SummaryThe mechanisms responsible for the transcriptional silencing of pluripotency genes in differentiated cells are poorly understood. We have observed that cells lacking the tumor suppressor p27 can be reprogrammed into induced pluripotent stem cells (iPSCs) in the absence of ectopic Sox2. Interestingly, cells and tissues from p27 null mice, including brain, lung, and retina, present an elevated basal expression of Sox2, suggesting that p27 contributes to the repression of Sox2. Furthermore, p27 null iPSCs fail to fully repress Sox2 upon differentiation. Mechanistically, we have found that upon differentiation p27 associates to the SRR2 enhancer of the Sox2 gene together with a p130-E2F4-SIN3A repressive complex. Finally, Sox2 haploinsufficiency genetically rescues some of the phenotypes characteristic of p27 null mice, including gigantism, pituitary hyperplasia, pituitary tumors, and retinal defects. Collectively, these results demonstrate an unprecedented connection between p27 and Sox2 relevant for reprogramming and cancer and for understanding human pathologies associated with p27 germline mutations.

BackgroundThe NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated.Methodology/Principal FindingsHere we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation.Conclusions/SignificanceWe characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline.

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...

A haploid genotype may be insufficient to support normal wild-type function. Such haplo-insufficiency has recently been documented for numerous tumour suppressor genes. p53 is a crucial tumour suppressor governing DNA repair, cell cycle arrest and apoptosis via its role as a stressresponsive transcription factor. p53 haplo-insufficiency has been observed in vivo with human familial cancer in Li-Fraumeni Syndrome (LFS) and in mouse p53-knockout models of LFS. The increased tumorigenesis associated with loss of one p53 allele has been attributed to reduced p53-dependent stress responses. However, the underlying biochemical basis for such attenuated responses in p53 þ /À cells remains unclear. Here we have determined basal p53 messenger RNA (mRNA) and protein levels, and compared the p53 stress response in p53 þ / þ , p53 þ /À and p53À/À isogenic clones derived from HCT116 cells. Basal expression of p53 in p53 þ /À cells was 25% relative to p53 þ / þ cells, and this differential was maintained following oncogenic stress. This deficiency was manifested at both p53 mRNA and protein levels and resulted in attenuated p53 stress responses, in particular for p21 waf1 upregulation and survivin downregulation, and reduced G1 arrest and apoptosis. These observations identify a molecular basis for wild-type p53 haplo-insufficiency, which may explain the attenuated tumour-suppressive phenotype observed in cells with a single wild-type p53 allele and in humans with LFS.

SummaryThe RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.

Previously, we have observed a link between p53 expression and histone H3 post-translational modifications. Here, we ask if specific post-translational modifications of p53 impact upon histone H3 modifications in a selective manner. We have also screened for internal co-operative effects within the repertoire of p53 modifications. Exogenous p53 constructs were expressed in HCT116 p53 À/À cells. Four mutant p53 constructs were used, with single 'phosphorylation' mutations at serines 15 and 37 (S15A, S15D, S37A and S37D) and compared with exogenously expressed wild-type p53. The results showed that the replacement of serine 15 with either alanine (S15A) or aspartic acid (S15D) induced phosphorylation at S33P, S37P and S46P. In contrast, phosphorylation mutants p53(S37A) and p53(S37D) were not phosphorylated on S33. S46 phosphorylation appeared specifically enhanced by p53(S37D) relative to p53(S37A). Distal induction of S392 phosphorylation was observed for each of the p53 N-terminal phosphorylation mutants. Analysis of endogenous histone H3 (from the transfected cells) revealed loss of di-methylated K9 following expression of wild type and mutant p53 constructs. Expression of p53 (S15A), (S15D) and (S37A) selectively induced acetylation at K9 and K14. In contrast, wt p53 and p53(S37D) had no effect upon K9 or K14 acetylation. K18 acetylation status was unaffected throughout.

A disordered to β-sheet transition was thought to drive the functional switch of Q/N-rich prions, similar to pathogenic amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) regions within yeast prion domains in amyloid formation. We show that many human prion-like domains (PrLDs) contain CC regions that overlap with polyQ tracts. Most of the proteins bearing these domains are transcriptional coactivators, including the Mediator complex subunit 15 (MED15) involved in bridging enhancers and promoters. We demonstrate that the human MED15-PrLD forms homodimers in solution sustained by CC interactions and that it is this CC fold that mediates the transition towards a β-sheet amyloid state, its chemical or genetic disruption abolishing aggregation. As in functional yeast prions, a GFP globular domain adjacent to MED15-PrLD retains its structural integrity in the amyloid state. Expression of MED15-PrLD in human cells promotes the formation of cytoplasmic and perinuclear inclusions, kidnapping endogenous full-length MED15 to these aggregates in a prion-like manner. The prion-like properties of MED15 are conserved, suggesting novel mechanisms for the function and malfunction of this transcription coactivator.

After reprogramming to naive pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naive PSCs cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human, and rhesus monkey naive PSCs and analyzed their ability to colonize rabbit and cynomolgus monkey embryos. Mouse embryonic stem cells (ESCs) remained mitotically active and efficiently colonized host embryos. In contrast, primate naive PSCs colonized host embryos with much lower efficiency. Unlike mouse ESCs, they slowed DNA replication after dissociation and, after injection into host embryos, they stalled in the G1 phase and differentiated prematurely, regardless of host species. We conclude that human and non-human primate naive PSCs do not efficiently make chimeras because they are inherently unfit to remain mitotically active during colonization.