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RIASSUNTI DEI PROGETTI DEL CORSO DI DOTTORATO DI RICERCA IN BIOLOGIA MOLECOLARE E CELLULARE

A.A. 2013 (XXIX CICLO)

1.Beltrame Monica: Deciphering the molecular network involving the transcription factor Sox18 in blood vascular and lymphatic development

2.Berruti Giovanna: An organ culture-approach to study the fertility gene product USP8/UBPy

3.Bertoni Giovanni (1): Detailing of small RNA-based regulatory networks by parallel transcriptomic-proteomic profiling

4.Bertoni Giovanni (2): Characterization of novel essential functions in the opportunistic pathogen Pseudomonas aeruginosa

5.Bolognesi Martino: Structure-based epitope discovery from B. pseudomallei antigens for vaccine Development

6.Briani Federica: Looking for novel Pseudomonas aeruginosa inhibitors: S1 ribosomal protein as an unexploited target for new antibacterial molecules

7.Caporali Elisabetta: Key cell wall modelling factors to design fruit shapes in Arabidopsis

8. Cappelletti Graziella, Francesco Demartin (Co-Tutor): Dissecting microtubule dynamics at the synapse

9.Caretti Giuseppina: Epigenetic mechanisms underlying skeletal muscle atrophy.

10.Cattaneo Elena: hES cells differentiation into striatal neurons by conditional over-expression of transcription factors

11.Colombo Lucia: Genetic and epigenetic control of seed number in Arabidopsis

12.Costa Alex: Functional characterization of Arabidopsis thaliana iGluRs (glutamate receptors) channels

13.Cotelli Franco: Characterization of the role played by numblike during vascular development of the brain and Cerebral Cavernous Malformations onset using zebrafish as model system

14.Duga Stefano: Whole-exome sequencing applied to the study of the genetic basis of Parkinson disease

15. Fornara Fabio: Molecular control of flowering in rice

16. Gissi Carmela: Evolutionary dynamics of nuclear genes involved in replication and repair of the mitochondrial genome in fast-evolving chordates

17.Gnesutta Nerina: NF-Y partners in the regulation of CCAAT promoters

18.Guerrini Luisa: Role of the p300 acetylase in regulating the activities of the p63 transcription factor

19.Kater Martin: Mining the Molecular Pathways Controlling Rice Reproductive Development

20.Lazzaro Federico: Determine the biological significance of the rNMPs incorporated in genomic DNA

21.Mantovani Roberto: NF-YA isoforms in ES cells and cancer

22.Marini Federica: Unravelling the role of the Fanconia anemia protein P/Slx4 and the DNA damage checkpoint factor 53BP1/Rad9 in responding to double strand DNA lesions

23.Messina Graziella: Identification of the mechanism(s) regulating Nfix expression during fetal Myogenesis

24.Messina Graziella: Genome-wide mapping of Nfix binding sites in skeletal muscle

25.Moroni Anna: The structural mechanism of KAT1 channel regulation by the cytoplasmic domain CNBHD

26.Muzi Falconi Marco: Haspin kinase: a new regulator of cell polarity and cell division

27.Nardini Marco: Structural analysis of transcription factor/DNA complexes

28.Pavesi Giulio: Development and Application of Bioinformatic Tools for the Analysis of ChIP-Seq Data

29. Petroni Katia: Role of anthocyanin-enriched diet on cardioprotection

30.Plevani Paolo/Giacomo Buscemi: DAXX protein and the human DNA damage response:chromatin remodeling, gene expression, genome stability and tumorigenesis

31.Pesaresi Paolo: Chromatin Remodeling Enzymes are at the basis of SVP transcription factor function

32.Ricagno Stefano: Structural-Based Drug Discovery against RNA-based VIRUSES

33.Riva Paola: Competitive endogenous RNA (ceRNA) Cross-Talk in Neurofibromatosis type 1 phenotype expression variability

34.Soldà Giulia: Identification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing

35.Tonelli Chiara/Conti Lucio: The role of ABA in the floral transition: site and mechanism of action

36.Zuccato Chiara: An in vivo study of the impact of ADAM10 dysfunction in Huntington’s Disease

Project leader: MONICA BELTRAME ([email protected])

Location: Department of Biosciences, University of Milan, Italy

RESEARCH PROJECT SUMMARY

Deciphering the molecular network involving the transcription factor Sox18 in blood vascular and lymphatic development

Our group is interested in gene expression regulation during embryonic development in vertebrates. We are studying a family of transcription factors, the Sox (Sry-related HMG box) proteins, which are found throughout the animal kingdom and play key roles in embryonic development. Mutations in several Sox genes have been shown to result in developmental anomalies, from fly to mammals. The emerging picture of SOX transcription factors is one of tissue-specific switches that induce changes in gene expression required for cell-type specification or differentiation.

Our interest is currently centered on SOX18, which is mutated in patients affected by the Hypotrichosis-Lymphedema-Telangiectasia syndrome. SOX18 is transiently expressed in the endothelial component of nascent blood and lymphatic vessels during embryonic development and in adult life, when neovascularization occurs.

Despite its relevance, relatively little is known about upstream factors and downstream targets involving SOX18 in endothelial cell differentiation and vascular development. We are addressing these questions using the zebrafish model system, as it provides several advantages over other vertebrate model organisms for the in vivo imaging of the vascular system and for the genetic or experimental manipulation of vascular development. We have shown that Sox18 and the closely related Sox7 protein (both belonging to the SoxF group) play redundant roles in arterio-venous differentiation of endothelial cells in zebrafish, while only Sox18 plays a conserved role in lymphatic development (Cermenati et al., 2008, Blood; Cermenati et al. 2013, ATVB). Gene expression profiling, at key developmental stages, under conditions of perturbed SoxF protein expression will serve as a basis to shed light on the molecular networks controlling blood vascular and lymphatic development. Loss-of-function and gain-of-function approaches in specific transgenic lines will be used to assess the functional relevance of interesting genes, whose expression is modified when SoxF proteins are perturbed. Given the pathological relevance of angiogenesis/lymphangiogenesis and lymphatic dysfunction in humans, the identification of new players might open up new therapeutic perspectives.

Project leader: GIOVANNA BERRUTI ([email protected])

Location: Department of Biosciences, University of Milan, Italy.


An organ culture-approach to study the fertility gene product USP8/UBPy

USP8/UBPy, recently found to be a candidate to 'male-fertility gene' [1], is a deubiquitinase preferentially expressed in the testis and the central nervous system, which acts as a regulator of the endocytic vesicle trafficking [2]. USP8 is involved in the biogenesis of the acrosome, an organelle essential to fertilization. USP8-KO is lethal, consequently functional studies in vivo are not feasible [3]. The purpose of this research is to investigate the role and importance of USP8 in spermiogenesis, using a system of organ culture developed recently that could allow us to recapitulate 'ex vivo' what happens in vivo [4]. As organ donors there will be used male mice Acr-GFP [5] that accumulate EGFP in the acrosome during its biogenesis, thus making it detectable at fluorescence microscopy. The initial phase of the research will be devoted to the development of organ culture conditions optimal for obtaining, from highly immature cells, spermatids that have achieved development stages corresponding to each of the 4 acrosomogenic phases (acrosomogenesis ‘in vivo’ requires about 2 weeks in the mouse). The cultured Acr-GFP testicles will assist to identify the phases in non-invasive manner. Analysis of confocal double-immunolabeling to highlight USP8 and classical markers of the biosynthetic and endocytic traffic will be performed to identify the type/s of traffic that carry the cargo protein to the acrosome in development. Acrosomogenesis is microtubule (MT)-dependent and USP8 has a MIT (microtubule interacting and trafficking) domain [2]; however, the MT-arrays involved in acrosomogenesis are transient structures devoid of any visible centrosomal foci. It will be investigated in the organ cultured cells whether the possible association USP8-MT is preferential with dynamic MTs (tyrosinataed tubulin) or more stable MTs (detyrosinated, acetylated tubulin, etc.); this will be performed by using confocal double-immunolabeling and in vitro protein-protein interaction assays (GST pull down). To silence the activity of USP8, we propose to produce a non-viral vector that replicates episomally and carries the expression cassette for a USP8-siRNA, in addition to the cassette for RFP as a reporter protein [6]. In this way we could see what effect the silencing of USP8 has on the development of Acr-GFP acrosome in the 'red' cells. Comparisons will be with the respective not silenced controls. As an alternative to silencing, the activity of USP8 could be blocked through transfection experiments by expression of a catalytically inactive variant of USP8, USP8C748A[7], fused with RFP (the recombinant construct has to be generated). In conclusion, this research combines two innovative approaches, i.e., the ‘organ culture’ and ‘non-viral vector-mediated’ silencing/inhibition of the USP8/UBPy enzymatic activity. If we succeed in both, it could be provided an experimental platform for studying not only USP8, but other key fertility gene products, for which the homologous deletion results in embryonic lethality, in a system that recapitulates the ‘in vivo’ situation.

1. Kosova et al, Amer J Hum Gen 90, 950-61, 20122. Berruti et al, Biol Reprod 82, 930-39. 20103. Nierdof et al., Mol Cell Biol 27, 5029-39, 20074. Sato et al, Nature 471, 504-7, 20115. Nakanishi et al. FEBS Lett 449, 277-83, 1999

6. Jenke et al, Hum Gen Ther 16, 533-9, 20057. Alwan & van Leeuwen, JBC 282, 1658-69, 2007

Project leader: GIOVANNI BERTONI (1) ([email protected])

Location: Department of Biosciences, University of Milan, Italy.


Detailing of small RNA-based regulatory networks by

parallel transcriptomic-proteomic profiling

Pseudomonas aeruginosa is an important opportunistic pathogen in immune-compromised and cystic fibrosis patients responsible for numerous acute and chronic infections. Crucial traits contributing to pathogenicity of P. aeruginosa are the production of a large assortment of virulence factors, biofilm formation and the ability to rapidly develop resistance to multiple classes of antibiotics. Expression of these traits is fine-tuned by a dynamic and intricate regulatory network [1], in which more than 50 regulatory proteins play key roles as transcription regulators. On the contrary, the involvement in this context of small RNAs (sRNAs), important regulatory molecules acting post-transcriptionally on target mRNAs and/or via interactions with proteins, has been studied to a lesser extent.

Preliminary results suggest that four novel P. aeruginosa sRNAs, which were identified in the proponent lab [2], are involved in the regulation of virulence traits in response to infection-relevant stimuli. In actual fact, overexpression of these sRNAs was shown to influence the expression of P. aeruginosa virulence descriptors such as motility, biofilm formation, secretion of proteases, toxic secondary metabolites and siderophores. In addition, it was observed that the expression of these sRNAs can be responsive to temperature shift from room to body temperature, oxygen availability, iron limitation, envelope stressors and quorum sensing, a system of stimulus and response correlated to bacterial cell density that coordinate the expression of several virulence genes. Focusing on these four sRNAs, the main aim of this research project is to unravel the cognate regulons, i.e. the set of genes which can be both direct and indirect targets of the sRNA-mediated regulation. This aim will be accomplished combining quantitative proteomics with transcriptomics. For virulence-relevant direct targets, the sRNA/target mRNA interaction will be characterized.

1. Balasubramanian D, Schneper L, Kumari H & Mathee K (2013) A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res, 41:1-20.

2. Ferrara S, Brugnoli M, De Bonis A, Righetti F, Delvillani F, Dehò G, Horner D, Briani F & Bertoni G (2012) Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs. PLoS One, 7:e36553.

