Manipulating the Mediator complex to induce naïve pluripotency

Abstract

Human naïve pluripotent stem cells (PSCs) represent an optimal homogenous starting point for molecular interventions and differentiation strategies. This is in contrast to the standard primed PSCs which fluctuate in identity and are transcriptionally heterogeneous. However, despite many efforts, the maintenance and expansion of human naïve PSCs remains a challenge. Here, we discuss our recent strategy for the stabilization of human PSC in the naïve state based on the use of a single chemical inhibitor of the related kinases CDK8 and CDK19. These kinases phosphorylate and negatively regulate the multiprotein Mediator complex, which is critical for enhancer-driven recruitment of RNA Pol II. The net effect of CDK8/19 inhibition is a global stimulation of enhancers, which in turn reinforces transcriptional programs including those related to cellular identity. In the case of pluripotent cells, the presence of CDK8/19i efficiently stabilizes the naïve state. Importantly, in contrast to previous chemical methods to induced the naïve state based on the inhibition of the FGF-MEK-ERK pathway, CDK8/19i-naïve human PSCs are chromosomally stable and retain developmental potential after long-term expansion. We suggest this could be related to the fact that CDK8/19 inhibition does not induce DNA demethylation. These principles may apply to other fate decisions.

Keywords: 2i; CDK8; Demethylation; Embryonic stem; MEK; Mediator; Naïve; Phase separation; Pluripotency; Primed; RNA polymerase; Reprogramming; Super-enhancers.

Graphical abstract

Fig. 1

The Mediator complex integrates multiple signals and bridges enahcers to promoters to regulate pluripotent cell identity. Mediator acts as a central hub which links enhancers and target promoters, integrates combinatorial signals, and recruits RNA Pol II, to guide the transcriptional program of cell identity. The core transcriptional machinery is shown simplified, within a phase-separated transcriptional condensate (light green, membrane-less region). The largest enhancers, known as super-enhancers emerge by coalescing multiple smaller constituent enhancer regions (purple thick lines), each enriched in lineage-specifying transcription factors, histone marks, and chromatin regulators (indicated by small coloured circles). Mediator associates with these many factors and receives upstream signaling inputs from pathways, including MEK-ERK. Mediator recruits RNA Pol II to the pre-initiation complex, a function which can be hindered by the CDK8/19 kinase-module, via structural or kinase-dependent activities of CDK8/19. In PSCs, the 2i inhibitor cocktail is known to rapidly alter the transcriptional program to promote naïve pluripotency, of which, MEK-inhibition is the key feature. However, how MEK-inhibition resets the transcriptional machinery to the naïve program remains to be clarified. In PSCs, we find that MEK-inhibition leads to global up-regulation of enhancer activity via increased RNA Pol II recruitment. We find that the reorganization of the transcriptional machinery by MEK inhibition operates largely via controling the activity of CDK8/19 downstream. In this way, both treatments induce a highly overlapping set of phospho-changes focused on the transcriptional machinery, hyper-activating enhancers and Mediator, triggering increased RNA Pol II recruitment, and promoting the transcriptional program of naïve pluripotency.

Fig. 2

A summary of CDK8/19 function in early embryo cell identity transitions. (A) Schematic overview of mouse early embryo development. Naïve PSCs can be isolated from the E4.5 epiblast, and they can retain this cell identity homogenously in vitro. Primed PSCs can be isolated from the E6.5 epiblast, and they can be maintained in the fluctuating and heterogenous primed state in vitro. The 2i inhibitor cocktail can stabilize naïve identity, and promote primed PSCs to transition into the naïve state. (B) Similar to small molecule 2i-treatment of PSCs in vitro, in the embryo, MEK-signaling is naturally repressed by down-regulation of the FGF receptor. We find that CDK8/19 kinase inhibition, by genetic or pharmacological methods, can phenocopy these effects of 2i: stabilizing naïve identity, and promoting primed cells to transition into the naïve state. Interestingly, we observe that in the embryo, CDK8 activity appears to be naturally repressed, with down-regulation of CDK8 levels and decreased nuclear availability of its essential binding partner Cyclin C. The table below summarizes the developmental phenotypes observed by genetic- or pharmacological-inhibition of CDK8 in the mouse. (C) A summary of the molecular and morphological differences reported for naïve and primed pluripotent states.

Fig. 3

Emergence and dissolution of transcriptional programs in the naïve to prime transition. A putative model of dominance and antagonism in transcriptional programs. Binary cell fate choices are common though development and regeneration. In the case of the naïve-primed developmental window of pluripotency, global hyperactivation of enhancers and Mediator-recruitment of RNA Pol II produces a coherent outcome: up-regulation and stabilization of naïve epiblast identity. Here, we suggest a putative mechanism. In primed identity, cells are plastic and heterogenous due to co-existence of lineage-specifying transcriptional programs for forward development into the 3 embryoinc germ layers, however, they are moderately expressed, antagonistic, and weakly established. Powerful naïve-specifiying transcription factors and super-enhancers have begun their decline, but are not yet decommissioned. Upon global hyperactivation of enhancers and Mediator-recruitment of RNA Pol II (by treatment with 2i or CDK8/19i), the naïve-specifiying transcription factors and super-enhancers become dominant, and suppress the nascent germ layer programs.

Fig. 4

Unified model of 2i and CDK8/19i naïve pluripotency. A summary of the signaling hierarchy between MEK, CDK8/19-kinase, and Mediator. A large proportion of the effect of MEK signaling channels through CDK8/19-Mediator to affect the transcriptional program of cell identity. CDK8/19-independent effects of MEK inhibition that we have identified include global DNA hypo-methylation in mouse and human PSCs, specification of the primitive endoderm in the mouse pre-implnatation blastocyst, and, in human PSCs, SSEA4 down-regulation.