Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML.

Key points• Dopamine's control over excitatory signals from the cortex to the nucleus accumbens is thought to underlie motor learning, behavioural reinforcement and drug dependence.• In this study, we combined optical recordings of presynaptic release with whole-cell electrophysiology in CB 1 receptor-null mice and bacterial artificial chromosome (BAC) transgenic mice with fluorescently labelled D1 and D2 receptor-expressing neurons to identify the specific interactions between dopamine and glutamate signalling at individual cortical terminals within the nucleus accumbens core.• Experiments showed that dopamine produces frequency-dependent filtering of low-probability release synapses. At low frequencies, D1 receptors excited striatal output neurons of the striatonigral and striatopallidal pathways, while D2 receptors specifically inhibited neurons of the striatopallidal pathway. At higher frequencies, the dopamine-dependent release of adenosine and endocannabinoids promoted further temporal filtering of cortical signals entering both output pathways.• These results help us understand how dopamine provides frequency and temporal filtering of cortical information by promoting activity through the striatonigral pathway, while inhibiting weak signals.Abstract Interactions between dopamine and glutamate signalling within the nucleus accumbens core are required for behavioural reinforcement and habit formation. Dopamine modulates excitatory glutamatergic signals from the prefrontal cortex, but the precise mechanism has not been identified. We combined optical and electrophysiology recordings in murine slice preparations from CB 1 receptor-null mice and green fluorescent protein hemizygotic bacterial artificial chromosome transgenic mice to show how dopamine regulates glutamatergic synapses specific to the striatonigral and striatopallidal basal ganglia pathways. At low cortical frequencies, dopamine D1 receptors promote glutamate release to both D1 and D2 receptor-expressing medium spiny neurons while D2 receptors specifically inhibit excitatory inputs to D2 receptor-expressing cells by decreasing exocytosis from cortical terminals with a low probability of release. At higher cortical stimulation frequencies, this dopaminergic modulation of presynaptic activity is occluded by adenosine and endocannabinoids. Glutamatergic inputs to both D1 and D2 receptor-bearing medium spiny neurons are inhibited by adenosine, released upon activation of NMDA and AMPA receptors and adenylyl cyclase in D1 receptor-expressing cells. Excitatory inputs to D2 receptor-expressing cells are specifically inhibited by endocannabinoids, whose release is dependent on D2 and group 1 metabotropic glutamate receptors. The convergence of excitatory and inhibitory modulation of corticoaccumbal activity by dopamine, adenosine and endocannabinoids creates subsets of corticoaccumbal inputs, selectively and temporally reinforces strong cortical signals through the striatonigral pathway while inhibiting the weak, and may provide a mechanism whereby conti...

Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell–induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes. These hotspots are enriched with histone H2A isoforms and RNA binding proteins and are associated with evolutionarily conserved elements of the human genome. These findings provide a basis for understanding genome integrity as it relates to aging and disease in the nervous system.

Constitutive JAK-STAT signaling drives the proliferation of most myeloproliferative neoplasms (MPN) and a subset of acute myeloid leukemia (AML), but persistence emerges with chronic exposure to JAK inhibitors. MPN and post-MPN AML are dependent on tyrosine phosphorylation of STATs, but the role of serine STAT1 phosphorylation remains unclear. We previously demonstrated that Mediator kinase inhibitor cortistatin A (CA) reduced proliferation of JAK2-mutant AML in vitro and in vivo and also suppressed CDK8-dependent phosphorylation of STAT1 at serine 727. Here we report that phosphorylation of STAT1 S727 promotes the proliferation of AML cells with JAK-STAT pathway activation. Inhibition of serine phosphorylation by CA promotes growth arrest and differentiation, inhibits colony formation in MPN patient samples and reduces allele burden in MPN mouse models. These results reveal that STAT1 pS727 regulates growth and differentiation in JAK-STAT activated neoplasms and suggest that Mediator kinase inhibition represents a therapeutic strategy to regulate JAK-STAT signaling.

