Depletion of Mediator Kinase Module Subunits Represses Superenhancer-Associated Genes in Colon Cancer Cells

In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by beta-catenin, which has previously been shown to associate with MED12. Importantly, beta-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.Peer reviewe

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. Pancreatic islets derived from stem cells are benchmarked against primary cells.Peer reviewe

Function of the Metazoan Mediator Kinase Module in Transcription

Precise control of transcription, the copying of the genetic DNA code to an expressed RNA molecule, is fundamental for all processes of an organism. Transcriptional regulation is dependent on the activity of proteins and protein complexes including general and cell specific transcription factors and co-activators. Studying how these transcriptional regulators function is crucial for understanding processes such as development and disease. This PhD thesis focuses on the function of one evolutionarily conserved transcriptional co-activator, called the Mediator complex, and in particular on the role of its kinase module. This kinase module consists of four proteins: Cdk8, cyclin C, Med12 and Med13 in lower metazoans and CDK8 or CDK19, cyclin C, MED12 or MED12L and MED13 or MED13L in vertebrates. The Mediator kinase module regulates transcription through various mechanisms, including association with transcription factors and regulation of enhancer-dependent transcription. Kinase module deregulation is implicated in developmental disorders and cancer, but the molecular mechanisms underlying these diseases remain poorly understood. CDK8 was found to be dispensable for cell-autonomous survival but required for mouse embryonic development at the pre-implantation stage. Cdk8-CycC-mediated regulation of transcription was dependent on Med12-Med13 while Cdk8 and CycC depletion caused distinct and even opposite effects on gene expression as compared to Med12 and Med13 depletion in fruit fly cells. This work identified highly similar effects on transcription after depletion of fruit fly Med12 or Med13, suggesting limited Med12-independent functions for Med13. Kinase module subunit hierarchy was conserved in human colon cancer cells where depletion of MED12 or double depletion of the redundant MED13 and MED13L also resulted in highly similar transcriptional responses. Med12 and Med13 were found to be activators of innate immunity genes that are dependent on the serpent/GATA transcription factor in fruit fly cells and larvae. In human colon cancer cells, MED12, MED13 and MED13L were disproportionally required for the expression of genes associated with cancer-acquired super-enhancers. MED12 or MED13 and MED13L depletion caused a dramatic decrease in the expression of the super-enhancer associated MYC oncogene and impaired proliferation of colon cancer cells, suggesting that targeting of these kinase module subunits is a possible future therapeutic opportunity. This thesis extends the present understanding of the relationships between Mediator kinase module subunits in metazoan species and identifies kinase module-dependent functions in development, immunity and cancer-acquired transcription. Further research should be directed at studying the molecular mechanisms of kinase module functions in vivo and at the potential to target this complex with the goal of treating human diseases such as cancer.Trots att det genetiska materialet, DNA, i alla celler i en flercellig organism så som människan är identiskt består en individ av hundratals olika typer av specialiserade celler. Detta är möjligt eftersom olika celler inte aktivt använder hela det genetiska materialet utan bara de gener som behövs för att upprätthålla just den celltypens funktioner. Då en gen är i aktiv användning, expresserad, översätts den genetiska DNA koden till RNA av ett polymerasenzym i en process som kallas transkription. En betydande del av generna transkriberas av RNA-polymeras II och kodar för budbärar-RNA, eller mRNA, som i sin tur innehåller information om hur cellen ska sammansätta proteiner. RNA-polymeras II enzymets aktivitet regleras av en mängd olika transkriptionsfaktorer och ko-faktorer för att kontrollera att cellen transkriberar de gener som behövs. En sådan viktig ko-faktor är Mediatorn, ett protein-komplex som består av runt 30 olika proteiner. Mediatorn är viktig för kommunikationen mellan RNA-polymeras II, cell-specifika transkriptions-faktorer och så kallade enhancer-områden, sekvenser i DNA som förstärker en gens transkriptionsaktivitet. Den här avhandlingen har fokuserat på Mediatorns kinas-komplex, som består av de fyra proteinerna Cdk8, Cyklin C, Med12 och Med13 (i ryggradsdjur CDK8 eller CDK19, Cyclin C, MED12 eller MED12L och MED13 eller MED13L). Mediatorns kinas-komplex har visats vara viktigt för transkription styrd av cellspecifika transkriptionsfaktorer och enhancer-områden i olika sammanhang. Mutationer i kinas-komplexets proteiner eller obalans i proteinernas mängd kan leda till olika sjukdomar som till exempel cancer. Avhandlingen strävade till att klargöra kinas-komplexets olika delproteiners funktion, identifiera gener som är beroende av kinas-komplexet och studera kinas-komplexets roll i levande organismer in vivo. Den här avhandlingen utökar vetskapen om Mediatorns kinas-komplex funktion i olika flercelliga organismer. Studien identifierade nya funktioner för kinas-komplexet under embryonalutvecklingen, i det medfödda immunförsvaret och i reglering av gener som är viktiga i tjocktarmscancerceller. Vidare klargjorde studien förhållanden mellan olika proteiner i kinas-komplexet och identifierade gener vars transkription är beroende av de olika delproteinerna. Vidare forskning bör inriktas på att klargöra de olika delproteinernas roll in vivo och möjligheten att manipulera kinas-komplexets funktion i medicinskt syfte, till exempel i vård av cancer