Project leader: GIOVANNI BERTONI (2) ([email protected])



Characterization of novel essential functions in the opportunistic pathogen Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen in compromised individuals, such as patients with cystic fibrosis, severe burns or impaired immunity. The proponent lab aimed to screen novel essential functions of P. aeruginosa by shotgun antisense identification, a technique that was developed a decade ago for the Gram-positive bacterium Staphylococcus aureus and was under-used in Gram-negative bacteria for a considerable period of time. This approach in P. aeruginosa generated a panel of about 20 novel essential candidate proteins that are suggested to take part in disparate cellular functions, including protein secretion, biosynthesis of cofactors, prosthetic groups, and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcriptional regulation. The essential role of two of these proteins, TgpA [1] and Gcp, was validated by means of insertional and conditional mutagenesis. The main aim of this research project is to unravel the cellular role of both TgpA and Gcp and identify protein partners.

1. Milani, A., Vecchietti, D., Rusmini, R. and Bertoni, G. (2012) TgpA, a protein with a eukaryotic-like transglutaminase domain, plays a critical role in the viability of Pseudomonas aeruginosa. PLoS ONE 7(11): e50323.

Project leader: MARTINO BOLOGNESI ([email protected])



Structure-based epitope discovery from B. pseudomallei antigens for vaccine development

Structure-based antigen engineering is frequently used in the vaccine development process to specifically modify protein antigens of a pathogen to enhance their immunogenic properties, with the aim of improving their protective efficacy. Such approaches may entail engineering epitope-containing regions of the protein, or simply the epitope sequences themselves in the form of synthetic peptides.

In this context, one of our lines of research regards structure-based epitope discovery, focusing on protein antigens from the Gram negative pathogen Burkholderia pseudomallei, which causes melioidosis, a severely debilitating, and often fatal disease, endemic in the subtropical and tropical regions of the word. Together with computational biologists and immunologists, we have constructed a structural vaccinology pipeline for epitope identification. Selected targets enter into a medium-throughput protein production pipeline involving bioinformatics (recombinant construct design), heterologous expression, purification and 3D structure determination (X-ray crystallography). 3D antigen structures form the basis for the application of in silico-based epitope predictions, combined with experimental validation and immunological testing. Our structural vaccinology network includes both national groups and international labs in the UK (immunologists), Spain (computational biologists) and Thailand (immunologists). The latter group carries out sera recognition tests with immune sera from melioidosis patients, to validate the reactivity of selected antigens and epitopes.

Overall, we aim to connect the understanding of structural properties at atomic resolution, to the reactivity properties of the protein (or specific epitopes) in an immunological context, with the scope of identifying candidate epitopes to be considered in a potential vaccine.

The success of our pipeline has been demonstrated for two known B. pseudomallei antigens (1, 2). Based on their crystal structures, consensus epitopes were successfully identified and, when synthesized as free peptides, were shown to possess interesting immunological properties in comparison with their full-length recombinant counterparts. Antibodies raised against one of the most reactive epitope peptides are presently being tested in passive immunization tests in mice.

The proposed project forsees the use of molecular biology, protein biochemistry and structural biology techniques, for the design, cloning and heterologous expression of protein antigens as recombinant fusion proteins, biophysical analyses (dynamic light scattering, thermofluorimetry), protein crystallization screening and 3D structure determination. Diffraction data are collected at the European Synchrotron Research Facility (ESRF, Grenoble, France) on a routine basis. Subsequent computational data elaboration and structural determination is carried out in-house. Generated 3D structures will serve as the starting point for entry into the above-mentioned structural vaccinology pipeline.

1. Gourlay LJ, et al. Exploiting the Burkholderia pseudomallei Acute Phase Antigen BPSL2765 for Structure-Based Epitope Discovery/Design in Structural Vaccinology. (2013) Chem. Biol. 20, 1147-

56.2. Lassaux P, et al. A structure-based strategy for epitope discovery in Burkholderia pseudomallei OppA antigen. (2013) Structure. 21, 167-75.

Project leader: FEDERICA BRIANI ([email protected])



Looking for novel Pseudomonas aeruginosa inhibitors: S1 ribosomal protein as an unexploited target for new antibacterial molecules

P. aeruginosa is a Gram negative, mesophilic bacterium, endowed with a noteworthy metabolic versatility reflected by a large genome. It can infect hosts as diverse as worms, flies and mammals. In humans it behaves as an opportunistic pathogen and it is responsible for a variety of serious nosocomial infections. Moreover, P. aeruginosa is the most common pathogen found in the lung of cystic fibrosis patients and the primary factor in pulmonary pathology. The diffusion of isolates of P. aeruginosa (and other pathogenic bacteria) multi-resistant to extant antibiotics makes urgently needed the development of new antibacterial molecules that may escape bacterial resistance.

A strategy that can be applied to search for new antibiotics is to tackle factors participating in essential cellular processes. We propose to explore P. aeruginosa ribosomal protein S1 as a potential target for new antibacterials. S1 is a ribosomal protein widely conserved among Gram negative bacteria and absent in mammals. In vivo this protein is required for translation of most E. coli mRNAs. Conversely, the protein is dispensable for initiation complex formation on leaderless mRNAs (1,2,3).

The general objective of this research will be pursued through the following activities:1. Assessing S1 essentiality in P. aeruginosaGenes encoding functions essential for cell survival or pathogenesis may represent good antibiotics target. It is likely that rpsA is an essential gene in P. aeruginosa as it is in E. coli and M. tuberculosis, but a formal demonstration is lacking. We will aim at this objective by constructing a conditional allele of rpsA. If conversely, the gene will result to be non-essential, we will perform phenotypic analyses of the mutant both in vitro and in Galleria mellonella larvae infection model.

2. Identification and characterization of S1 and translation initiation inhibitors

We have developed a whole-cell fluorescent screening for specific inhibitors of S1-dependent translation. The theoretical bases of our screening rest on differential S1 requirement exhibited by different transcripts in E. coli. This screening will be used to test a large collection of chemical compounds already available in our lab. Specific inhibitors of S1-dependent translation will be further characterized to elucidate their mechanism of action and their effect on P. aeruginosa.

1. Sørensen,M.A., Fricke,J. and Pedersen,S. (1998) Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo. J.Mol.Biol., 280, 561-569.2.Tedin,K., Resch,A. and Bläsi,U. (1997) Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence. Mol.Microbiol., 25, 189-199.

3. Delvillani,F., Papiani,G., Dehò,G. and Briani,F. (2011) S1 ribosomal protein and the interplay between translation and mRNA decay. Nucleic Acids Res., 39, 7702-7715.

Project leader: ELISABETTA CAPORALI ([email protected])



Key cell wall modelling factors to design fruit shapes in Arabidopsis]

Morphogenesis is the remarkable process by which a developing plant acquires its shape. Underlying the architectural complexity of plants are diverse cell types that easily reveal relationships between cell structure and specialized functions. Much less obvious are the mechanisms by which the cellular growth machinery and mechanical properties of the cell wall interact to dictate cell shape that lead to an organ formation. Fully developed fruits have a complex primary cell wall matrix, and are exposed to a highly precise regulation required to determine the final size and shape of the organ. Although progress is being made in identifying and characterizing the genes required for the synthesis of fruit cell wall matrix components, little is known about how the production and accumulation of wall components are regulated at different levels: transcriptional control, biochemical control, or both. It remains a central challenge for developmental biology how much this regulation contributes to produce the diversity of cell shapes and functions that characterize the formation of a seed or a silique.

We propose an innovative investigation of morphogenesis to address how these elements are controlled at the molecular and cell level, and how the mechanical properties of these elements lead to specific growth patterns using seed and fruit development in Arabidopsis as a model system. The outcome of this project not only will discuss the unique geometric properties and physical processes that regulate seed and fruit organogenesis in Arabidopsis thaliana, but also will lead us to develop testable mathematical models that improve our understanding of how genetic networks, protein motors, and extracellular polymer properties of these elements lead to specific seed/fruit growth patterns.

Project leader: GRAZIELLA CAPPELLETTI ([email protected])


Co-Tutor: Francesco Demartin

Dipartimento di Chimica, Via Venezian 21, Milano


Dissecting microtubule dynamics at the synapse

Microtubules (MTs) are highly dynamic polymers that control many aspects of neuronal function: they

provide a scaffold to sustain axonal and dendritic architecture and supply the railway for axonal transport.

Far from being mere structural elements, MTs are emerging as key determinants of neuronal polarity. In

spite of the fact that the regulation of MT organization and dynamics has been extensively studied during

axon and dendrite formation and maintenance, much less is known about the regulation of MT dynamics at

synaptic terminals. Recently, we have unravelled the role of a synaptic protein, namely -synuclein, in

regulating MTs.

The goal of the present project is to investigate the role of -synuclein in controlling MT behaviour at the

synapse by focusing on MT nucleation that determines where, when and how polymerization of new MTs is

initiated. In the first part of the work, the PhD fellow will perform light and electron microscopy analyses on

primary neuronal cultures obtained from mice embryos. In these cultures, -synuclein will be overexpressed

or knocked-down. Next, taking advantage of the very high resolution afforded by 3D EM tomography, a

detailed analysis of the structure of MTs nucleated at the synapse will be carried out. The labelling with

immuno-gold particle will allow localizing -synuclein into the 3D structures. For image analysis will be

used a software developed by the Department of Chemistry (University of Milan). A better understanding of

MT regulation at the synapse and novel insights into the physiological role of -synuclein is the expected

outcome.

mailto:[email protected]

Project leader: GIUSEPPINA CARETTI ([email protected])



Epigenetic mechanisms underlying skeletal muscle atrophy

We are interested in studying the epigenetic regulation of factors involved in skeletal muscle wasting. Muscle wasting occurs in association with different conditions such as atrophy of disuse, muscular dystrophies, sarcopenia of aging, and cachexia secondary to other diseases (as cancer, heart diseases or chronic obstructive lung disease). In all these circumstances, muscle wasting manifests with overlapping features and similar molecular mechanisms (Ruegg and Glass, 2010). One of the key factors involved in muscle wasting is myostatin, which is a negative regulator of skeletal muscle mass and is up-regulated in atrophic conditions. Myostatin loss of function in knockout mice or in naturally occurring mutants leads to a significant increase in muscle mass, known as “double muscling” (Huang et al., 2011).

We have recently shown that the histone-methylase SMYD3 and the bromodomain protein BRD4 positively regulate transcription of the myostatin gene, both in muscle homeostasis and during muscle atrophy. Importantly, myostatin levels can be reduced in in vitro cultured myotubes by a recently developed epigenetic drug, called JQ1, which associates with the bromodomains of BRD4 and blocks its function on the chromatin (Proserpio et al., 2013).The specific aims of the project are: 1) to study the ability of JQ1 to block myostatin and muscle loss in vivo, using different models of muscle wasting. 2) To investigate the effect of the small inhibitor JQ1 on the function of adult skeletal muscle stem cells, also known as satellite cells. 3) To identify the genes altered by BRD4 blockade and by SMYD3 reduction in normal skeletal muscle and in atrophic conditions.

References:Ruegg MA and Glass DJ. Molecular Mechanisms and Treatment Options for Muscle Wasting Diseases. Annu. Rev. Pharmacol. Toxicol. 2011.51:373-395

Huang Z, Chen X, Chen D. Myostatin: a novel insight into its role in metabolism, signal pathways, and expression regulation. Cell Signal 2011;23(9):1441-6.