Objective-Prenatal cocaine exposure (PCE) can cause persistent neuropsychological and motor abnormalities in affected children, but the physiological consequences of PCE remain unclear. Conclusions drawn from clinical studies can sometimes be confounded by poly-substance abuse and nutritional deprivation. However, existing observations suggest that cocaine exposure in utero, as in adults, increases synaptic dopamine and promotes enduring dopamine-dependent plasticity at striatal synapses, altering behaviors and basal ganglia function.Methods-We used a combination of behavioral measures, electrophysiology, optical imaging, and biochemical and electrochemical recordings to examine corticostriatal activity in adolescent mice exposed to cocaine in utero.Results-We show that PCE caused abnormal dopamine-dependent behaviors, including heightened excitation following stress and blunted locomotor augmentation to repeated treatment with amphetamine. These abnormal behaviors were consistent with abnormal GABA interneuron function, which promoted a reversible depression in corticostriatal activity. PCE hyperpolarized and reduced tonic GABA currents in both fast-spiking and PLTS-type GABA interneurons to increase tonic inhibition at GABA B receptors on presynaptic corticostriatal terminals. While D2 receptors paradoxically increased glutamate release following PCE, normal corticostriatal modulation by dopamine was reestablished with a GABA A R antagonist.Interpretation-The dynamic alterations at corticostriatal synapses that occur in response to PCE parallel the reported effects of repeated psychostimulants in mature animals, but differ in being specifically generated through GABA. Our results indicate that approaches which normalize GABA and D2 receptor-dependent synaptic plasticity may be useful for treating the behavioral effects of PCE and other developmental disorders that are generated through abnormal GABAergic signaling.

Several recent studies have demonstrated substantial biological differences between cord blood (CB)- and adult peripheral blood (PB)-derived megakaryocytes (MKs). Specifically, neonatal (CB) progenitors proliferate at a much higher rate than adult (PB) progenitors, and generate 10-fold more MKs per progenitor when cultured with thrombopoietin (Tpo). The highly proliferative neonatal MKs undergo cytoplasmic maturation without polyploidization, which ultimately generates large numbers of small, low ploidy, but fully mature MKs. Adult MKs, in contrast, undergo successive rounds of endomitosis to reach much higher ploidy levels, and maturation is coupled with polyploidization, so that MKs with the highest ploidy levels are also the most mature. The molecular mechanisms underlying these developmental differences are just beginning to be elucidated. Here, we investigated the effects of cortistatin A (CA), a highly specific small molecule inhibitor of cyclin-dependent kinase 8 (CDK8) and its paralog CDK19, on megakaryopoiesis. CDK8 and CDK19, together with CCNC (Cyclin C), MED12/MED12L and MED13/MED13L, form "CDK modules" which can associate with Mediator, a 26-subunit complex that acts as a bridge between transcription factors and the transcriptional machinery to coordinate gene expression. The Mediator complex has been implicated in developmental disorders and cancer. CB-derived MK progenitors treated with CA from day 7 to day 14 of culture exhibited a dose-dependent reduction in proliferation (6.2±1.7 vs. 24.9±2.2 fold expansion in treated vs. control cultures; p=0.003), accompanied by an increase in ploidy levels to those comparable to adult PB-derived MKs (34±6% vs. 8.9±0.9% MKs with ploidy ≥8N in treated vs. control cultures; p=0.014). MK maturation, evaluated by CD42b surface expression level, also increased with advancing ploidy in CA-treated MKs, in a manner similar to that observed in adult MKs, and CA-treated mature MKs were capable of pro-platelet formation in vitro. These changes were not observed when undifferentiated CB-derived CD34+ or CD41-negative cells were treated, indicating that the effect was specific to committed MK progenitors. CA treatment induced the expected decrease in STAT-1 phosphorylation at Serine 727, a specific site of CDK8-mediated phosphorylation, confirming effective CDK8 inhibition in treated MKs. Next, we used microarray to evaluate the gene expression profile of CB-derived MKs following CA treatment for 4 and 8 hours vs. untreated cells. These studies revealed significant CA-induced changes in the MK gene expression profile. By Gene Set Enrichment Analysis (GSEA), CA treatment significantly upregulated genes that were downregulated in CB- vs. PB-derived MKs. Furthermore, genes upregulated by CA in CB-derived MKs were also upregulated in the megakaryoblastic cell line SET2 (Pelish, Liau et al., Nature, in press), suggesting that the gene expression program affected in SET2 cells is likely the same one affected in neonatal MKs. At the protein level, we observed time- and dose-dependent increases in RUNX-1 in CA-treated vs. control MKs. In summary, this study demonstrated that treatment with CA induced a phenotypic switch from neonatal to adult-like megakaryopoiesis, accompanied by changes in the MK gene expression profile. These findings indicate a novel role for Mediator kinases in the regulation of megakaryopoiesis, and potentially on the developmental differences between neonatal and adult MKs. These studies open the door to a better understanding of and to potential novel therapies for a number of developmental stage-specific megakaryocyte and platelet disorders, which exclusively or more severely affect neonates and infants, including the thrombocytopenia/absent radius (TAR) syndrome and the transient myeloproliferative disorder associated with trisomy 21 and GATA1s mutations. Disclosures No relevant conflicts of interest to declare.