Elena Manzoni

5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.

Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier.

Epigenetic Erasing and Pancreatic Differentiation of Dermal Fibroblasts into Insulin-Producing Cells are Boosted by the Use of Low-Stiffness Substrate

Several studies have demonstrated the possibility to revert differentiation process, reactivating hypermethylated genes and facilitating cell transition to a different lineage. Beside the epigenetic mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces in supporting cell plasticity and boosting differentiation. Here, we describe epigenetic erasing and conversion of dermal fibroblasts into insulin-producing cells (EpiCC), and demonstrate that the use of a low-stiffness substrate positively influences these processes. Our results show a higher expression of pluripotency genes and a significant bigger decrease of DNA methylation levels in 5-azacytidine (5-aza-CR) treated cells plated on soft matrix, compared to those cultured on plastic dishes. Furthermore, the use of low-stiffness also induces a significant increased up-regulation of ten-eleven translocation 2 (Tet2) and histone acetyltransferase 1 (Hat1) genes, and more decreased histone deacetylase enzyme1 (Hdac1) transcription levels. The soft substrate also encourages morphological changes, actin cytoskeleton re-organization, and the activation of the Hippo signaling pathway, leading to yes-associated protein (YAP) phosphorylation and its cytoplasmic translocation. Altogether, this results in increased epigenetic conversion efficiency and in EpiCC acquisition of a mono-hormonal phenotype. Our findings indicate that mechano-transduction related responsed influence cell plasticity induced by 5-aza-CR and improve fibroblast differentiation toward the pancreatic lineage.

Social comparison and risk taking behavior

We study theoretically and experimentally decision making under uncertainty in a social environment. We introduce an interdependent preferences model that assumes that the decision maker evaluates monetary outcomes in relation both with his individual and his social reference point. In the experiment we reproduce a workplace environment whereby subjects interact in an effort task, earn (possibly) different wages from this task and then undertake a risky decision that may give them an extra bonus. Controlling for intrinsic risk attitudes, we find that both downward and upward social comparison strongly influence risk attitudes and that they both generate more risk-loving behavior. Moreover, we find that a propension to envy counterposes such effect, by increasing risk aversion.

Methylation mechanisms and biomechanical effectors controlling cell fate

Mammalian development and cell fate specification are controlled by multiple regulatory mechanisms that interact in a coordinated way to ensure proper regulation of gene expression and spatial restriction, allowing cells to adopt distinct differentiation traits and a terminal phenotype. For example, cell potency is modulated by changes in methylation that are under the control of methyltransferases and ten-eleven translocation (TET) enzymes, which establish or erase a phenotype-specific methylation pattern during embryo development and mesenchymal to epithelial transition (MET). Cell plasticity is also responsive to extracellular factors, such as small molecules that interact with cell fate definition and induce a transient pluripotent state that allows the direct conversion of an adult mature cell into another differentiated cell type. In addition, cell-secreted vesicles emerge as powerful effectors, capable of modifying cell function and phenotype and delivering different signals, such as octamer-binding transcription factor-4 (Oct4) and SRY (sex determining region Y)-box 2 (Sox2) mRNAs (implicated in the preservation of pluripotency), thus triggering epigenetic changes in the recipient cells. In parallel, mechanical properties of the cellular microenvironment and three-dimensional rearrangement can affect both cell potency and differentiation through marked effects on cytoskeletal remodelling and with the involvement of specific mechanosensing-related pathways.

Last Minute Policies and the Incumbency Advantage

This paper models a purely informational mechanism behind the incumbency advantage. In a two-period electoral campaign with two policy issues, a specialized incumbent and an unspecialized, but possibly more competent challenger compete for election by voters who are heterogeneously informed about the state of the world. Due to the asymmetries in government responsibility between candidates, the incumbents statement may convey information on the relevance of the issues to voters. In equilibrium, the incumbent sometimes strategically releases his statement early and thus signals the importance of his signature issue to the voters. We fi nd that, since the incumbents positioning on the issue reveals private information which the challenger can use in later statements, the incumbents incentives to distort the campaign are decreasing in the quality of the incumbent, as previously documented by the empirical literature. However, we show that this implies a non-monotonicity in the distortions that arise in equilibrium.

196 USE OF A MICRO-BIOREACTOR TO PROMOTE 3-DIMENSIONAL CELL REARRANGEMENT AND INDUCE, MAINTAIN, AND STABILIZE HIGH PLASTICITY IN EPIGENETICALLY ERASED FIBROBLASTS

Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948-8953; Brevini et al. 2014 Stem Cell Rev. 10, 633-642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroethylene powder; Sigma 430935, St. Louis, MO; Group B). Both groups were erased with 5-aza-CR and cultured in embryonic stem cell medium for 28 days. Morphological analysis was carried out for the entire length of the experiment. The OCT4, NANOG, and REX1 expression levels were assessed by real-time PCR at different time points. Exposure to 5-aza-CR induced a dramatic change in morphology in Group A fibroblasts. Cells became rounded, with larger and granulated nuclei and retained a monolayer distribution for the entire length of the experiment. The same changes in cell and nuclear morphology were observed also in cells encapsulated in liquid marble (Group B). In addition, these cells formed 3D spherical structures that were stably maintained until Day 28. These morphological rearrangements were accompanied by the active expression of the pluripotency markers, OCT4, NANOG, and REX1, in both groups. However, while Group A cells progressively down-regulated their expression by Day 6, Group B cells steadily transcribed these genes until Day 28, when cultures were arrested. Altogether, the data confirm that epigenetic erasing induces a high plasticity state in terminally differentiated fibroblasts with the expression of pluripotency related genes. Striking morphological changes accompanied the removal of epigenetic marks. These were influenced by the use of an adequate 3D in vitro culture system, with the induction of distinctive cell rearrangements and the formation of spherical structures that boosted and maintained cell plasticity. These results suggest a correlation between the mechanotransduction pathways induced by the micro-bioreactor culture system and the epigenetic regulation of cell phenotype.