Proserpio V., Fittipaldi R., Ryall J.G, Sartorelli V., Caretti G. The Methyltransferase SMYD3 Mediates the Recruitment of Transcriptional Elongation Factors at the Myostatin and c-Met Genes and Regulates Skeletal Muscle Atrophy. Genes & Development. 2013; 27; 1299-1314

http://en.wikipedia.org/wiki/Chronic_obstructive_lung_disease

Project leader: ELENA CATTANEO ([email protected]; [email protected])

Location: Department of Biosciences (Via Viotti 3, University of Milan, Italy


hES cells differentiation into striatal neurons byconditional over-expression of transcription factors

Huntington’s disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder that usually onsets in midlife. It is characterized by motor, cognitive, and psychiatric symptoms. Once symptomatic, patients are rapidly disabled and require increasing multidisciplinary care. HD is a tremendous burden for medical, social, and family resources. The symptoms and the progression of HD can be linked to its neuropathology, which is characterized by loss of specific neuronal populations in many brain regions. Several studies have shown that medium spiny neurons (MSN) are severely affected in HD. MSN are inhibitory projection neurons and are the primary source of striatal projections.

The laboratory is actively involved in international research programmes aiming at deriving specific and robust differentiation protocols for the generation of MSN. Most recently we have developed a protocol to obtain such neurons from human embryonic stem (hES) or from induced pluripotent stem cells (hiPS) using a defined in vitro neural induction system and quantitative assessment tools (Delli Carri, 2013).

In this project we aim at developing strategies to further improve the recovery and quality of fully functional human MSN from hES/hiPS cells with the goal of future transplantation studies in HD. We will combine morphogens treatment with transcription factors inducible over-expression. We plan to develop doxycycline-inducible hES lines that over-express critical combinations of transcription factors (TFs) known to be important for striatal specification and differentiation. Changes in gene transcriptional profiling and expression of positional markers will be used to verify the identity acquired by the implemented cells as they progress along neuronal differentiation. Cell sorting will be employed to further select for suitable neural progenitors. Quality of the neurons obtained at the end of the differentiation protocol will be verified by a convergence of features such as expression of neuronal markers as well as neurochemical and bioelectrical properties.

In conclusion this project aims at (i) developing new hES cell lines over-expressing critical striatal TFs (ii) characterizing the identity of the neural progenitors and post-mitotic neurons derived from differentiation studies. Moreover, the project will also focus on refining existing protocols for making striatal neurons, by considering developing cell sorting strategies and small molecules treatments.


Project leader: LUCIA COLOMBO ([email protected])



Genetic and epigenetic control of seed number in Arabidopsis

The understanding of the genetic control of organ primordia formation and differentiation are one of the most challenging aspect of the developmental biology. In this project we combine the study of a very fascinating and still unknown process with its possible application for improving plant yield.

The seed are formed from the ovule upon fertilization. Several important events are required for successful seed settings: the ovule primordia have to be formed, followed by pattern formation and morphogenesis and finally after fertilization seed has to develop.

The seeds number is one of the most important trait in plant breeding. Yield enhancement is required to meet increasing food demand. In addition, there is an increasing demand for plant-derived products for non-food purposes, such as energy production.

The genetic networks controlling ovule number and fertility are strictly connected and a restricted number of master genes control these developmental processes such as SEEDSTICK (STK) and CUP SHAPED COTYLEDONS (CUCs) encode for transcription factors. This proposal aims to study the molecular network underpinning the ovule number and fertility using Arabidopsis as model. The objectives will be reached using an integrative approach base on advance technology such as laser microscopy, genome wide target identification and genome wide expression profile.

Project leader: ALEX COSTA ([email protected])



Functional characterization of Arabidopsis thaliana iGluRs (glutamate receptors) channels

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate neurotransmission in animal nervous systems. Homologous proteins in plants (20 members in Arabidopsis, Lacombe et al., 2001 Science) have been implicated in root development, ion transport, and several metabolic and signaling pathways. A recent work demonstrate the involvement of two members of the plant GLR family, GLR3.3 and GLR3.6, in long-distance wound signaling (Mousavi et al., 2013 Nature). Moreover, another member of this family, the GLR1.2 is specifically expressed in pollen and its activity is crucial for proper Ca2+ fluxes (formation of the Ca2+ tip gradient), and ultimately for the proper pollen tube growth and fertility (Michard et al., 2011, Science). Analyses of animal iGluRs, analyzed in heterologous expression system such as Xenopus oocytes have shown that these channels have varying conductances for sodium Na+, potassium K+ and Ca2+. They were therefore classified as non-selective cation channels (NSCC). Studies conducted thus far suggest that also plant iGluRs are NSCCs. Importantly, in a recent study Vincill et al. established HEK cells as a system for studying iGluR function (Vincill et al., 2012 Plant Physiol). In their work they found that AtGLR3.4 is an amino acid gated channel that is suggested to be selective to Ca2+ and is capable of inducing cytosolic Ca2+ peaks in response to asparagine, glycine, or serine. A second work using a different heterologous expression system, Xenopus oocytes, found that another Arabidopsis iGluR homolog, AtGLR1.4, functioned as a ligand-gated, nonselective, Ca2+-permeable cation channel that responded to an even broader range of amino acids, none of which are agonists of animal iGluRs (Tapken et al., 2013 Sci Signal). At the electrophysiological level only few members of this large plant iGluR family have been functionally characterized. In this regard future research is needed to analyze in detail conductance properties of different plant iGluRs.

The aim of this project will be the identification, molecular cloning, functional characterization and study of the physiological role of different Ca2+ permeable iGluR channels from Arabidopsis. Public microarray data show that at least 13 members of the iGluR family are expressed in the different tissues of the root tip cells with, in some cases distinct expression patterns. Arabidopsis plants expressing the Ca2+ probe Cameleon will be treated with the 20 standard amino acids, and the Ca2+ responses will be monitored in root tip cells by using both an wide field fluorescence microscopy (for an initial screening) and the SPIM-FRET set up (recently developed in our lab; Costa et al., 2013 PLOS ONE) for single cell analyses. This analyses will allow to correlate the amino acids-induced cytosolic Ca2+ rises with the expression patterns of the 13 members the root expressed iGluRs. This series of experiments will provide useful information about which are the effective iGluRs ligands and narrow down the number of interesting candidates. The predicted plasma membrane localized iGluRs will be cloned in the pcDNA3 expression vector (for mammalian cells) and in order to study their functionality in the heterologous mammalian system (HEK293T cells) will be co-expressed individually with the Ca2+ probe Cameleon for imaging analyses. The cells co-expressing the different channels together with the Cameleon will be treated with the amino acids reported to induce cytosolic Ca2+ rises in root cells. This approach will enable a first fast screening of the iGluRs candidates. Only those, in which the treatment with the different amino acids, will show a cytosolic Ca2+ increase will be further electrophysiologically

characterized. In this latter case, a combined imaging and electrophysiological set up will also permit to perform at the same time and in the same cell a detail electrical characterization of the channel and the study of its Ca2+ permeability. Finally the identified and selected channels will be also studied in Xenopus oocytes in which a more detailed analysis of the selectivity and other properties will be carried out. A second part of the project will be devoted to the study of the physiological role of the functional iGluR channels. For this aim the in planta expression pattern of the channel/s will be performed by qRT-PCR and through the cloning of promoter/s region/s (in order to generate GUS transgenic reporters lines). The plasma membrane localization will be confirmed by the generation of GFP fusion proteins. Isolation and phenotypic characterization of knock out mutant/s for any given channel/s, will then shed light on the physiological role played by the channel/s. In the isolated KO mutant/s the cytosolic localized Cameleon probe will be introduced and the response to the aminoacid evaluated.

Project leader: Franco COTELLI ([email protected])



Characterization of the role played by numblike during vascular development of the brain and Cerebral Cavernous Malformations onset using zebrafish as model system

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities composed of enlarged thin-walled capillary clusters, which may cause several neural deficits. In human, the onset of this disorder has been related to sporadic or heritable mutations in, at least, one of these three genes, KRIT1 (CCM1), OSM (CCM2) and PDCD10 (CCM3) [1]. A specific mutation in the gene KRIT1 is responsible for the majority of malformation and in vitro experiments revealed that its silencing inactivate the NOTCH pathway [2,3].

In the last years zebrafish has been well established as a great model organism for studying embryonic development, human illness and heritable disorders. This is due to light transparency, very small size and genetic tractability of the embryos. Zebrafish has also proven to be a good model for comparative studies, which demonstrated a striking degree of anatomical and functional conservation between zebrafish and mammals [4,5].

Thanks to all these features it turned out to be a powerful model system to study CCMs. Previous works characterized in zebrafish loss-of-function mutations of ccm1 and functional knockdown of ccm3, which result in dilation of thin-walled vessels and over-branching of cranial vessels [6-8]. All these defects may be related to alterations of the Notch pathway.

Since preceding data have shown that Numblike is able to interact with Notch and Shh and influence their signalling pathway [9,10], the aim of this project is to verify the possibility that the onset of the CCMs is linked to alterations of numblike expression. Our goals will be, in the first instance, to confirm alteration in cranial vasculature due to functional knockdown of Numblike. Then we propose to investigate any relationship between ccm1/ccm3 and numblike. Finally we plan to improve and optimize flow-OPT technique and use it to collect more data about vascular alteration of the cranial net in our samples [11].

[1] Labauge P, Denier C, Bergametti F, Tournier-Lasserve E (2007). Genetics of cavernous angiomas. Lancet Neurol; 6:237-244.

[2] Eerola I, Plate KH, Spiegel R, Boon LM, Mulliken JB, Vikkula M (2000). KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation. Hum Mol Gen; 9:1351–1355.

[3] Wüstehube J, Bartol A, Liebler SS, Brütsch R, Zhu Y, Felbor U, Sure U, Augustin HG, Fischer A (2010). Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling. Proc Natl Acad Sci U S A; 107:12640-14645.

[4] Zon LI (1999). Zebrafish: a new model for human disease. Genome Res; 9:99-100.

[5] Vogel AM, Weinstein BM (2000). Studying vascular development in the zebrafish. Trends Cardiovasc Med; 10:352-360.

[6] Kleaveland B, Zheng X, Liu JJ, Blum Y, Tung JJ, Zou Z, Sweeney SM, Chen M, Guo L, Lu MM, Zhou D, Kitajewski J, Affolter M, Ginsberg MH, Kahn ML (2009). Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous

malformation protein pathway. Nat Med; 15:169-176. Erratum in: Nat Med;15:584. Sweeney, Shawn M [added].

[7] Hogan BM, Bussmann J, Wolburg H, Schulte-Merker S (2008). ccm1 cell autonomously regulates endothelial cellular morphogenesis and vascular tubulogenesis in zebrafish. Hum Mol Genet; 17:2424-2432.

[8] Yoruk B, Gillers BS, Chi NC, Scott IC (2012). Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease. Dev Biol; 362:121-131

[9] Gulino A, Di Marcotullio L, Screpanti I (2010). The multiple functions of Numb. Exp Cell Res; 316:900-906.

[10] Liu L, Lanner F, Lendahl U, Das D (2011). Numblike and Numb differentially affect p53 and Sonic Hedgehog signaling. Biochem Biophys Res Commun; 413:426-431.

[11] Bassi A, Fieramonti L, D'Andrea C, Mione M, Valentini G (2011). In vivo label-free three-dimensional imaging of zebrafish vasculature with optical projection tomography. J Biomed Opt; 16: 100502.

Project leader: STEFANO DUGA (s [email protected] )

Location: Department of Medical Biotechnology and Translational Medicine, via Viotti 3/5, 20133 Milano


Whole-exome sequencing applied to the study of the genetic basis of Parkinson disease

Parkinson disease (PD) is a degenerative disorder of the central nervous system, characterized by the progressive death of dopaminergic neurons in the substantia nigra, associated with resting tremor, bradykinesia, postural instability, and rigidity. PD is a complex disorder caused by the combination of so-far largely unidentified environmental factors and of predisposing susceptibility genetic components. Despite extensive studies, only a minority of such genetic factors are known.

