Jmjd 2 c / Kdm 4 c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation

Jmjd2/Kdm4 H3K9-demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we uncover that Jmjd2c facilitates this process by stabilizing the assembly of Mediator-Cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2cknockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2, independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and furthermore destabilizes loading of the Mediator and Cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c-G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with impact on timely gene activation. D ev el o pm en t • A dv an ce a rt ic le Introduction Pluripotency, the ability of a cell to generate all somatic lineages, is transiently acquired in vivo during mammalian pre-implantation development. Upon blastocyst formation, pluripotent cells develop within the inner cell mass (ICM), a mosaic of cells surrounded by an extra-embryonic layer the trophectoderm. By the time of implantation, a second extra-embryonic lineage, the primitive endoderm, emerges at the ICM surface. Concurrently, the ICM maintains its pluripotency as it matures into the epiblast but ultimately goes on to form the three primary germ layers and germ cells upon gastrulation (Boroviak and Nichols, 2014; Rossant, 2008). Pluripotent mouse embryonic stem cells (ESCs) are derived from ICM cells, and can self-renew and faithfully maintain an undifferentiated state in vitro in the presence of leukaemia inhibitory factor (LIF) and serum components while preserving their multi-lineage differentiation capacity (Evans and Kaufman, 1981; Martin, 1981; Niwa et al., 1998; Ying et al., 2003). Most recently, stem cell lines with similar lineage potential were established from other developmental stages (Chung et al., 2006; Tesar, 2005), including a number of post-implantation epiblast-derived stem cells (EpiSCs) (Brons et al., 2007; Osorno et al., 2012; Tesar et al., 2007). While ESCs are thought to represent an immature (pre-implantation) phase of pluripotency, EpiSCs exist in a more advanced state on the verge of differentiation (Nichols and Smith, 2009). Moreover, ESCs can stably transit into self-renewing EpiSCs, acquiring characteristics of post-implantation epiblast-like cells (Guo et al., 2009). ESC abilities depend on the potent expression of self-renewal genes and transcriptional priming of silent, lineage-affiliated genes – a critical balance of gene expression maintained through crosstalk between transcriptional factors and chromatin regulators (Azuara et al., 2006; Bernstein et al., 2006; Chen and Dent, 2014; Ng and Surani, 2011; Stock et al., 2007). Remarkably, both active (ESC-specific) and primed (lineagespecific) genes are expressed in a heterogeneous manner, a feature long considered a hallmark of ESC cultures to safeguard the swift response to differentiation cues (Efroni et al., 2008; Torres-Padilla and Chambers, 2014). Yet, it is now possible to derive and maintain ESCs with reduced D ev el o pm en t • A dv an ce a rt ic le heterogeneity and transcriptional gene priming through chemical inhibition of two differentiation-associated pathways Mek and Gsk3 (2i conditions), capturing a naïve pluripotent state in vitro (Marks et al., 2012; Ying et al., 2008). Gene promoter regions enriched in CpG islands and H3K4me3 function as genomic platforms for the recruitment of transcription factors and co-regulators as well as the basal transcriptional machinery (Deaton and Bird, 2011; Illingworth and Bird, 2009). Moreover, distal DNA elements such as enhancers play a significant role in potentiating gene expression being typically decorated by H3K4me1 and bound by pioneer transcription factors (Calo and Wysocka, 2013; Gibcus and Dekker, 2013; Spitz and Furlong, 2012). For example, the core pluripotency factor Oct4 was commonly shown to mark both active and poised enhancers in ESCs and EpiSCs (Buecker et al., 2014; Calo and Wysocka, 2013). Enhancer activity and robust ESCspecific gene expression entail long-range DNA interactions with the transcriptional apparatus at promoters, involving the cooperative action of Mediator-Cohesin complexes (Kagey et al., 2010). Yet, relatively little is known about the identity of proteins that stabilize the formation of such assemblies. Histone demethylases have emerged as key players in the control of cell identity and development mainly through modulation of the chromatin environment of tissue-specific genes (Nottke et al., 2009). Recently, additional roles for these molecules independent of their enzymatic activity were reported (Shpargel et al., 2012; Wang et al., 2012; Yang et al., 2010), especially in regulating the recruitment of Polycomb repressive complexes (PRC) and poised RNA polymerase II to the promoter regions of developmental genes in ESCs (Farcas et al., 2012; Wu et al., 2013). Jmjd2c/Kdm4c is a member of the Jmjd2/Kdm4 gene family initially identified as H3K9me2/3 and/or H3K36me2/3 histone demethylases (Chen et al., 2006; Klose et al., 2006; Whetstine et al., 2006). Jmjd2c is highly expressed in the early embryo and in ESCs (Boroviak et al., 2015; Burton et al., 2013; Loh et al., 2007; Wang et al., 2010), and RNA interference-mediated depletion of the protein was shown to impair cleavage-stage development and ESC integrity as well as inhibiting somatic cell reprogramming (Das et al., 2014; Loh et al., D ev el o pm en t • A dv an ce a rt ic le 2007; Wang et al., 2010). Jmjd2c-null ESCs and mice could, however, be generated via gene trap approaches (Pedersen et al., 2014), in agreement with functional redundancy between Jmjd2 gene family members to support cell proliferation and survival (Pedersen et al., 2016). At the genomic level, Jmjd2c was shown to preferentially target H3K4me3-rich promoter regions of active and developmental-associated genes in ESCs via its Tudor domains (Das et al., 2014; Pedersen et al., 2014), where Jmjd2c was proposed to assist Jmjd2b-Nanog and PRC2 in transcriptional activation and repression, respectively (Das et al., 2014). In this study, we uncover a previously unrecognized link between Jmjd2c recruitment to lineage-specific enhancers and the establishment of a functionally primed state for differentiation in vitro. We show that, in the absence of Jmjd2c, ESC differentiation is severely impeded at an early postimplantation epiblast-like stage. While Jmjd2c-knockout ESCs can transit into self-renewing EpiSCs, these cells fail to form derivatives of the three primary germ layers, as revealed by their inability to initiate appropriate gene expression programs. In contrast, Jmjd2c-knockout cells remain capable of adopting extra-embryonic endoderm-like phenotypes under permissive conditions. Mechanistically, we show that Jmjd2c is re-distributed to lineagespecific enhancers in primed (serum/LIF) as opposed to naïve (2i/LIF) ESCs. Strikingly, Jmjd2c-bound enhancers are co-occupied by the antagonistic enzyme G9a/Ehmt2, independently of its silencing H3K9-modifying activity. We show that Jmjd2c-G9a co-occupancy coincides with the formation of activating, Med1-containing enhancer complexes. Loss of Jmjd2c abrogates G9a recruitment at Jmjd2c-bound distal sites, and this correlates with inefficient loading of Mediator-Cohesin complexes in ESC-derived EpiSCs with impact on gene activation upon lineage specification. Collectively, these data reveal that Jmjd2c is required for successful gene transcription and somatic differentiation in pluripotent stem cells, and propose a novel regulatory role for Jmjd2c in stabilizing the assembly of essential enhancerprotein complexes at the onset of ESC differentiation. D ev el o pm en t • A dv an ce a rt ic le Results Jmjd2c is required for embryonic stem cell differentiation towards somatic lineages Combining genetic deletion and functional assays, we tested whether Jmjd2c plays a role at the exit of pluripotency. Jmjd2c mutant and wild-type JM8-ESCs were obtained through the EUCOMM/IKMC repository (Bradley et al., 2012; Skarnes et al., 2011), and targeting of both Jmjd2c alleles in Jmjd2c-knockout (E2 and E3) ESC clones confirmed by long-range PCR and GFP expression (Fig. S1A-C). Western blotting validated that full-length Jmjd2c protein expression was completely abolished in Jmjd2c-knockout samples (Fig. 1A). This was accompanied by a notable increase in bulk H3K9me2 levels relative to wild-type (Fig. 1B, S1D); an effect attributed to Jmjd2c loss itself in the continued expression of other H3K9-demethylases and methyltransferases (Fig. S1D-E). Constitutively Jmjd2c-depleted E2 and E3 ESC clones grew normally in medium supplemented with serum plus LIF (Fig. S1F), in agreement with a similar conditional knockout model (Pedersen et al., 2014). When plated at low density these cells also displayed comparable proportion of undifferentiated colonies relative to their wild-type counterparts (Fig. 1C), despite reduced expression levels of pluripotencyassociated factors (Fig. S1G). Jmjd2c depletion did not, however, result in general de-repression of differentiation-associated genes (Fig. S1H), including those co-bound by Jmjd2c and PRC complexes at their promoter regions (Das et al., 2014). These