Cdk8 Is Essential for Preimplantation Mouse Development▿†

The Cdk8 kinase and associated proteins form a nonessential transcriptional repressor module of the Mediator in the budding yeast Saccharomyces cerevisiae. Genetic analyses of this module have demonstrated functions ranging from environmental responses in budding yeast to organogenesis and development in worms, flies, and zebrafish. Here we have investigated the function of mammalian Cdk8 using mice harboring a gene trap insertion at the Cdk8 locus inactivating this kinase. No phenotypes were noted in heterozygote Cdk8+/− mice, but intercrossing these did not produce homozygous Cdk8−/− offspring. Developmental analysis demonstrated a requirement for Cdk8 prior to implantation at embryonic days 2.5 to 3.0. Cdk8−/− preimplantation embryos had fragmented blastomeres and did not proceed to compaction. As Cdk8 deficiency in cultured metazoan cells did not affect cell viability, the results suggest that transcriptional repression of genes critical for early-cell-fate determination underlies the requirement of Cdk8 in embryogenesis

Restaurants and Carbon Foodprint Mission Zero Foodprint Workbook

The significance of responsibility, and environmentally friendly and meaningful actions will increase as as competitive factors in restaurants, and customer awareness and values will add pressure for this. All sectors have their own environmental impacts, but for restaurants, these are quite large. According to the Natural Resources Institute Finland, over one fifth of carbon footprints are created from food production and consumption. Reducing the carbon footprint of operations does not necessarily require you to make huge changes in what you do; everyday choices can play a major role in the environment. You can continue to create experiences for your customers with good food, drink and service, but in a more sustainable way and more cost-effectively. This is workbook is made for you and everyone involved in the restaurant sector by Mission Zero Foodprint project. The workbook contains all useful pieces of information for climate action and effective measures in the restaurant sector. The workbook also gives you tips on how to communicate about your climate actions to your customers and partners. Be at the forefront rather than lagging behind. In the end, everyone will participate in the reduction of emissions. We hope you will find these tools for a better future useful! Mission Zero Foodprint was funded by European Regional Development Fund and Uusimaa Regional Council.Julkaisun suomekielinen versio https://urn.fi/URN:ISBN:978-951-799-627-

Ravintolat hiilijalanjäljillä Mission Zero Foodprint -työkirja

Vastuullisuus, ympäristöystävällisyys ja merkityksellisyys ravintoloiden kilpailukykytekijänä kasvaa ja asiakkaiden valistuneisuus ja arvot lisäävät tähän painetta. Kaikilla toimialoilla on omat ympäristövaikutuksensa, mutta ravintoloilla ne ovat varsin suuret. Luonnonvarakeskuksen mukaan reilu viidennes hiilijalanjäljestä syntyy ruoantuotannosta ja –kulutuksesta. Toiminnan hiilijalan pienentäminen ei välttämättä vaadi suuria muutoksia toiminnassasi, arjen valinnoilla voi olla suuri merkitys ympäristölle. Voit jatkaa elämysten tuottamista asiakkaillesi hyvän ruoan, juoman ja palvelun parissa, mutta kestävämmin ja kustannustehokkaammin. Tässä on Sinulle ja kaikille ravintola- alan toimijoille Mission Zero Foodprint -hankkeen työkirja, johon on koottu kaikki hyödyllinen tieto ilmaston hyväksi tehtäville, tehokkaille toimenpiteille ravintola-alalla. Työkirjasta saat myös vinkkejä, miten viestit ilmastoteoistasi asiakkaillesi ja yhteistyökumppaneillesi. Ole mieluummin etujoukoissa, kuin jälkijunassa. Loppujen lopuksi kaikki tulevat osallistumaan päästöjen vähennystalkoisiin. Toivomme, että hyödynnät näitä työkaluja paremman tulevaisuuden eteen

Aberrant metabolite trafficking and fuel sensitivity in human pluripotent stem cell-derived islets

Pancreatic islets regulate blood glucose homeostasis through the controlled release of insulin; however, current metabolic models of glucose-sensitive insulin secretion are incomplete. A comprehensive understanding of islet metabolism is integral to studies of endocrine cell development as well as diabetic islet dysfunction. Human pluripotent stem cell-derived islets (SC-islets) are a developmentally relevant model of human islet function that have great potential in providing a cure for type 1 diabetes. Using multiple 13C-labeled metabolic fuels, we demonstrate that SC-islets show numerous divergent patterns of metabolite trafficking in proposed insulin release pathways compared with primary human islets but are still reliant on mitochondrial aerobic metabolism to derive function. Furthermore, reductive tricarboxylic acid cycle activity and glycolytic metabolite cycling occur in SC-islets, suggesting that non-canonical coupling factors are also present. In aggregate, we show that many facets of SC-islet metabolism overlap with those of primary islets, albeit with a retained immature signature.Peer reviewe

Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.Peer reviewe

Human IP3 receptor triple knockout stem cells remain pluripotent despite altered mitochondrial metabolism

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER Ca2+-release channels that control a broad set of cellular processes. Animal models lacking IP3Rs in different combinations display severe developmental phenotypes. Given the importance of IP3Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP3R and triple IP3R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP3R isoforms, IP3R1, IP3R2, IP3R3, failed to generate Ca2+ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP3Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP3Rs in human cell types of interest.Peer reviewe