015). Althoughboth cohesin and the Mediator complex are essential for chromatinarchitecture and pluripotency gene expression in ESCs (Kagey

et al., 2010), a number of studies have highlighted additional factorsthat play a role.

CTCFThe dimerizing sequence-specific factor CTCF is significantlyenriched at TAD boundaries and occupies a large subset of cohesin-bound sites (Fig. 7A). Acute depletion of CTCF in ESCs usingauxin-induced degron systems triggers rapid loss of TAD insulation(Nora et al., 2017), without a loss of higher order chromosomefolding into open and closed compartments (A/B compartments).Importantly, loss of TAD insulation results in prominent changes ingene expression, with upregulated genes more likely to bewithin thesame TAD and the majority of downregulated genes having a CTCFbinding site nearby. Interestingly, even though CTCF binding canbe inhibited by DNA methylation (Phillips and Corces, 2009), arecent study demonstrated that the absence of DNA methylation inESCs does not alter global CTCF binding, TAD structure or higherorder chromatin folding (Nothjunge et al., 2017). Thus, other factorslikely collaborate with CTCF to allow for the precise establishmentof TAD boundaries and higher order chromatin folding in ESCs.Future studies should aim to identify these factors.

SWI/SNF

TF

TIP60-P400

NuRD

Naïve pluripotencyand ESC survival

Self-renewalESC differentiation

Pluripotency genemodulation

Prevention ofEpiLC features inS/L-grown ESCs

Regulatory element

TF

Regulatory element

BRG1

SMARCAD1

Cit

me3

R26

K9

H3

+

Regulatory element

TIP60

CHD4HDAC1/2

TF

TF

TF

TF

P400

Fig. 6. Nucleosome remodeling complexes in ESCs. Three major families of nucleosome remodeling complexes function in ESCs. The SWI/SNF complexmember BRG1 promotes the recruitment of pluripotency TFs and is therefore required for naïve pluripotency and ESC survival. SMARCAD1 links SWI/SNFcomplexes to deimination of H3R26 and repression of H3K9me3. SMARCAD1 prevents acquisition of EpiLC features. The TIP60-P400 complex counteracts TFrecruitment by decreasing chromatin accessibility, and thereby is required for ESC self-renewal and differentiation. Finally, the NuRD complex can transientlydecrease chromatin accessibility at transcriptionally active promoters, which allows for reestablishment of altered TF binding patterns. NuRD modulatespluripotency gene expression under naïve conditions and during exit from naïve pluripotency.

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YY1The specific enrichment of CTCF at TAD boundaries raised thequestion of whether there is an analogous dimerizing factormediating enhancer-promoter loops. Two recent studies identifiedthe TF YY1 as such a factor, demonstrating that YY1 binding atpromoters and enhancers is necessary for looping and propercognate gene expression in naïve ESCs and during ESC to NPCdifferentiation (Beagan et al., 2017;Weintraub et al., 2017). The factthat ubiquitously expressed YY1 occupies only a subset of all itspotential motifs at enhancers and promoters in ESCs suggests thatother factors such as lineage-dependent TFs may promote localaccessibility of YY1 to specific regulatory elements to shape celltype-specific gene expression. These results highlight YY1 as anovel defining feature of enhancer-promoter loops in addition tocohesin and Mediator (Fig. 7B).

PRC1/2A recent unbiased screen (using promoter-capture HiC) forregulatory elements contacting promoters through long-rangeinteractions in ESCs revealed an enrichment for PRC1-boundpromoters (Schoenfelder et al., 2015a). Given the positivereinforcement of PRC1 and PRC2 binding, these results are inagreement with previous results implicating the PRC2 core subunitEED in the formation of distinct chromatin interaction domains inESCs (Denholtz et al., 2013). Subsequent analyses in S/L-culturedESCs showed that promoters of all four HOX gene clusters andearly developmental TF genes are enriched among these PRC1-bound interaction sites (Schoenfelder et al., 2015b). Removal ofRING1A and RING1B results in dissolution of these interactions(Fig. 7C). Almost all PRC1-bound interaction sites also show PRC2occupancy. However, in comparison with RING1B loss, most of thetested HOX gene cluster interactions remained detectable, albeitreduced, upon EED depletion. Similarly, a previous study showed areduction but no loss of long-range interactions of HOX geneclusters upon EED loss (Joshi et al., 2015). RING1B-bound HOXgene clusters are enriched for contacts with poised enhancers, manyof which are also occupied by RING1B. Surprisingly though, and incontrast to RING1B-bound promoter-promoter interactions, thesepromoter-enhancer interactions remain intact in the absence ofRING1A/B (Schoenfelder et al., 2015b). At the level oftranscription, loss of RING1B-bound promoter-promoter contactsresults in upregulation of cognate genes without a significant gain of