Even though most forms of PD are sporadic, some rare monogenic forms, with a clear “Mendelian” inheritance, have been reported, leading to the identification of several loci for familial PD. However, for some of them, the specific gene remains elusive. Intriguingly, the clinical presentations and neuropathological findings of hereditary forms of PD are often indistinguishable from the sporadic ones, raising the possibility that common pathophysiologic mechanisms underlie both hereditary and sporadic PD.

In the past few years, the study of the genetic variants predisposing to sporadic PD took advantage of genome wide association studies (GWAS). However, the 5 published GWAS did not add much to what was previously known, explaining only a very small portion of the total heritability of the disease. This suggests that the missing heritability could be, at least in part, due to rare higher-impact variants. In this frame, the rapid advancement of sequencing technologies has made it possible to capture, by targeted hybridization and deep-sequencing, all genetic variations within exonic regions of the genome, including splice junctions and miRNAs: the so called “whole-exome” approach.

This project aims to discover new genes involved in recessive Mendelian form of PD by a next-generation sequencing approach. The specific aims are: 1) to capture and sequence the exome of PD probands of recessive families (only affected siblings with consanguineous parents) and to select the variants potentially causative; 2) to verify if selected variants are specific for PD analyzing a large panel of cases and controls; 3) to search the candidate genes in a group of potentially recessive PD cases; 4) to evaluate the functional role of identified mutations

To accomplish theses goals, we will focus on 10 PD patients derived from 7 families with consanguineous parents and 2 or more affected siblings selected from the DNA Bank of the Parkinson Institute of Milan, which is based in our laboratory. Such cases are highly suggestive of a recessive form of PD and represent a valuable resource for the identification of PD causative genes as their inbred nature overcomes several of the limitations that exist in the use of exome sequencing. The exome-sequencing experiments will be performed in collaboration with Prof. John Landers of the University of Massachusetts Medical School, Worcester.


To distinguish potentially pathogenic mutations from other benign and unrelated variants, all identified variations will be subjected to a filtering process including the following criteria: i) absence in databases; ii) type and position of the mutation; iii) cosegregation in the pedigree; iv) predicted functional consequences; and v) evolutionary conservation. This filtering scheme should drastically restrict the number of potentially causative variations. The candidate mutations/genes will be screened in a large available cohort of PD cases and controls (a cohort of 3000 patients and 1500 controls is already available) and the identified mutations will be functionally characterized by a multifaceted approach, including gene expression analysis, in-vitro studies, and over-expression/knock-down experiments in animal models (zebrafish or mouse).

Project leader: FABIO FORNARA ([email protected])



Molecular control of flowering in rice

Rice is a tropical plant that flowers when exposed to short day lengths (SDs), typical of tropical regions. However, many varieties are known that can be grown in temperate areas of the world, including Mediterranean Europe. Upon perception of a favourable photoperiod, leaves express proteins belonging to the Phosphatidylethanolamine Binding family. Such proteins, encoded by Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), act as mobile signals that move through the vascular system to the shoot apical meristem, where they induce profound developmental reprogramming of the stem cell population present at the apex. Our laboratory is interested in understanding the molecular mechanisms responsible for reprogramming a group of undifferentiated cells to become an inflorescence. To this aim, we generated transgenic rice plants that express Hd3a and RFT1 under the control of a meristem-specific and inducible promoter. With such a tool we are able to trigger developmental reprogramming, independently of the day length conditions in which plants are grown.The candidate will use these tools as basis for his/her PhD project that will follow this rationale.

1. Monitoring flowering at the phenotypic level by measuring flowering time of induced and non-induced plants, and the robustness of the system.

2. Monitoring induction at the molecular level, assaying expression of candidate genes, known to be targets regulated by Hd3a and RFT1 proteins.

3. Sampling meristematic cells, including the stem cell population, of induced and non-induced plants to perform global transcript profiling of genes differentially expressed during reprogramming. Profiling will be performed through next generation sequencing technologies.

4. Validate candidate genes by independent methods of expression analysis.5. Study relevant candidates in transgenic rice lines, silencing or ectopically expressing the

genes of interest.

Project leader: CARMELA GISSI ([email protected])



Evolutionary dynamics of nuclear genes involved in replication and repair of the mitochondrial genome in fast-evolving chordates

The mitochondrial genome (mtDNA) of Metazoa is the molecule of choice in animal phylogenetic reconstructions but isalso regarded as a model system for studying the processes governing the evolution of an entire genome (Gissi et al. 2008). As peculiarity, this genome is characterized by co-evolution with the nuclear genome, in fact the biogenesis and maintenance of mitochondria depends on tightly regulated interactions between the nuclear and mt genetic systems (Garesse and Vallejo 2001; Cannino et al. 2007). For example, the mtDNA of metazoans encodes only for some subunits of the respiratory complexes and for few components of the mt protein synthesis machinery, while the overwhelming majority of mt proteins are encoded by the nucleus, including those involved in replication, transcription and repair of the mtDNA as well as in the formation of the mt nucleoid. In general, we can expect that these nuclear-encoded mt proteins will evolve in different way depending on the details of the mtDNA organization and functionality in the different taxa, and then on the overall mtDNA evolutionary trends. At present, the mtDNA has been completely sequenced in more than 2000 metazoan species belonging to the most diverse phyla, from sponges to mammals. Interestingly, among Chordata, the mtDNA of vertebrates shows low evolutionary rate and almost frozen structural and compositional features, while the mtDNA of Tunicata, the sister taxon of vertebrates, is characterized by fast nucleotide substitution rate, hypervariability of the gene order (with genes nevertheless all located on the same strand), apparent absence of a major regulatory region for transcription and replication, and strong variability of base composition and asymmetry (Gissi et al. 2010; Rubinstein et al. 2013).

The aim of this project is to study the evolutionary dynamics of the above-mentioned nuclear-encoded gene categories in representative of vertebrates and tunicates, and in amphioxus (the only representative of Cephalochordata), in order to predict which proteins and protein-regions are mainly responsible of the differences observed between Tunicata, Vertebrata and Cephalochordata in the mt genome organization and functionality. This study will also allow the candidate to participate to new genome and transcriptome projects of tunicate species.

References

Cannino G, Di Liegro CM, Rinaldi AM (2007) Nuclear-mitochondrial interaction. Mitochondrion. 7: 359-366. Epub 2007 Aug 2002.

Garesse R, Vallejo CG (2001) Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes. Gene. 263: 1-16.

Gissi C, Iannelli F, Pesole G (2008) Evolution of the mitochondrial genome of Metazoa as

exemplified by comparison of congeneric species. Heredity 101: 301-320Gissi C, Pesole G, Mastrototaro F, Iannelli F, Guida V, Griggio F (2010) Hypervariability of

ascidian mitochondrial gene order: exposing the myth of deuterostome organelle genome stability. Mol Biol Evol. 27: 211-215.

Rubinstein ND, Feldstein T, Shenkar N, Botero-Castro F, Griggio F, Mastrototaro F, Delsuc F, Douzery EJ, Gissi C, Huchon D (2013) Deep sequencing of mixed total DNA without barcodes allows efficient assembly of highly plastic ascidian mitochondrial genomes. Genome Biol Evol. 5: 1185-1199

Project leader: NERINA GNESUTTA ([email protected])



NF-Y partners in the regulation of CCAAT promoters

Transcriptional regulation is at the heart of all biological process, and it is governed by transcription factors -TFs- which bind to discrete genomic regions. Many protooncogenes and tumor suppressors are TFs and their disregulation leads to changes in gene expression patterns that result in uncoltrolled cell growth and cancer. The CCAAT box is a DNA element which is found enriched in promoters of growth controlling genes, and is specifically bound by the trimeric transcription factor NF-Y. Recently, the locations of several TF binding sites has been mapped in vivo at the genomic level by the ENCODE consortium project. Bioinformatic analyses of these data, performed by our group and others’, have shown that NF-Y locations significantly overlap, within short distances, with a few set of other TFs, among which the protooncogenes Myc and Fos. Such analyses suggest that specific configurations of the relative binding sites in promoters, can underlie the rules of biochemical and functional interactions of NF-Y with its partners to control gene expression. Such information, together with the knowledge of the crystal structure of NF-Y bound to DNA, recently solved by our group in collaboration with proff Nardini and Bolognesi, is the foundation of the proposed project.

The research project aims at understanding, at the biochemical, structural and functional levels, the interactions of NF-Y with TFs Myc (Myc/Max), and other E-box binding TFs (Max and USF1), and with Fos (Fos/Jun), based on “prototypical” CCAAT promoters bound by NF-Y and its genomic partners. Such informations will allow us to analyse larger sets of promoters, and could provide useful information to understand and predict TFs interactions in the regulation of CCAAT promoter genes. Specific aims of the project will include: in vitro biochemical analyses of purified proteins to evaluate TFs cooperativity in DNA binding by EMSA; isolation of TFs ternary complexes with DNA for SAXS structural analyses, to visualise surfaces involved in protein interactions; in vivo studies of promoter occupancy by ChIP, following TFs inactivation, to understand possible hierarchy in DNA binding; in vivo studies by transient expression of wt and mutant proteins with gene promoter-reporter assays to evaluate functional interactions in gene expression regulation.

References:-Dolfini D, Gatta R, Mantovani R. NF-Y and the transcriptional activation of CCAAT promoters. Crit. Rev. Biochem. Mol. Biol. 2012; 47: 29-49.-Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R. Struhl R. NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. Genome Res 2013; 23:1195-209-Nardini M, Gnesutta N, Donati G, Gatta R, Forni C, Fossati A, Vonrhein C, Moras D, Romier C, Bolognesi M, Mantovani R. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination. Cell 2013; 152: 132-143.

Project leader: LUISA GUERRINI ([email protected])



Role of the p300 acetylase in regulating the activities of the p63 transcription factor

The transcription factor p63 is a key regulator of ectodermal, orofacial and limb development. In particular, it plays a critical role in epithelial biology, contributing to development and maintenance of the stratified epidermis (1). Dominant mutations in the p63 gene are causative of several human hereditary syndromes, such as AEC, EEC, LMS and SHFM-IV (2). Unequivocal establishment of the role of p63 in the pathogenesis of these human hereditary syndromes is complicated by the fact that this protein exists in multiple isoforms with different, often contradictory, biological activities. Despite increasing knowledge about the biological function of p63 in the tissues in which is expressed, relatively little is known about the mechanisms governing the expression levels of the p63 proteins. The function played by p63 in ectodermal differentiation and stratified epithelial progenitor-cell maintenance is well assessed, however, further studies are required to determine the interplay of p63 with other signalling pathways including those regulating the other p53 family members.

It is likely that interrelationships between p53 and specific p63 isoforms play an essential role into the proliferation and differentiation program of developing cells. A current opinion is that p63 and/or p73 reside with p53 in larger transcriptional complex in which each sibling may regulate the activity of the others (3). To this respect, mutations affecting the relative stability of specific isoforms might grossly alter the fine tuning of p53 and p63 activity on specific promoters.

It has already been demonstrated that one component of the principal p53 regulatory pathway, the p300 acetylase, is clearly involved also in the control of p73 isoforms (4, 5) with very little being known about its role on p63 isoforms (6).

We are now interested in unravelling the role of the p300 acetylase on p63 regulation during keratynocites differentiation and during mouse limb development.

The p300 acetylase has a critical role in regulating p53 and p73 protein stability and transcriptional activity. Recently, a new p300 site has been described in p53, lysine 164, which integrity is essential for p53 activities (7) that is perfectly conserved in p63 and is found mutated to glutamic acid (K193E) in patient affected by the SHFM-IV syndrome. Interestingly, we have made preliminary observations suggesting that the effects of p300 on p63 stability could be affected by this natural mutation.

