Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases

Abstract

Pluripotent stem cells (PSCs) transition between cell states in vitro and reflect developmental changes in the early embryo. PSCs can be stabilized in the naïve state by blocking extracellular differentiation stimuli, particularly FGF-MEK signaling. Here, we report that multiple features of the naïve state in human and mouse PSCs can be recapitulated without affecting FGF-MEK-signaling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 kinases removes their ability to repress the Mediator complex at enhancers. Thus CDK8/19 inhibition increases Mediator-driven recruitment of RNA Pol II to promoters and enhancers. This efficiently stabilizes the naïve transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition.Moreover, naïve pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naïve pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity. RESULTS Inhibition of Mediator kinase stabilizes mouse naïve pluripotencyGFP knock-in reporters at key stem cell marker genes such as Nanog represent well-established and precise indicators of the naïve (GFP high ) and primed states (GFP low ) 18,22,29 . For example, in 2i-naïve state, Nanog promoter activity is enhanced, yielding a characteristically homogenous Nanog-GFP high cell expression pattern and uniform dome-shaped colonies (Fig. 1A-C, and Extended Data Fig. 1A). In contrast, the Nanog promoter is metastable in primed state PSCs, reversibly oscillating between high and low activity, presenting a heterogeneous Nanog-GFP expression pattern and flattened diffuse colonies, indicative of a general underlying switch in transcriptional program 18,20,23,29,30 . The BRD4 inhibitor JQ1 destabilizes enhancers and resulted in colony flattening and GFP low status (Fig. 1A), as reported [26][27][28] . In this experimental setting, we tested the effect of manipulating the transcriptional cyclin-dependent kinases (CDK7, CDK8/19 and CDK9) with a panel of small molecule inhibitors. Several potent Lynch et al., submitted 19 19 and structurally-unrelated CDK8/19 inhibitors had a positive effect, inducing the formation of homogenous dome-shaped colonies, and upregulating both the Nanog-GFP reporter and endogenous Nanog expression, similar to PSC in the 2i-naïve state (Fig. 1A-E; Extended Data Fig. 1A; Supplementary Table 1), while inhibition of CDK7 or CDK9 did not. Potency and selectivity of CDK8/19inhibitors, commercially available or developed in-house, were assessed by multiple methods: (i) selectivity was suggested by a KinomeScan panel of 456 kinases; (ii) Lanthascreen assays demonstrated inhibitory activity at nanomolar concentrations against pure recombinant CDK8/CCNC and CDK19/CCNC; (iii) luciferase reporter cell assays (TOP-FLASH); and (iv) potent inhibition of STAT1-Ser727 phosphorylation in human PSCs, a well-documented CDK8 t...

Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

“…Cultures were passaged every 5-7 days manually using 0.5 µM EDTA/1xPBS (Gibco, Grand Island, NY, USA). To reset hPSCs to CDK8/19i-naïve state, cells were maintained on Matrigel (Corning #356231 or #354277) using mTeSR1 (Stem Cell Technologies, Vancouver, Canada); the basal media was supplemented with 20 ng/mL of rhLIF (Peprotech, Rocky Hill, NJ, USA) plus 0.4 µM of CNIO-CDK8/19 inhibitor (ETP-47799), as reported [20]. Differentiation of OCT4-∆PE hPSCs as control for the FACS experiments was induced with 10 µM retinoic acid (RA, Sigma, St. Louis, MO, USA) for 5 days [7].…”

“…For short-term pre-exposure, hPSCs were exposed for 4 days to naïve conditions. Naïve cocktails used were: CDK8/19i [0.4 µM of CNIO-CDK8/19 inhibitor and 20 ng/mL hrLIF (Peprotech)], as reported [20]; or the 2i-based PXGL [1 µM PD0325901 (Axon Medchem, Reston, VA, USA), 2 µM Gö6983 (Selleckchem, Houston, TX, USA), 2 µM XAV939 (Selleckchem) and 20 ng/mL hrLIF (Peprotech)] [8]. PGCs-like derivation was performed as previously reported [22].…”

“…Gene set enrichment analysis was performed using the pre-ranked GSEA function as implemented by the Broad Institute [28]. First, we generated two gene signatures of differentially expressed genes (DEGs), either up-regulated or down-regulated, in shortterm cultured human PSCs (D2#2, D2#4, HERVH and H1) (raw data reported in [20] and accessible at GSE127186). We defined gene signatures as DEGs with adjusted FDR q-value lower than 0.1 and log2 fold changes larger than 1 (for up-regulated genes) or lower than −0.5 (for down-regulated genes).…”

“…We showed that the simple addition of one chemical, a CDK8 and CDK19 inhibitor (CDK8/19i), is sufficient to induce the primed to naïve transition in mouse and human PSCs. Interestingly, the inhibition of CDK8 and CDK19 does not deplete genomic DNA methylation and, thereby, does not result in karyotypic abnormalities in human PSCs [20]. Moreover, CDK8/19inaïve human PSCs efficiently form embryoid bodies and teratomas in mice [20], a property that is generally compromised in 2i-naïve human PSCs [12].…”

“…Recently, we reported that the naïve state can be induced and stabilized by stimulating the Mediator complex at enhancers through the inhibition of its kinase repression module, composed of the highly similar kinases CDK8 and CDK19 [20,21]. We showed that the simple addition of one chemical, a CDK8 and CDK19 inhibitor (CDK8/19i), is sufficient to induce the primed to naïve transition in mouse and human PSCs.…”