contacts with active enhancers. Similarly, the RING1B-boundgenes in contact with poised enhancers get upregulated uponRING1A/B deletion, alongside a gain in active enhancer marks(Schoenfelder et al., 2015b). A candidate factor that may restrict andshape PRC-dependent long-range interactions could be MLL2; arecent study showed that loss of MLL2 results in increased PRC1/2occupancy at bivalent promoters and redistribution of their long-range interactions, including those at HOX genes (Mas et al., 2018).Together, these studies highlight PRC1, PRC2 andMLL2 as centralplayers in the formation and maintenance of defined long-rangepromoter-promoter interactions, which are enriched at promoters ofmost HOX gene clusters and other developmental genes. Futurestudies should address whether the formation of such PRC-dependent promoter-promoter contacts are a general feature ofPRC-mediated gene repression in ESCs and subsequent stages ofdevelopment.

A recent study also discovered that some of the poised enhancersin ESCs are near genes that gain activity in anterior neuralprogenitors (AntNPCs), correlating with accumulation of H3K27acat those enhancers (Cruz-Molina et al., 2017). Using circularizedchromosome conformation capture (4C) analysis, this study showedthat those enhancers are looped to their target gene promoters inboth ESCs and AntNPCs. The deletions of selected poisedenhancers, or of EED, results in failure to induce target genes inAntNPCs. In addition, 4C analysis of EED-null ESCs suggests arequirement for PRC2 for the looping of these poised enhancers totarget promoters. CG-rich motifs are overrepresented in poisedversus active enhancers, and deletion of selected poised enhancersharboring such elements results in locally diminished H3K27me3levels. These results implicate PRC2 in the formation of poisedenhancer-promoter contacts, which become active enhancer-promoter loops later in development. However, given the broadepigenetic changes upon EED deletion, it is not clear whetherlooping of the selected poised enhancer elements in ESCs isrequired for the activation of their cognate genes in AntNPCs.Similar to PRC2, PRC1 is enriched at poised enhancers (Cruz-Molina et al., 2017), however its requirement for AntNPC targetgene induction and loop formation was not tested. Overall, theseresults extend the physiologically relevant spectrum of PRC2-dependent long-range interactions to poised enhancer-promotercontacts. These studies highlight that inactive regulatoryelements adapt complex looped 3D structures in ESCs that

Cohesin

A TAD boundary

CTCF

Cohesin

B Enhancer-promoter

YY1

Cohesin?

C Repressed promoter-promoter

RING1B

++

Activation ofubiquitous genes

Gene activation Gene repressionInsulation

Fig. 7. Chromatin looping factors in ESCs. Three factors were recently shown to be responsible, together with cohesin, for the formation of distinctchromatin 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 loopsregulate gene transcription more directly. (C) RING1B plays a role in controlling contacts between repressed promoters, thereby contributing to repression ofcognate genes.

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might be permissive of cell type-specific gene expression upondifferentiation cues.

Enhancer switchingMarked changes in enhancer activity occur in the transition fromnaïve to formative pluripotency, with comparably fewer pronouncedchanges in cognate gene expression (Fig. 8). TFs are essentialregulators of enhancer activity and are likely involved in thisrewiring of enhancer networks. An overview of these changes wasprovided by a study of core pluripotency factors (Galonska et al.,2015). This study revealed that exiting naïve pluripotency isassociated with widespread differential OCT4, SOX2 and NANOGbinding, which correlates with differential enhancer activity atselected sites. Mechanistically, reorganization of OCT4 enhancerbinding depends on collaboration with the EpiLC-specific TFOTX2 (Buecker et al., 2014). Even in the absence of differentiationcues, ectopic OTX2 expression is sufficient to redirect OCT4 toEpiLC-specific sites, highlighting the context-dependent nature ofTF enhancer selection. Similarly, another study demonstrated thatGRHL2, a TF that is upregulated in EpiLCs, activates newenhancers upon exit of naïve pluripotency to maintain, not alter,expression of a subset of expressed ESC genes (Chen et al., 2018).These same genes are regulated by distinct TFs at alternativeenhancers in the naïve ESC state. Given that GRHL2 is involved indownstream differentiation to epithelial lineages, its maintenance ofexpression of a subset of ground-state pluripotency genes couldcontribute to a network that is permissive to cues driving epithelialfate as EpiLCs differentiate and diversify into the germ layers.Together, these findings highlight that, in general, lineage-

specifying and signal-dependent TFs are likely involved in therewiring of pluripotency enhancer-promoter networks, creating atranscriptional environment permissive to lineage diversification.