We intend now to better investigate the mechanisms through which p300 regulates p63 expression since, depending on their relative expression levels, p300 can mediate either p63 protein stabilization or p63 protein degradation. This dual role of p300 has already been described for the

p53 protein (8).

Our studies should contribute to the identification of the molecular mechanisms and the cellular players involved in the control of epithelial and limb development and homeostasis.

References1) Barbieri CE, et al (2006) p63 and epithelial biology. Exp Cell Res 312, 695-706

2) Brunner HG, et al (2002) P63 gene mutations and human developmental syndromes. Am J Med Genet 112, 284-290

3) King KE, Weinberg WC.(2007) p63: defining roles in morphogenesis, homeostasis, and neoplasia of the epidermis. Mol Carcinog 46,716-244) Barlev NA,et al . Acetylation of p53 Activates Transcrition trough Recruitment of Coactivators/Histone AcetylTransferase. Mol Cell 2001; 8: 1243-1254

5) Mantovani F, et al Pin1 Links the Activities of c-Abl and p300 in regulating p73 Function. Mol Cell 2004; 14:625-636

6) MacPartlin M, et al. p300 regulates p63 Trasc-riptional Activity. Jour Biol Chem 280: 30604-30610.

7) Tang Y, et al. Acetylation Is Indispensable for p53 Activation. Cell 2008; 133:612-626

8) Grossman SR, et al.. Polyubiquitination of p53 by a ubiquitin Ligase activity of p300. Science 2008; 300:342-344.

Project leader: MARTIN KATER ([email protected])



Mining the Molecular Pathways Controlling Rice Reproductive Development

Rice is one of the major food crops in the world. However, rice is also a model species for molecular genetics research in monocot species. The lab of Prof. Martin Kater is working for the last 15 years on rice reproductive development. The research is focuses on the molecular control of flower, ovule and seed development (see for instance Dreni et al., 2007, 2011, 2013; Yun et al., 2013; Li et al., 2011). The proposed PhD research project will be integrated in a research line that focuses on the molecular control of ovule and seed development. Recently, the Kater group identified OsMADS13 as a key regulator of ovule and seed development (Dreni et al., 2007). The PhD fellow will be involved in the identification of the regulatory pathways that are controlled by OsMADS13. This research has an unique starting point since a subset of candidate genes were already identified very recently by using a RNA-seq transcriptome analysis approach. For this analysis we used laser microdissected ovule primordia of wild-type and osmads13 mutant plants. Furthermore, we also profiled genome-wide expression of developing seeds in the osmads13 mutant and higher order mutants of genes (OsMADS3, OsMADS58 and OsMADS21) that act redundantly with OsMADS13 during seed development. The research program will focus on the identification of direct targets and their functional characterization. This research should finally provide deep insight into the molecular mechanisms that control the formation of seeds in monocot species.

References:Dreni, L., Jacchia, S., Fornara, F., Fornari, M., Ouwerkerk, P., An, G., Colombo, L., Kater, M.M. (2007). The D-lineage MADS-box gene OsMADS13 Controls Ovule Identity in Rice. Plant J. 52, 690-699.

Li, H., Liang, W., Hu, Y., Zhu, L., Yin, C., Xu, J, Dreni, L., Kater, M.M., and Zhang, D. (2011). Rice MADS6 Interacts with the Floral Homeotic Genes SUPERWOMAN1, MADS3, MADS58, MADS13, and DROOPING LEAF in Specifying Floral Organ Identities and Meristem Fate. Plant Cell 23, 2536-2552.

Dreni, L., Pilatone, A., Yun, D., Erreni, S., Pajoro, A., Caporali, E., Zhang, D., and Kater, M.M. (2011). Functional Analysis of all AGAMOUS Subfamily Members in Rice Reveals their Roles in Reproductive Organ Identity Determination and Meristem Determinacy. Plant Cell 23, 2850-2863.

Dreni, L, Osnato, M. and Kater M.M. (2013). The Ins and Outs of the Rice AGAMOUS Subfamily. Mol. Plant 6, 650-664.Yun, D., Liang, W., Dreni, L., Yin, C., Zhou, Z., Kater, M.M. and Zhang, D. (2013). OsMADS16 genetically interacts with OsMADS3 and OsMADS58 in specifying floral patterning in rice. Mol. Plant (in press)

Project leader: FEDERICO LAZZARO ([email protected])



Determine the biological significance of the rNMPs incorporated in genomic DNA

Ribonuclease H (RNase H) are enzymes capable of removing the RNA moiety in RNA:DNA hybrid molecules. These enzymes are evolutionary conserved and in eukaryotic cells type 1 and 2 enzymes are present. Mutations in the RNase H2 enzyme, are found in a subset of patients suffering of a rare genetic disease, called Aicardi-Goutières Syndrome (AGS). AGS is a genetic encephalopathy whose clinical features mimic congenital viral infection. Initiation of autoimmunity is caused by interferon (IFN)-stimulatory nucleic acids derived from exogenous (e.g. viral infection) or endogenous sources (i.e. DNA replication, repair or retrotranscription) (Cerritelli & Crouch, 2009). It has been found that replicative DNA polymerases can incorporate rNTPs in place of dNTPs during DNA replication with an unexpected high frequency (~ 1/1000 nt) (McElhinny, Kumar, et al., 2010a; McElhinny, Watts, et al., 2010b). rNMPs embedded in chromosomal DNA represent an imprint, positioned in S-phase, that regulates DNA transactions (Dalgaard, 2012). RNase H enzymes are crucial for the removal of these rNMPs from genomic DNA and for the maintenance of chromosome integrity. Recently we have found that impairment of RNase H1 and RNase H2 in yeast causes rNMPs accumulation in the genome and chronic activation of the post-replication repair (PRR) system which is becoming essential for cell survival (Lazzaro et al., 2012).The high rate of rNTPs mis-incorporation observed under normal conditions (1/1000 dNTPs) suggests possible physiological functions for the presence of rNMPs in newly replicated DNA. In a collaborative study we recently demonstrated that the presence of rNMPs during leading strand DNA synthesis acts as a strand discrimination signal for the Mismatch DNA repair machinery(Ghodgaonkar et al., 2013). In this project we will focus our studies on: 1) mapping of specific regions where DNA replicative polymerases could incorporate rNMPs; 2) analysis of the structural effects of the presence of rNMPs within a DNA molecule using AFM studies in collaboration with Dr. Podestà (Dep. of Physics, Univ. Milano). (3) We will study the effects of rNMPs incorporation on chromatin structures and the biological consequences on DNA repair, transcription, silencing.

Cerritelli, S. M., & Crouch, R. J. (2009). Ribonuclease H: the enzymes in eukaryotes. FEBS J, 276(6), 1494–1505. doi:10.1111/j.1742-4658.2009.06908.x

Dalgaard, J. Z. (2012). ScienceDirect.com - Trends in Genetics - Causes and consequences of ribonucleotide incorporation into nuclear DNA. Trends Genet, 1–6. doi:10.1016/j.tig.2012.07.008

Ghodgaonkar, M. M., Lazzaro, F., Olivera-Pimentel, M., Artola-Borán, M., Cejka, P., Reijns, M. A., et al. (2013). Ribonucleotides Misincorporated into DNA Act as Strand-Discrimination Signals in Eukaryotic Mismatch Repair. Molecular Cell, 50(3), 323–332. doi:10.1016/j.molcel.2013.03.019

Lazzaro, F., Novarina, D., Amara, F., Watt, D. L., Stone, J. E., Costanzo, V., et al. (2012). RNase H

and postreplication repair protect cells from ribonucleotides incorporated in DNA. Mol Cell, 45(1), 99–110. doi:10.1016/j.molcel.2011.12.019

McElhinny, S. A. N., Kumar, D., Clark, A. B., Watt, D. L., Watts, B. E., Lundstr o m, E.-B., et al. (2010a). Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol, 6(10), 774–781. doi:10.1038/nchembio.424

McElhinny, S. A. N., Watts, B. E., Kumar, D., Watt, D. L., Lundstr o m, E.-B., Burgers, P. M. J., et al. (2010b). Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A, 107(11), 4949–4954. doi:10.1073/pnas.0914857107

Project leader: ROBERTO MANTOVANI ([email protected])



NF-YA isoforms in ES cells and cancer

Regenerative medicine has taken the center stage in medical sciences since the discovery of embryonic stem cells (ES). ES cells express “stemness” genes, many of which code for transcription factors. NF-Y is a trimeric CCAAT-binding factor, composed of NF-YA, NF-YB and NF-YC (1). We recently showed that one of the splicing isoform of NF-YA plays a crucial role in maintaining the mouse ES stemness potential (2). The mechanisms are related to the capacity to connect with the circuitry of stem cells transcription factors and their regulated genes. In general, two splicing isoforms -long and short- are produced from the NF-YA locus, and their expression is apparently quite regulated. They differ in 28 AA in the Q-rich transcriptional activation domain. Somewhat surprisingly, it has recently emerged that the two isoforms have different, often opposing roles in important cellular processes.

The aim of the project will be to investigate the mechanistic role of NF-YA isoforms in different cellular contexts, by overexpression and functional inactivation. (i) The expansion of the stem cells compartment(s) has been associated to NF-YAs, and it is possible that NF-YAl is involved in differentiation. We will evaluate the interplay, in terms of protein-protein interactions and coregulated genes, of NF-YA with important ES regulators, by analysis of profilings and ChIP-Seq data. (ii) The CCAAT box is often present in promoters of genes overexpressed in different types of cancer, and it is believed that NF-Y plays an important role in mediating high level expression. Several indirect evidence suggests that NF-YA isoforms might play a role in cancer progression. We will therefore assess this aspect, by overexpressing the isoform in non transformed and transformed cells, to assess their pro-proliferative and transforming potential. The expected results are a better understanding of the molecular mechanisms that lead to differentiation or the expansion of the stem cells pools, as well as the interplay between NF-Y and other TFs, ES TFs and oncogenes, on common targets.

1. Nardini M., Gnesutta N., Donati G., Gatta R, Forni C., Fossati A., Vonrhein C., Moras D., Romier C., Bolognesi M., Mantovani R. NF-Y is a sequence-specific transcription factor displaying histone-like DNA binding and H2B-like ubiquitination. Cell, 152, 132-143 (2013).

2. Dolfini D. and Mantovani R. Targeting the Y/CCAAT box in cancer: YB-1 or NF-Y?Cell Death and Differentiation, 20, 676-685 (2013).

Project leader: FEDERICA MARINI ([email protected])



Title: Unravelling the role of the Fanconia anemia protein P/Slx4 and the DNA damage checkpoint factor 53BP1/Rad9 in responding to double strand DNA lesions

Chromosomes maintenance and stability are essential goals for all the organisms in order to transfer the correct genetic information to the progeny. Double Strand Breaks (DSBs) are deleterious lesions that can be a serious threat for the cell. In fact, defects in DSBs repair leads to chromosomes instability and tumorigenesis, and DSBs are frequently accumulated in several genetic disorders and senescent cells. These lesions are processed by several nucleases, leading to the formation of a 3’ end single strand DNA filament, through a finely regulated process called DSB resection. This process can be divided in an initial step orchestrated by the Mre11 complex together with CtIP/Sae2 and a later, processive, step dependent on Exo1 and Bloom helicase/Sgs1. Mutations in the corresponding human othologs of Mre11, Sae2 and Sgs1 lead to severe disorders (ataxia telangiectasia-like, Seckel, Jawad and Bloom syndromes), characterized by genomic instability and cancer predisposition (1).

