Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST.

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.

Role of the Transcription Factor Sox2 in the Osteogenic Lineage

The Sox2 transcription factor is modified by sumoylation at the K247 position although the addition of SUMO1 and Pias1 promotes the sumoylation of Sox2 at the additional K123 site. The role of sumoylation on Sox2 biological functions was analyzed by comparing the activity of WT and sumoylation mutants on the transcription of the FGF4 gene in HeLa cells and on the downregulation of the Wnt pathwayvin 293T cells. When SUMO1 and PIAS1 promote the sumoylation of WT Sox2, the transcriptional activity of the FGF4 promoter is inhibited showing that Sox2 sumoylation is necessary for the repression function. However, there is no effect of Sox2 sumoylation on β-Catenin activity. Since we were interested in osteoblast differentiation we set up an inducible system for Sox2 in primary osteoblasts. Following Sox2 doxycycline induction, 158 genes were differentially expressed: 120 up-regulated and 38 down-regulated. We annotated as direct Sox2 targets a number of genes involved in osteoblast biology and we further analyzed 3 of them involved in the BMP pathway. The results show that Sox2 regulates the BMP pathway without affecting SMAD phosphorylation, and that Sox2 sumoylation is not necessary for this function. We also found that genes involved in the Hippo pathway were direct Sox2 targets. As the Hippo pathway is activated by Sox2 and Sox2 interacts with the NF2 promoter, we checked the effect of Sox2 on the expression of NF2. We showed that Sox2 down-regulates the transcriptional activity of the NF2 promoter, allowing the transcription of the YAP/TEAD genes in osteoblasts, thus acting as an upstream regulator of the Hippo pathway. We conclude that Sox2 induction in osteoblasts triggers FGF dependent inhibition of the BMP, Wnt and Hippo pathways.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Complexity of N-Myc transcriptional function in childhood neuroblastoma

Myc is a transcription factor that can activate transcription of several hundreds genes by direct binding to their promoters at specific DNA sequences (E-box). However, recent studies have also shown that it can exert its biological role by repressing transcription. Such studies collectively support a model in which c-Myc-mediated repression occurs through interactions with transcription factors bound to promoter DNA regions but not through direct recognition of typical E-box sequences. Here, we investigated whether N-Myc can also repress gene transcription, and how this is mechanistically achieved. We used human neuroblastoma cells as a model system in that N-MYC amplification/over-expression represents a key prognostic marker of this tumour. By means of transcription profile analyses we could identify at least 5 genes (TRKA, p75NTR, ABCC3, TG2, p21) that are specifically repressed by N-Myc. Through a dual-step-ChIP assay and genetic dissection of gene promoters, we found that N-Myc is physically associated with gene promoters in vivo, in proximity of the transcription start site. N-Myc association with promoters requires interaction with other proteins, such as Sp1 and Miz1 transcription factors. Furthermore, we found that N-Myc may repress gene expression by interfering directly with Sp1 and/or with Miz1 activity (i.e. TRKA, p75NTR, ABCC3, p21) or by recruiting Histone Deacetylase 1 (Hdac1) (i.e. TG2). In vitro analyses show that distinct N-Myc domains can interact with Sp1, Miz1 and Hdac1, supporting the idea that Myc may participate in distinct repression complexes by interacting specifically with diverse proteins. Finally, results show that N-Myc, through repressed genes, affects important cellular functions, such as apoptosis, growth, differentiation and motility. Overall, our results support a model in which N-Myc, like c-Myc, can repress gene transcription by direct interaction with Sp1 and/or Miz1, and provide further lines of evidence on the importance of transcriptional repression by Myc factors in tumour biology.

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

Different addictive drug exposure induces selective alterations of the Endogenous Opioid System : modulation of transcription and epigenetic mechanisms

Drug addiction manifests clinically as compulsive drug seeking, and cravings that can persist and recur even after extended periods of abstinence. The fundamental principle that unites addictive drugs is that each one enhances synaptic DA by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. Our attention has focused on the study of phenomena associated with the consumption of alcohol and heroin. Alcohol has long been considered an unspecific pharmacological agent, recent molecular pharmacology studies have shown that acts on different primary targets. Through gene expression studies conducted recently it has been shown that the classical opioid receptors are differently involved in the consumption of ethanol and, furthermore, the system nociceptin / NOP, included in the family of endogenous opioid system, and both appear able to play a key role in the initiation of alcohol use in rodents. What emerges is that manipulation of the opioid system, nociceptin, may be useful in the treatment of addictions and there are several evidences that support the use of this strategy. The linkage between gene expression alterations and epigenetic modulation in PDYN and PNOC promoters following alcohol treatment confirm the possible chromatin remodeling mechanism already proposed for alcoholism. In the second part of present study, we also investigated alterations in signaling molecules directly associated with MAPK pathway in a unique collection of postmortem brains from heroin abusers. The interest was focused on understanding the effects that prolonged exposure of heroin can cause in an individual, over the entire MAPK cascade and consequently on the transcription factor ELK1, which is regulated by this pathway. We have shown that the activation of ERK1/2 resulting in Elk-1 phosphorylation in striatal neurons supporting the hypothesis that prolonged exposure to substance abuse causes a dysregulation of MAPK pathway.