Crosstalk between transcription factors and chromatin-modifying enzymesTFs are sequence-specific DNA-binding proteins that can recruitchromatin-modifying enzymes and thus create enhancer andpromoter states that are either permissive or repressive fortranscription (Fig. 9). For example, NANOG-dependentrecruitment of TET1 enhances establishment of pluripotencyduring reprogramming (Costa et al., 2013). A subset of TFsnamed ‘pioneer factors’ are thought to bind nucleosome-boundDNA and facilitate the binding of other collaborating TFs and

chromatin remodeling enzymes (Long et al., 2016). An exemplarpioneer factor is the core pluripotency factor OCT4. Indeed, thebinding of OCT4 to DNA is required for the binding of BRG1, thecatalytical subunit of the SWI/SNF chromatin-remodeling complex,resulting in open chromatin at co-bound sites (King and Klose,2017). These open chromatin conditions are required for the bindingof other pluripotency TFs including SOX2 and NANOG. Similarly,the binding of FOXD3, a TF essential for early embryonicdevelopment (Hanna et al., 2002), increases chromatinaccessibility at its newly bound sites in EpiLCs, potentiallythrough its interaction with BRG1 (Krishnakumar et al., 2016).FOXD3 also interacts with HDAC1/2 and LSD1, suggesting it canrecruit these factors to simultaneously suppress its target genes(Krishnakumar et al., 2016; Respuela et al., 2016).

Alternatively, TFs can promote silencing of specific loci.Activity-dependent neuroprotective protein (ADNP), a proteinassociated with neurological disorders, was recently shown to beessential for naïve pluripotency (Ostapcuk et al., 2018). Thisfunction is based on its sequence-specific binding and recruitmentof heterochromatin protein 1 (HP1, also known as CBX5) andCHD4 to euchromatic H3K9me3-depleted regions, leading todiminished chromatin accessibility and cognate gene expression.These data highlight the necessity of TFs to actively suppresschromatin accessibility, thereby preventing premature expression oflineage-specifying genes. Another unexpected mechanism of TFregulation by epigenetic enzymes was demonstrated by a recentstudy showing the requirement of lysyl oxidase-like 2 (LOXL2)-dependent oxidation of lysine residues in the transcription initiationfactor TFIID subunit TAF10 during ESC differentiation (Iturbideet al., 2015). In this context, oxidation results in degradation ofTAF10, release of core TFIID complex members from corepluripotency promoters and, consequently, decreased transcription.

Conclusions and future directionsTranscriptional regulation of maintenance and exit of pluripotencyexemplifies the requirement of the interplay between sequence-specific TFs and chromatin-modifying enzymes to regulate geneactivation and repression. This interplay is often transient andmultivalent, requiring experimental approaches with appropriatetemporal and molecular resolution in order to dissect andcharacterize outstanding key questions (Box 1). A prerequisite toreliably apply such approaches is to account for transcriptionalheterogeneity derived from cell-to-cell variation and variation

Cohesin

Naïve Formative

Maintenance of pluripotency gene expressionFormative-specific gene expression

Naïve pluripotency gene expression

Cohesin

Naïve-expressed TF

Formative-induced TF

Collaborating TF

Target gene promoter

Naïve-active enhancer

Formative-active enhancer

Key

Fig. 8. Enhancer switching between ESC pluripotencystates. The transition from naïve to formative pluripotency isassociated with numerous changes in enhancer activity, TFand cohesin occupancy. Naïve-expressed TFs collaborate todrive naïve-specific gene expression. Upon exit from naïvepluripotency they can be redirected through collaboration withformative-specific TFs to activate other enhancers and induceformative-specific genes or contribute to maintenance ofexpression of certain pluripotency genes.