DSB resection allows the recruitment onto the lesion of both the checkpoint and the recombination factors. In our laboratory it has been demonstrated that the checkpoint factor Rad9 (53BP1 in human) binds near the lesion and counteracts the resection process, limiting the formation of ssDNA (2). A similar inhibitory role in DSB resection has been recently shown for 53BP1 in human cells. Interestingly, down-regulation of 53BP1 restores homologous recombination and DSB repair in cells with mutations in the breast cancer gene BRCA1 (3). Therefore, the studying of the functional role of Rad9/53BP1 and the regulation of the DSB resection is fundamental to understand why defects in this process lead to chromosome rearrangements and cancer.

Recently it has been shown that the Slx4 protein counteracts Rad9 binding near a DNA lesion, leading to DNA damage checkpoint inactivation (4). SLX4 is functionally highly conserved from yeast to humans and participates in many different DNA repair pathways such as resolving replication fork blocks, homologous recombination and inter-strand crosslink repair. The main function of SLX4 is to act as a scaffold for several nucleases involved in different steps of DSB repair. Furthermore, SLX4 was recently shown to be a component of the Fanconi anemia pathway (FA), a rare recessive disorder characterized by chromosomal instability, increased cancer susceptibility, developmental of abnormalities, bone marrow failure, and childhood cancers (5).

The PhD student will use both yeast and human cell lines as model systems to investigate the role of Slx4 in the maintenance of genomic stability. He/She will study the kinetic of a DSB resection and repair by Southern blotting, the recruitment of checkpoint and recombination factors by ChIP and indirect immunofluorescence. The functional interaction between Slx4 and 53BP1/Rad9 will be

investigated through genetic and biochemical approaches. Furthermore, He/She will set up specific screening to identify novel genes and factors involved in DSB repair.

1) Jackson, S. P. & Bartek, J. (2009) Nature 461(7267), 1071--1078.2) Lazzaro, F. et al. (2008) EMBO J 27(10), 1502--1512.3) Zimmermann M, de Lange T. (2013) Trends Cell Biol. doi: 10.1016/j.tcb.2013.09.003. 4) Ohouo PY et al. (2013) Nature. 3;493(7430):120-4. 5) Kim Y, et al. (2013) Blood 3;121(1):54-63.

Project leader: GRAZIELLA MESSINA ([email protected])



Identification of the mechanism(s) regulating Nfix expression during fetal myogenesis

Skeletal muscle is the tissue responsible for posture, locomotion and diaphragmatic breathing. The molecular mechanisms regulating muscle differentiation and maturation are quite well characterized 1-4. Interestingly, skeletal myogenesis, like hematopoiesis, occurs in successive, distinct though overlapping developmental stages that involve different cell populations and expression of different genes. Skeletal muscle is, in fact, a heterogeneous tissue composed of individual muscle fibres, diversified in size, shape and contractile protein content, to fulfil the different functional needs of the vertebrate body. This heterogeneity derives and depends at least in part upon distinct classes of myogenic progenitors, i.e. embryonic and fetal myoblasts and satellite cells. In particular embryonic and fetal myoblasts control the differentiation of the pre-natal musculature, whereas satellite cells (SCs) are responsible for post-natal muscle growth and regeneration following muscle damage or injury 5. Myoblast fusion into multinucleate muscle fibres begins at around E11 in the mouse and characterizes “embryonic” or primary myogenesis necessary to establish the basic muscle pattern. Fetal myogenesis is characterized by growth and maturation of each muscle anlagen and by the onset of innervation. This second wave of myogenesis (also called secondary myogenesis) takes place between E14.5 and E17.5 and involves the fusion of fetal myoblasts either with each other to form secondary fibres (initially smaller and surrounding primary fibres) or, at a minor extent, with primary fibres. A genome wide expression analysis carried on purified embryonic and fetal myoblasts 6 identified many differentially expressed genes, clearly revealing that embryonic and fetal myoblasts are intrinsically different populations of myoblasts with distinct genetic programs. I have recently demonstrated the pivotal role of the transcription factor Nuclear Factor IX, Nfix, in driving the transcriptional switch from embryonic to fetal myogenesis 7. This work provided the first evidence that a single factor is responsible for the differential gene expression that transforms the primary primitive musculature (due to embryonic myogenesis) into a more mature and organized muscle (fetal myogenesis). Interestingly no evidences in literature describe the possible mechanism(s) which regulate Nfix during development. This project will address this import point that is still missing in the scenario of pre-natal muscle development.

References

1. Davis, R.L., Cheng, P.F., Lassar, A.B. & Weintraub, H. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell 60, 733-746 (1990).

2. Weintraub, H. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75, 1241-1244 (1993).

3. Relaix, F., Rocancourt, D., Mansouri, A. & Buckingham, M. A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435, 948-953 (2005).

4. Hutcheson, D.A., Zhao, J., Merrell, A., Haldar, M. & Kardon, G. Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for beta-catenin. Genes & development 23, 997-1013 (2009).

5. Biressi, S., Molinaro, M. & Cossu, G. Cellular heterogeneity during vertebrate skeletal muscle development. Developmental biology 308, 281-293 (2007).

6. Biressi, S. et al. Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells. Developmental biology 304, 633-651 (2007).

7. Messina, G. et al. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell 140, 554-566

Project leader: GRAZIELLA MESSINA ([email protected])



Genome-wide mapping of Nfix binding sites in skeletal muscle

Skeletal muscle is the tissue responsible for posture, locomotion and diaphragmatic breathing. The molecular mechanisms regulating muscle differentiation and maturation are quite well characterized 1-4. Interestingly, skeletal myogenesis, like hematopoiesis, occurs in successive, distinct though overlapping developmental stages that involve different cell populations and expression of different genes. Skeletal muscle is, in fact, a heterogeneous tissue composed of individual muscle fibres, diversified in size, shape and contractile protein content, to fulfil the different functional needs of the vertebrate body. This heterogeneity derives and depends at least in part upon distinct classes of myogenic progenitors, i.e. embryonic and fetal myoblasts and satellite cells. In particular embryonic and fetal myoblasts control the differentiation of the pre-natal musculature, whereas satellite cells (SCs) are responsible for post-natal muscle growth and regeneration following muscle damage or injury 5. In particular, embryonic myoblasts fuse into multinucleate muscle fibres and characterize “embryonic” or primary myogenesis necessary to establish the basic muscle pattern. The second wave of myogenesis takes place between E14.5 and E17.5 and involves the fusion of fetal myoblasts either with each other to form secondary fibres (initially smaller and surrounding primary fibres) or, at a minor extent, with primary fibres. A genome wide expression analysis carried on purified embryonic and fetal myoblasts 6 identified many differentially expressed genes, clearly revealing that embryonic and fetal myoblasts are intrinsically different populations of myoblasts with distinct genetic programs. We have demonstrated the pivotal role of the transcription factor Nuclear Factor IX, Nfix, in driving the transcriptional switch from embryonic to fetal myogenesis 7. Interestingly, satellite cells express high levels of Nfix and we have very interesting results on the role of Nfix even during post-natal muscle regeneration 8.

Since we demonstrated that Nfix is able to directly bind the promoters of different genes crucial and/or involved in proper skeletal muscle development and regeneration, this project will aim to perform a genome-wide ChiP-seq analysis for Nfix in order to identify other possible targets of Nfix in skeletal muscle and therefore to discover other and new pathways in which Nfix may play new functions.

References

1. Davis, R.L., Cheng, P.F., Lassar, A.B. & Weintraub, H. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell 60, 733-746 (1990).

2. Weintraub, H. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75, 1241-1244 (1993).

3. Relaix, F., Rocancourt, D., Mansouri, A. & Buckingham, M. A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435, 948-953 (2005).

4. Hutcheson, D.A., Zhao, J., Merrell, A., Haldar, M. & Kardon, G. Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for beta-catenin. Genes & development 23, 997-1013 (2009).

5. Biressi, S., Molinaro, M. & Cossu, G. Cellular heterogeneity during vertebrate skeletal muscle development. Developmental biology 308, 281-293 (2007).

6. Biressi, S. et al. Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells. Developmental biology 304, 633-651 (2007).

7. Messina, G. et al. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell 140, 554-566

8. Giuliana Rossi, Stefania Antonini, Chiara Vezzali, Giulio Cossu and Graziella Messina The transcription factor Nfix regulates the proper timing of muscle regeneration and the progression of Muscular Dystrophy, in preparation

Project leader: ANNA MORONI ([email protected])



The structural mechanism of KAT1 channel regulation by the cytoplasmic domain CNBHD

The KAT1 family of voltage-dependent potassium channels (AKT1, SKOR, GORK) are important regulators of nutrient uptake and osmotic responses in plant cells. KAT1 in particular plays a key role in stomata function, thus controlling carbon dioxide uptake for photosynthesis and water balance of the entire organism. The KAT1 channel activity depends on membrane voltage as well as on extra- and intracellular modulatory factors which act by shifting the voltage range of KAT1 activation. KAT1 channels have two large intracellular regions that underlie the specialized gating and regulation of this channel family (Marten and Hoshi, PNAS 94, 3448-3453,1997). Indeed, voltage-dependent gating depend on the N and C- termini in a manner that is reminiscent of mammalian KCNHD channels. Moreover, the carboxy-teminal region of KAT1 channels domain is structurally and functionally related to those of mammalian KCNH channels that contains a cyclic nucleotide- binding homology domain (CNBHD) which is connected to the pore through a C-linker domain (Haitin et al., Nature 501, 444-448, 2013). The CNBHD however does not bind cyclic nucleotide and presumably regulate channel gating in a cyclic nucleotide-independent manner. We propose in this project to solve the structure of the CNBHD of KAT1 and to dissect its functional role by a combination of X-ray crystallography and cell electrophysiology.

Project leader: MARCO MUZI FALCONI ([email protected])



Haspin kinase: a new regulator of cell polarity and cell division

Haspin is an atypical serine-threonine protein kinase which is evolutionarily conserved in all eukaryotes (Higgins, 2003). The structure of human haspin has been determined, but very little is known regarding its substrates and its role in vivo. In human cells, Haspin is involved in mitotic phosphorylation of histone H3-Thr3 and in protecting centromeric cohesion during mitosis (Dai et al., 2005; Dai et al., 2006; Yamagishi et al., 2010; Kelly et al., 2010; Wang et al, 2010). Budding yeast contains two haspin-coding genes (ALK1 and ALK2) and their gene products are cell cycle regulated both at the level of protein stability and phosphorylation (Nespoli et al. 2010). Since in yeast H3-Thr is not phosphorylated this model system could be exploited to identify new functions for Haspin. We recently showed that Alk1 and Alk2 play a role in modulating cell polarization, actin distribution and mitotic spindle alignment (Panigada et al. 2013). In this project we propose to investigate the molecular mechanisms involved in this new function of Haspin. Preliminary evidence suggest a genetic interaction between Haspin and Cdc42. Moreover, Alk2 physically interacts with the Cdc42, a GTPase controlling cell polarity (Etienne-Manneville, 2004), and elevated Alk2 levels reduce phosphorylation of the Cdc42 activator Cdc24. These findings suggest that haspin may impact on cell polarity by regulating Cdc42 activity or one of its several effectors. Through the combined use of cell biology, biochemistry, molecular biology and genetics we will analyze how haspin affects Cdc42 localization, activation/inactivation, interaction with downstream effectors and how haspin modulates cell polarization, and asymmetric cell division.