RNA non-codificanti indotti da danno al DNA regolano il fattore di trascrizione HIF-1α

Recenti analisi sull’intero trascrittoma hanno rivelato una estensiva trascrizione di RNA non codificanti (ncRNA), le quali funzioni sono tuttavia in gran parte sconosciute. In questo lavoro è stato dimostrato che alte dosi di camptotecina (CPT), un farmaco antitumorale inibitore della Top1, aumentano la trascrizione di due ncRNA antisenso in 5’ e 3’ (5'aHIF-1α e 3'aHIF-1α rispettivamente) al locus genico di HIF-1α e diminuiscono i livelli dell’mRNA di HIF-1α stesso. Gli effetti del trattamento sono Top1-dipendenti, mentre non dipendono dal danno al DNA alla forca di replicazione o dai checkpoint attivati dal danno al DNA. I ncRNA vengono attivati in risposta a diversi tipi di stress, il 5'aHIF-1α è lungo circa 10 kb e possiede sia il CAP in 5’ sia poliadenilazione in 3’ (in letteratura è noto che il 3'aHIF-1α è un trascritto di 1,7 kb, senza 5’CAP né poliadenilazione). Analisi di localizzazione intracellulare hanno dimostrato che entrambi sono trascritti nucleari. In particolare 5'aHIF-1α co-localizza con proteine del complesso del poro nucleare, suggerendo un suo possibile ruolo come mediatore degli scambi della membrana nucleare. È stata dimostrata inoltre la trascrizione dei due ncRNA in tessuti di tumore umano del rene, evidenziandone possibili ruoli nello sviluppo del cancro. È anche noto in letteratura che basse dosi di CPT in condizioni di ipossia diminuiscono i livelli di proteina di HIF-1α. Dopo aver dimostrato su diverse linee cellulari che i due ncRNA sopracitati non potessero essere implicati in tale effetto, abbiamo studiato le variazioni dell’intero miRnoma alle nuove condizioni sperimentali. In tal modo abbiamo scoperto che il miR-X sembra essere il mediatore molecolare dell’abbattimento di HIF-1α dopo trattamento con basse dosi di CPT in ipossia. Complessivamente, questi risultati suggeriscono che il fattore di trascrizione HIF-1α venga finemente regolato da RNA non-codificanti indotti da danno al DNA.

Molecular characterization of human CD4+ IL-10-producing regulatory cells

Previous studies in the group led to the identification of CD4+FOXP3- cells with regulatory functions in human blood that coproduce IL-10 and IFN-gamma. These cells do not belong to the Treg cell lineage since they are Foxp3- but they show some similarities with Th1 cells since they express CCR5, T-bet and produce high levels of IFN-gamma. Thus, they share relevant characteristics with both T regulatory type I cells (Tr1) and Th1 cells and we called them Th1-10 cells. In this study we presented a molecular characterization of Th1-10 cells that includes a gene expression and a microRNA profiling and performed functional studies to assess Th1-10 cells regulatory properties. We demonstrated that Th1-10 cells have a high regulatory potential being able to block the proliferation of activated CD4 naïve T cells to a similar extent as conventional Treg cells, and that this suppression capacity is at least partially mediated by secreted IL10. We showed also that Th1-10 cells are closely related to Th1 effector memory cells and express genes involved in cytotoxicity. In particular, they express the transcription factor EOMES and the cytotoxic effector molecules GZMA and GZMK, and they release cytotoxic granules upon stimulation. Moreover, we found that Eomes regulates cytotoxic functions in CD4+ T cells. We demonstrated that miR-92a, selectively downregulated in Th1-10 cells, directly targets the 3’UTR of EOMES.and this finding identifies miR-92a as a possible mediator of Th1-10 cytotoxicity. Th1-10 cells retain some proliferative capacity when sorted ex vivo and activated in vitro via their TCR, and this effect is markedly enhanced by IL-15, which also had a pro-survival effect on Th-10 cells. Thus, in contrast to conventional cytotoxic T cells, Th1-10 cells have cytotoxic and regulatory functions and are not terminally differentiated, since they retain proliferative capacity.