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between culture conditions. The extent of this heterogeneity can beaddressed by single cell analyses. For example, single cell RNAsequencing shows that S/L-cultured ESCs have a higher degree oftranscriptional heterogeneity than 2i-cultured ESCs (Guo et al.,2016). Therefore, it is hard to interpret whether differences inpopulation-based assays using S/L culture conditions co-occur inthe same cells or occur in distinct subsets of cells. Althoughtranscriptional heterogeneity might have important functions duringearly embryonic development, such as during the naïve-to-formative pluripotency transition, 2i-cultured ESCs correlatebetter with the transcriptome of early epiblast cells, making 2i amore biologically meaningful culture method (Hayashi et al., 2011;Nakamura et al., 2016). However, long-term culture in 2i can lead toa loss of imprints (Choi et al., 2017; Yagi et al., 2017). Similarly,EpiLCs are more similar to late epiblast cells than EpiSCs are(Hayashi et al., 2011). In general, the field would profit from morestandardized culture conditions (Mulas et al., 2019) that wouldfacilitate data comparisons across multiple studies. The ultimate

goal of these culture models should be to develop trulyphysiologically relevant models of epigenetic control oftranscriptional regulation that better inform our understanding ofpluripotency and early cell fate decisions in the embryo.

Besides more standardized culture conditions, several recenttechnological advances have proven suitable to address themechanisms underlying the dynamic interplay of chromatinstructure and transcriptional regulation. These includeimplementations of single cell-based analyses of chromatinaccessibility, structure and epigenetic marks (reviewed by Ludwigand Bintu, 2019), which could be applied to early stages of ESCdifferentiation. In addition, tools such as the auxin-inducible degronsystem allow for increased temporal resolution and have beenfundamental in highlighting the essential structural role of CTCF inTAD boundary maintenance (Nora et al., 2017). Such tools could beapplied to analyze other complexes that, when deleted, areassociated with deleterious phenotypes, such as the SWI/SNFcomplex. In addition, multi-subunit complexes with changingsubunit composition and no apparent sequence specificity arefrequently localized to defined loci, raising the question of whatprovides the specificity. Locus-specific mass spectrometryapproaches such as ChIP-SICAP or CAPTURE have proven to bevaluable in specifically identifying novel factors and complexcompositions associated with known chromatin-associated factors(Liu et al., 2017; Rafiee et al., 2016). In addition, imagingtechnologies such as live-cell super-resolution imaging and electrontomography are valuable to resolve the structural dynamicsassociated with different ESC states (Cho et al., 2018; Ou et al.,2017).

Overall, the combination of optimized culture conditions with therapidly evolving technologies for chromatin structure analysismakes the ESC culture system ideal to address the impact oftranscriptional control, epigenetic modifications and nucleararchitecture on developmental cell fate decisions.

NRCs

MLLs

P300/CBP

JMJDs

PRC1

PRC2

HDACs

H3K9MTs

TF TF TF

H3K4me1

H3K4me3

H3K27ac

H3K9me2/3

H3K27me3

Enhancer

Promoter

TF TF

TF TF

eRNA expression

Recruitment

Recruitment

Target gene expression

Key

/ /

/ /

Fig. 9. Regulation of enhancer and promoter activity through TF-mediated recruitment of chromatin-modifying enzymes. Collaborative bindingof multiple TFs creates an environment favoring the recruitment of chromatin-modifying enzymes. Enzymes catalyzing modifications that correlate with enhancerand gene activity include nucleosome remodeling complexes (NRCs), MLLs, P300/CBP and JMJDs. Enzymes catalyzing modifications that correlate withenhancer and gene repression include PRC1/2, HDACs and H3K9methyltransferases (H3K9MTs). Active enhancers and promoters typically correlate with localchromatin accessibility and H3K27ac signal. In addition, active enhancers and promoters carry H3K4me1 and H3K4me3, respectively. Inactive enhancersand promoters typically lose chromatin accessibility and gain H3K9 and H3K27 methylation.

Box 1. Key questions• Is MLL-dependent H3K4me1 and P300/CBP-dependent H3K27ac

required for maintenance of active ESC enhancers and/or de novoenhancer activation during differentiation?

• What is the impact of differential DNA methylation and PRC activity onthe activity of enhancer and gene sets, and what is the link tomaintenance and exit of pluripotency?

• What is the epistatic relationship of nucleosome remodeling, histoneand DNA modifications, and TF binding during de novo enhanceractivation and decommissioning of active enhancers?

• How do changes in chromatin architecture link to transcriptionalactivity in pluripotency and differentiation, and which factors areinvolved?

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AcknowledgementsWe thank Ryan Boileau, Bryan Marsh and Brian DeVeale for critical reading of themanuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingD.G. is supported by a research fellowship from the DeutscheForschungsgemeinschaft and R.B. is supported by grants from the NationalInstitutes of Health (R01GM125089-01 and R01GM122439-01). Deposited in PMCfor release after 12 months.

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