Dai et al., (2005) Genes Dev 19:472Dai et al (2006) Dev Cell 11:741Etienne-Manneville (2004) J Cell Sci 17:1291 Higgins J., (2003) Cell Mol Life Sci 60:446Kelly et al. (2010) Science 330:235Nespoli et al., (2006) Cell Cycle 5:1464Panigada et al., (2013) Dev Cell 26:483Wang et al. (2010) Science 330:231Yamagishi et al (2010) Science 330:239

Project leader: MARCO NARDINI ([email protected])]



Structural analysis of transcription factor/DNA complexes

One of the key issues in biology is how the genetic information is transferred to biological functions. Binding of transcription factors (TFs) to discrete sequences in gene promoters and enhancers, is crucial to the process, which needs to interface with chromatin, whose fundamental unit is the nucleosome, formed by core histones wrapped by 146 bp of DNA. Binding of TFs entails the recruitment of histone modifying and chromatin remodeling machines, thus helping to define the chromatin status (“euchromatin” vs “heterochromatin”). TFs fall in essentially two categories: (i) “pioneer” TFs, with intrinsic chromatin association capacity; (ii) "activating" TFs, binding to a favorable chromatin landscape pre-set by pioneers.

In this context, the present PhD project focus on NF-Y, a histone-like TF that binds and activates the CCAAT box [1], and on MYC, a proto-oncogene that binds the E-box (5’-CACGTG-3’), whose altered expression transforms cells [2]. NF-Y and MYC are deemed to be paradigms of pioneer and activating factors, respectively, and indeed they were shown to interact directly. Furthermore, the availability of the 3D structures for both NF-Y and MYC [3, 4] makes both TFs potential targets for development of anti-cancer drugs. Because of their direct binding, the strong correlation between the NF-Y and MYC loci in vivo, and the fact that regulation of MYC and CCAAT genes is crucial to oncogenic transformation, understanding of their interplay at the structural level will further increase druggable surfaces.

The present PhD project will be carried out in the Nardini’s (structural biology) lab as a continuation of an ongoing research project that led recently to the successful determination of the X-ray structure of the NF-Y in complex with its target DNA [3]. The project will pursue analyses of NF-Y in complex with DNA containing multiple CCAAT boxes, and the NF-Y/MYC/MAX-DNA complex, both by X-ray crystallography and solution scattering methods (Small/Wide Angle X-ray Scattering, SAXS/WAXS). The SAXS/WAXS experiments, being performed on solution samples, are always practicable, provided that the sample is sufficiently pure and monodisperse. For the X-ray crystallography approach, the Nardini lab experience in growing protein-DNA complex crystals will be crucial for the achievement of this step: several E-box and CCAAT-containing fragments will be designed and tested in an approach that proved successful for the NF-Y/CCAAT complex [3].

The 3D structure of the NF-Y/DNA complex, will also allow to search rationally for potential inhibitors. As a part of the PhD project, inhibitor/NF-Y docking simulations will be carried out to screen virtual chemical libraries of low molecular weight compounds, searching for inhibitors of the NF-Y quaternary assembly and of its DNA-binding capacity. X-ray crystallography will be then applied to characterize the structure of the complexes between NF-Y and the best inhibitors. A similar approach will be eventually applied to interfere with the

interactions between NF-Y and MYC/MAX. Potential inhibitors selected through the in silico approaches will be cross-validated in vitro through Thermal shift and electrophoretic mobility shift assay (EMSA) experiments, and in cells by ChIPs.

[1] Dolfini D, Gatta R, Mantovani R. Crit Rev Biochem Mol Biol. (2012) 47: 29-49.[2] Prendergast GC, Lawe D, and Ziff EB. Cell (1991) 65: 395-407.[3] Nardini M, Gnesutta N, Donati G, Gatta R et al. Cell (2013) 152: 132-143.[4] Nair SK, Burley SK. Cell (2003) 112: 193-205.

Project leader: GIULIO PAVESI ([email protected])



Development and Application of Bioinformatic Tools for the Analysis of ChIP-Seq Data

The modern “high-throughput” or “next-generation” sequencing techniques have opened new avenues in every aspect of genetics and genomics research. In particular, when applied downstream of experiments like chromatin or riboprotein immunoprecipitation (ChIP-Seq, RIP-Seq, respectively), they allow researchers to study at a unprecedented level of detail the mechanisms of regulation of transcription (epigenetic mechanisms or interactions between transcription factors and DNA) and translation (RNA binding proteins – RNA interactions). These experiments have become quite affordable in the last few years, and this fact has yielded an explosion in the data produced, where bioinformatics analysis plays a key role in order to obtain significant results.

Our research group has been and is currently involved in different studies in co-operation with other groups in this field, that produce data related to histone modifications, transcription factors and RNA binding proteins in different organisms (see for example [1,2]). In this framework, our task concerns the analysis of the data, either through the fine tuning and adaptation of existing methods, but in particular with the development of novel tools [3,4]. Thus, the goal of the project will be twofold: on one hand it will involve more basic research on the development and testing of novel bioinformatic methods, for which the state of the art is still far from having reached standard analysis procedures, and on the other their application to real analyses and case studies. The PhD student will thus perform both the analyses, by using tools developed by us or by others to be applied to the different case studies, but more importantly will be involved in the development and testing of novel methods for different steps of the typical analysis pipelines for ChIP-Seq data, and their implementation in suitable software tools.

1: Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R, Struhl K. NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. Genome Res. 2013 Aug;23(8):1195-209.

2: Mihailovic M, Wurth L, Zambelli F, Abaza I, Militti C, Mancuso FM, Roma G, Pavesi G, Gebauer F. Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR. RNA. 2012 Jan;18(1):53-64.

3: Zambelli F, Pesole G, Pavesi G. PscanChIP: Finding over-represented transcription factor-binding site motifs and their correlations in sequences from ChIP-Seq experiments. Nucleic Acids Res. 2013 Jul;41:W535-43.

4: Zambelli F, Prazzoli GM, Pesole G, Pavesi G. Cscan: finding common regulators of a set of genes by using a collection of genome-wide ChIP-seq datasets. Nucleic Acids Res. 2012 Jul;40W510-5.

Project leader: PAOLO PESARESI ([email protected])



Chromatin Remodeling Enzymes are at the basis of SVP transcription factor function

Reproductive success in plants is dependent on the timing of the switch from vegetative to reproductive phase coinciding with optimal environmental and developmental conditions. This is a key step that dramatically influences plant productivity, one of most important aspects in agriculture. Plants have evolved an elaborate regulatory network that integrates endogenous and environmental signal to ensure that flowering start when conditions are most favourable. During the last two decades, functional genomics studies have revealed the existence of a complex network of genetic interactions responsible of integrating the different types of signals, both internal and external that plant receives and that define when plants can enter into the reproductive phase (Liu et al., 2009). However, the molecular characterization of the floral transition process is far from being completed, therefore new studies flanked by new research methods are needed to identify and characterize the proteins and protein complexes that play key roles in the transition to the reproductive phase (Jang et al., 2009).

One of key player in floral transition is SVP (Short Vegetative Phase), a MADS-Box transcritpion factor studied for a long time at genetic level. It has been reported (Liu et al., 2007) that the ectopic expression of this transcription factor causes late flowering, whereas plants lacking SVP flowers earlier than WT. In addition, based on data obtained via yeast-2-hybrid assay (Gregis et al., 2009), it appears that SVP regulates floral transition through the association to a multi-subunit protein complex, however evidences of such a protein complex have not been provided, yet, in planta. During the last two years, in our laboratory we have developed a protocol that allows to coimmunoprecipitate nuclear protein complexes in their native state, followed by Mass-spectrometry analyses to identify the different immune-precipitated subunits. Such a strategy has been applied to SVP, and the obtained data indicate that SVP interacts with a large set of enzymes involved in acetylation, deacetylations and metylation of histones (Cohen et al., 2009; Berr et al., 2010). Based on these findings, and in agreement with recent publications in this field (Smaczniak et al., 2012), it appears that SVP controls flowering time in Arabidopsis by chromatin remodeling-dependent expression of key genes involved in floral transition.

The aim of this project is to validate the interaction of SVP with such enzymes and to dissect their direct involvement in flowering time determination through functional studies in the model species Arabidopsis thaliana.

References

1. Liu C, et al., Dev. Cell. 2009; 16 (5): 711-22.2. Jang S, et al., Plant J. 2009; 60: 614-25.

3. Liu C, et al., Development 2007 134, 1901-1910.

4. Gregis V, et al., The Plant J. 2009; 60: 626-37.

5. Cohen R, et al.,Planta. 2009; 230(6): 1207-21.

6. Berr A, et al., The Plant Cell. 2010; 22: 3232-48.

7. Smaczniak C, et al., PNAS. 2012; 109: 1560-5

Project leader: KATIA PETRONI ([email protected])



Role of anthocyanin-enriched diet on cardioprotection

Dietary flavonoids have received considerable attention since epidemiological studies have suggested that regular consumption of flavonoid-rich foods or beverages is associated with a decreased risk of cardiovascular mortality [1–3], attributed primarily to their antioxidant properties and by modulating cell signaling and metabolic pathways. Among the different classes of flavonoids, anthocyanins are the most recognized, visible members, that contribute to the cardioprotection. In these last years, recent studies have suggested that dietary flavonoids, and more specifically regular anthocyanin consumption, induce a state of myocardial resistance evidenced by a reduced infarct size following regional ischemia and reperfusion [4] that is related, at least in part, to an improvement in the antioxidant defenses of the heart (i.e. cardiac glutathione). Moreover, there are increasing evidences that seem to confirm that many biological effects of anthocyanins are related not only to their antioxidant properties but also to their ability to modulate mammalian cell signaling pathways. For instance, recent studies in rats have shown that an anthocyanin-rich diet modulate the metabolism of (n-3) PUFA and to induce a marked increase in plasma EPA and DHA, fatty acids known to be protective against heart disease complication [5,6].

Aim of this research proposal is to study the cardioprotective effects that an anthocyanin-enriched diet has on the myocardial muscle and also its role in the prevention of drug cardiotoxicity such as in the case of many antitumor drugs. With this aim, we will investigate as a dietary strategy whether using functional foods, as anthocyanin-rich corn, can have muscle protective properties and can reduce the incidence and prognosis of myopathies [6-7].

The project will be divided in three different tasks, including i) the role of dietary anthocyanins from corn in the prevention of cardiotoxicity induced by chemotherapic agents, ii), to investigate the effects of dietary anthocyanins on specific microRNAs involved in cardiac regeneration and aging, iii) to establish the molecular mechanism underpinning the cardioprotective action of anthocyanins in murine cardiomyocytes.

With these activities, we expect to contribute to the understanding of how and why anthocyanins contribute to promote cardioprotection.

References[1] Lancet 342:1007-11, 1993.[2] BMJ 312: 478-81, 1996[3] Am J Clin Nutr 85:895-909, 2007.[4] FEBS J 273:2077-2099, 2006.[5] J Nutr 138:747-752, 2008.[6] J Nutr 141:37-41, 2011.[7] Lancet 374:1849-56, 2009.

Project leaders: PAOLO PLEVANI and GIACOMO BUSCEMI ([email protected]);

([email protected])



DAXX protein and the human DNA damage response: chromatin remodeling, gene expression, genome stability and tumorigenesis

The DNA damage response (DDR) functions in cells to detect, signal and repair lesions to the nuclear DNA structure. The apical transducer of the DDR in human cells is the kinase ATM, which in response to double strand breaks (DSBs) phosphorylates a broad range of targets impacting on DNA repair, cell cycle arrest, senescence or apoptosis. ATM gene is mutated in ataxia telangiectasia, a rare disease characterized by neurological and immunological features and by cancer predisposition (1). Since DNA repair takes place within the complex organization of the chromatin, the DDR must be able to detect lesions within nucleosome-DNA template, remodel the local chromatin architecture for processing and repair lesions and restore the initial organization.