Natural compounds Camptothecin and Triptolide: highly specific enzyme inhibitors and tools to dissect transcriptional functions

With this work I elucidated new and unexpected mechanisms of two strong and highly specific transcription inhibitors: Triptolide and Campthotecin. Triptolide (TPL) is a diterpene epoxide derived from the Chinese plant Trypterigium Wilfoordii Hook F. TPL inhibits the ATPase activity of XPB, a subunit of the general transcription factor TFIIH. In this thesis I found that degradation of Rbp1 (the largest subunit of RNA Polymerase II) caused by TPL treatments, is preceded by an hyperphosphorylation event at serine 5 of the carboxy-terminal domain (CTD) of Rbp1. This event is concomitant with a block of RNA Polymerase II at promoters of active genes. The enzyme responsible for Ser5 hyperphosphorylation event is CDK7. Notably, CDK7 downregulation rescued both Ser5 hyperphosphorylation and Rbp1 degradation triggered by TPL. Camptothecin (CPT), derived from the plant Camptotheca acuminata, specifically inhibits topoisomerase 1 (Top1). We first found that CPT induced antisense transcription at divergent CpG islands promoter. Interestingly, by immunofluorescence experiments, CPT was found to induce a burst of R loop structures (DNA/RNA hybrids) at nucleoli and mitochondria. We then decided to investigate the role of Top1 in R loop homeostasis through a short interfering RNA approach (RNAi). Using DNA/RNA immunoprecipitation techniques coupled to NGS I found that Top1 depletion induces an increase of R loops at a genome-wide level. We found that such increase occurs on the entire gene body. At a subset of loci R loops resulted particularly stressed after Top1 depletion: some of these genes showed the formation of new R loops structures, whereas other loci showed a reduction of R loops. Interestingly we found that new peaks usually appear at tandem or divergent genes in the entire gene body, while losses of R loop peaks seems to be a feature specific of 3’ end regions of convergent genes.

Studies on the Hippo Pathway: new insights about a multifaceted signalling

The Hippo pathway is a well-known master regulator of cell growth and proliferation. Many studies have shed light on the centrality of Hippo functions, as this signalling is able to respond to different stimuli and translate them into distinct transcriptional outputs. Therefore, it is clearly implicated in a number of important processes, which alteration has consequences on the correct specification of the single cell, as well as the whole tissue. Even if the core of the signalling has been extensively characterized, it remains unclear which are the “co-workers” that permit the Hippo pathway to answer to so many different stimuli and act as a coordinator of the growth/differentiation balance. Taking advantage of the Drosophila model, which has witnessed most of the discoveries on this signalling pathway, this thesis aims to add some new knowledge about the Hippo pathway molecular mechanisms in different contexts, from development to disease. In the first part I studied the dynamics of the Hippo core kinase protein Warts in the development of the pupal eye. I have found out a critical time point in which the expression and the localization of Warts change suddenly, suggesting the intervention of upstream regulators modulating its activity in an extremely narrow time window. The second goal was investigating the role of the Hippo pathway in the neurodegenerative Gaucher disease. Indeed, I have produced some preliminary results which demonstrate a growth deficit associated with a massive reduction of some Yki targets, supporting a Hyper-Hippo condition underlying this neuropathic syndrome. Finally, I have evaluated the transcription factor Orthodenticle as a co-factor of Yorkie in driving tissue overgrowth, and my findings support a model of interaction of these two molecules based on Yki conformational changes. Altogether, my results lay the foundation for new important studies on the molecular mechanisms ruling Hippo pathway activity.

Evaluation of the transcriptomic response of an Alzheimer's disease murine model induced at different ages: searching for predictors

Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disease, characterized by the impairment of mnesic and cognitive functions, that represents the most frequent type of dementia in older people worldwide. Aging is the most important risk factor for the sporadic form of the pathology and it is associated to the progressive impairment of the proteostasis network. The endoplasmic reticulum (ER), the main cellular actor involved in proteostasis, appears significantly compromised in AD due to the accumulation of β-amyloid (Aβ) protein and phosphorylated-tau protein. Increasing proteins misfolding activates a specific cellular response known as Unfolded Protein response (UPR) which orchestrates the recovery of ER function. The aim of the present study was to investigate the role of UPR and aging process in a murine model of AD induced by intracerebroventricular (i.c.v.) injection of Aβ1-42 oligomers at 3 or 18 months. The oligomers injection in aged animals caused the increased of memory impairment, oxidative stress, and the depletion of glutathione reserve. Furthermore, the RNA-sequencing analysis was performed and the bioinformatic analysis showed the enrichment of several pathways involved in neurodegeneration and protein regulations. The following analysis highlighted the significant dysregulation of the three branches of the UPR, the protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF-6). In turn, ER stress affected the PI3K/Akt/Gsk3β and MAPK/ERK pathways, highlighting Mapkapk5 as a potential marker of the neurodegenerative process, which regulation could lead to the definition of new pharmacological and neuroprotective strategies to counteract AD.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.