Do The Right Thing. Incentives for policy selection in presidential and parliamentary systems

Constitutional structures shape politicians’ behaviour and hence policy outcomes through the dierent incentives schemes they generate. In this paper we analyze these mechanisms in parliamentary and presidential systems. The comparison is carried out by analyzing how the two systems may select the ecient policy in presence of asymmetric information. Presidential and parliamentary systems dier in that the policy proposed by the executive in the parliamentary system is condence-depende nt and observable. The main ndings suggest that the parliament responds better to the incentive scheme in the presidential system due to the lower uncertainty legislators face over their term limit. However, the parliamentary system generates a more ecient behavior of the executive due to selection and disciplining eects.

Mountain high and valley deep: epigenetic controls of pluripotency and cell fate

All the somatic cells composing a mammalian organism are genetically identical and contain the same DNA sequence. Nevertheless, they are able to adopt a distinct commitment, differentiate in a tissue specific way and respond to developmental cues, acquiring a terminal phenotype. At the end of the differentiation process, each cell is highly specialized and committed to a distinct determined fate. This is possible thanks to tissue-specific gene expression, timely regulated by epigenetic modifications, that gradually limit cell potency to a more restricted phenotype-related expression pattern. Complex chemical modifications of DNA, RNA and associated proteins, that determine activation or silencing of certain genes are responsible for the ‘epigenetic control’ that triggers the restriction of cell pluripotency, with the acquisition of the phenotypic definition and the preservation of its stability during subsequent cell divisions. The process is however reversible and may be modified by biochemical and biological manipulation, leading to the reactivation of hypermethylated pluripotency genes and inducing cells to transit from a terminally committed state to a higher plasticity one. These epigenetic regulatory mechanisms play a key role in embryonic development since they drive phenotype definition and tissue differentiation. At the same time, they are crucial for a better understanding of pluripotency regulation and restriction, stem cell biology and tissue repair process.

Erase and Rewind: Epigenetic Conversion of Cell Fate

The potential of cell therapy in regenerative medicine has greatly expanded thanks to the availability of sources of pluripotent cells. In particular, induced pluripotent stem cells (iPS) have dominated the scenario in the last years for their ability to proliferate and differentiate into specific cell types. Nevertheless, the concerns inherent to the cell reprogramming process, limit iPS use in therapy and pose questions on the long-term behavior of these cells. In particular, despite the development of virus-free methods for their obtainment, a major and persisting drawback, is related to the acquisition of a stable pluripotent state, that is un-physiological and may lead to cell instability. The increased understanding of epigenetic mechanisms has paved the way to the use of “small molecules” and “epigenetic modifiers” that allow the fine tuning of cell genotype and phenotype. In particular, it was demonstrated that an adult mature cell could be directly converted into a different cell type with the use of these chemicals, obtaining a new patient-specific cell, suitable for cell therapy. This approach is simple and direct and may represent a very promising tool for the regenerative medicine of several and diverse degenerative diseases.

5-azacytidine reactivates pluripotency gene expression, affects TET2 and histone transcription and modifies chromatin organization and morphology of mammal skin fibroblasts

Phenotype expression is controlled by epigenetic regulations that guide cells through differentiation. The process is reversible and cells can be driven back to a higher plasticity state with the use of epigenetic modifiers. In this work we exposed skin fibroblasts to the demethylating agent 5-azacytidine (5-aza-CR), which is a well-known DNA methyltransferase inhibitor, and has been recently shown to increase cell plasticity and facilitate phenotype changes in different cell types (Pennarossa, 2013; Brevini, 2014; Pennarossa, 2014; Brevini, 2016). Although many aspects controlling its demethylating action have been widely investigated, the mechanisms through which 5-aza-CR acts on cell plasticity are still poorly understood. At the end of 5-aza-CR treatment, cells were divided in three experimental groups and cultured for 24 and 48 hours: A) cells were returned in standard fibroblast medium; B) cells were cultured in medium specific for pluripotency maintenance; C) cells were placed in a medium encouraging pancreatic differentiation. At the end of the culture period, as expected, we could observe a global demethylating effect. In parallel, however we detected a transient upregulation of the pluripotency genes OCT4, NANOG and REX1. Increased transcription of TET2 and histones belonging to the 1,2,3 and 4 families, together with changes in the expression of enzymes controlling histone acetylation were also appreciated. Interestingly, these results were accompanied by morphological and ultrastructural changes as well as by chromatin structure modifications. All together our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state involves novel cellular targets that activate multiple epigenetic regulatory pathways.