DAXX is a chromatin-associated factor involved in transcriptional regulation working together with the DNA helicase ATRX, as a dedicated chaperone for the replication-independent deposition of the histone H3.3 variant onto gene regulatory regions as well as pericentric and telomeric heterochromatin (2). Mutations in DAXX, ATRX and H3.3 underlie paediatric/adult glioblastoma (3) and can associate in cancer with alternative lengthening of telomeres (ALT). Furthermore, DAXX interacts with several transcription factors, directly regulating gene expression.

DAXX was initially linked to DNA damage for its role in p53 regulation and apoptosis (4), but the recent description of H3.3 deposition at UV damaged DNA sites by HIRA (5), an histones chaperone, suggest that DAXX histones chaperon activity could be involved in the DDR.

Coherently, we have found that ATM and its mediator, Chk2, both phosphorylate DAXX on multiple residues, mutations of which impinge on DAXX activities, including DAXX/H3.3 interaction, suggesting that the ATM/Chk2/DAXX/H3.3 axis plays a role in chromatin dynamics in DDR, with potential implications for cancer pathogenesis.

This project aims to provide a deep understanding of the molecular bases and functionality of DAXX in the cellular response to DNA DSBs. We will explore the hypothesis that DAXX could be targeted by DDR kinases redirecting its activity to modify chromatin and transcription at DNA damage sites and/or to modulate transcription of genes involved in DDR. This could occur through H3.3 deposition or directly through the functional interaction with specific transcription factors.

Initially, we will determine how DAXX loss and expression of DAXX phospho-mutants affect genome stability, proliferation and survival after genotoxic stress. A specific role at telomeres or centromeres will also be assessed. Successively, we will evaluate if DAXX phosphorylations affect protein stability or localization and post-transcriptional modifications at other sites relevant for the activity of this protein. Finally, we will evaluate H3.3 deposition and chromatin modifications at


DSBs or other genomic regions (i.e. telomeres). In the meantime human cell lines knock in for DAXX mutants will be produced to approach gene expression studies in presence of DNA damage.

References:

1. Shiloh Y, Ziv Y. Nat Rev Mol Cell Biol. 2013 Apr;14(4):197-210.

2. Drané P, Ouararhni K, Depaux A, Shuaib M, Hamiche A. Genes Dev. 2010 Jun;24(12):1253-65.

3. Schwartzentruber J, et al., Nature. 2012 Jan 29;482(7384):226-31

4. Tang J, et al. Nat Cell Biol. 2006 Aug;8(8):855-62

5. Adam S, Polo SE, Almouzni G. Cell. 2013 Sep 26;155(1):94-106.

Project leader: STEFANO RICAGNO ([email protected])



Structural-Based Drug Discovery against RNA-based VIRUSES

The number of fatalities that occur worldwide as the result of infections by RNA viruses is in the order of many millions annually. Increasing human and animal population density combined with increasing mobility, commercial transport, land exploitation and climate change, all have an impact on virus emergence and epidemiology. Over the past 3 decades, many RNA virus threats were identified or “re-discovered”, including a variety of pathogenic flaviviruses. The flavivirus group includes several pathogens of global medical importance, i.e. dengue viruses (DENV); Infections by these neurotropic viruses may result in life-threatening aseptic encephalitis, with high risk of life-long debilitating neurologic sequels. About 500,000 people with severe dengue require hospitalization each year, a large proportion of which are children. WHO currently estimates there may be 50-100 million dengue infections worldwide every year. There is an increasing number of cases as the disease spreads to new areas, causing severe outbreaks. The threat of a possible outbreak of dengue fever now exists in Europe and local transmission of dengue, in addition to several imported cases, was reported for the first time in 2010. Vaccine development has proven particularly challenging because of the existence of 4 dengue serotypes and antibody dependent enhancement of infection. Hence neither vaccines nor antiviral therapy (or prophylaxis) are available today. We urgently need advanced levels of preparedness with which to confront and ultimately control these viral pathogens.

The infective enveloped flavivirus contains a (+) single-stranded RNA genome encoding 7 non-structural proteins involved in viral replication. The non-structural proteins NS3 and NS5 play key roles in the replication cycle, therefore are ideal antiviral targets. For discovering small-molecule inhibitors of viral replication, our approach relies on the three-dimensional structure of target proteins (“structure-based drug discovery”). The key outcomes of the project will be discovery and optimization of novel inhibitors that bind viral replicative enzyme domains and/or viral replication complexes. Inhibitors active against dengue may possibly also be active against other flaviviruses, including West Nile virus, Yellow fever and Japanese encephalitis virus.

This provides a unique opportunity to fully address the "Societal challenges" issue of the Horizon 2020 program, with a clear emphasis on world population health and well-being.

Project leader: PAOLA RIVA ([email protected])

Location: Department of Medical Biotechnology and Translational Medicine, via Viotti 3/5, 20133 Milano


Competitive endogenous RNA (ceRNA) Cross-Talk in Neurofibromatosis type 1 phenotype expression variability

Neurofibromatosis type 1 (NF1 [OMIM + 162200]) is a common autosomal dominant disorder affecting 1/3500 individuals and caused by the point mutations or deletion of NF1, a tumor-suppressor gene with a Ras-GTPase activity, encoding neurofibromin. NF1 is characterized by a highly variable expressivity with multisystemic symptoms that may manifest at birth and evolve during lifetime. The clinical signs, including café au lait spots, axillary and inguinal freckling, dermal or plexiform neurofibromas, iris Lisch nodules, but also an increased risk of other benign and malignant tumors, highlight altered developmental pathways and provide insights into the close relationship between development and cancer. Central to these interconnected aspects is neurofibromin defective deregulation and the resulting hyperactivation of the Ras signal transduction machinery. The heterogeneous clinical expression is hardly conceivable on the basis of a great majority of NF1 deletions or truncating mutations. We will focus our interest on the identification of mechanisms leading to dermal (DNF) rather than plexiform neurofibromas (PNF) in patients carrying the same lesions of NF1 gene. Given that the NF1 constitutional mutation is a necessary condition to develop the two above kinds of benign tumors and that no tumor-associated mutations have been till now demonstrated, the involvement of modifier genes and/or further pathogenetic mechanisms have been proposed, even if the basis of this variable expressivity is currently unknown. Considering that a new emerging layer of gene regulation, based on competitive endogenous RNA (ceRNA) activity, plays important roles in the development of different diseases including cancer, we hypothesize that this mechanism may be involved in the expression regulation of the functional copy of NF1 gene and have a consequence on the variability of NF1 clinical signs, comprising the development of DNF or PNF. The recent discovery of competitive endogenous RNAs (ceRNA), natural decoys that compete for a common pool of miRNAs, provides a framework explaining the activity of (miRNA) response elements (MRE)-harboring non-coding RNAs, small or long non coding RNAs and transcribed pseudogenes, in relation to the target protein-coding mRNA regulation. Functional interactions in ceRNA networks aid in coordinating a number of biologic processes and, when perturbed, contribute to disease pathogenesis. If little is known about the NF1 gene post-transcriptional regulation, only a few targeting miRNAs have been validated, the possible involvement of NF1 mRNA in ceRNA cross talk has never been investigated. Knowing that several NF1 pseudogenes are differently expressed in different tissues, the aims of the proposed project are 1) to investigate the possible involvement of NF1 mRNA in ceRNA cross talk identifying possible interferences of specific NF1 pseudogenes with the interactions between NF1 mRNA and the validated targeting miRNAs, 2) to study the expression profile of miRNA targeting NF1 mRNA, NF1 transcript and specific NF1 pseudogenes by a comparison of their levels in both constitutional and tumor mRNAs from patients with DNFs and PNFs. The first goal will be pursued

first by identifying NF1 pseudogenes sharing the same binding sites of miRNA targeting the NF1-coding mRNA by bioinformatic analysis. The expression profile of the selected pseudogenes and NF1-coding mRNA will be defined by interfering with specific miRNAs and pseudogenes in cell lines from different tissues. The detection of both NF1-coding and non coding mRNA levels consistent with the interference by the same miRNA, will indicate that the NF1-coding mRNA is involved in ceRNA cross talk. The data obtained will address the expression studies of specific NF1 pseudogenes and miRNAs in a current casuistry of 8 DNFs and 9 PNFs patients that will be increased. This project will provide new insights on NF1 post-transcriptional regulation and might open new perspectives on identification of mechanisms leading to the characteristic variable NF1 phenotype.

Project leader: GIULIA SOLDA’ ([email protected])

Location: Department of Medical Biotechnologies and Translational Medicine, via Viotti 3/5, 20133 Milano


Identification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing

Inherited nonsyndromic sensorineural hearing loss (NSHL) shows an extremely high genetic heterogeneity, with more than 70 genes already associated with NSHL, and many others still to be discovered (http://hereditaryhearingloss.org).

We are currently applying whole-exome sequencing (WES) as a cost- and time-effective strategy to search for pathogenic variants underlying deafness. Indeed, this technique has already proven to be helpful for the discovery of novel genes/mutations responsible for recessive NSHL (DFNB82 and DFNX4 loci) [1-3]. Seven NSHL families, with a clear recessive (autosomal or X-linked) inheritance pattern and at least two affected individuals, have been already selected for WES; additional families are being recruited. The WES of the first 16 patients has already been performed through external NGS services at BGI (Beijing Genomics Institute, China), and Yale Genome Center (New Haven, USA).

The proposed PhD project will involve a combination of both in-silico and wet-lab approaches in order to:

1) Develop data analysis pipelines to efficiently detect and prioritize candidate variants;2) Functionally characterize by in-vitro and in-vivo studies novel genes/mutations.The data analysis will include, among others, the implementation of the detection of splicing

mutations, indels, and structural variants. Putative pathogenic mutations identified by WES will be tested to evaluate their segregation with the disease within the probands’ families and their recurrence in sporadic and familial NSHL cases. In this frame, the availability of a large non-syndromic deafness series (about 1300 individuals) of patients/families will be a key resource in the validation step, to screen for the identified mutations and to search for additional genetic defects in the candidate genes pointed out by WES. A in-house database of all variants identified by WES in a wide (>3000) cohort of Italian subjects is also available to the study, to identify population-specific polymorphisms.

We are currently analyzing the putative pathogenic role of a novel variation in a candidate NSHL-causing gene both at the mRNA and at the protein level, by expression experiments in eukaryotic cell lines. In addition, the function of this newly identified gene in the auditory system is being tested in zebrafish, by adopting the CRISPR-Cas system cutting-edge technology to selectively and stably inactivate the gene of interest [4], thus allowing a life-long analysis of its roles in ear development and homeostasis. Similar approaches will be adopted for the characterization of additional genes/mutations derived by WES data analysis.

References1. Walsh T et al. 2010. Whole exome sequencing and homozygosity mapping identify mutation

in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87:90-4.

2. Schraders M, et al. 2011. Next-generation sequencing identifies mutations of SMPX, which encodes the small muscle protein, X-linked, as a cause of progressive hearing impairment. Am J Hum Genet 88:628-34.

3. Huebner AK, et al. 2011. Nonsense mutations in SMPX, encoding a protein responsive to physical force, result in X-chromosomal hearing loss. Am J Hum Genet 88:621-7.

4. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227-9.

Project leader: CHIARA TONELLI ([email protected]); ([email protected])

Tutor: LUCIO CONTI



The role of ABA in the floral transition: site and mechanism of action

Drought stress triggers an increase in abscisic acid (ABA) levels, leading to an acceleration of the floral transition, a phenomenon known as drought escape response (DE). Our current understanding of the DE response posits that ABA promotes the upregulation of the florigen genes FT and TSF under long day phototoperiodic conditions. Conversely, under a short day photoperiod ABA negatively regulates flowering, independently of the florigen genes. The overarching questions of the proposed project are: how does ABA participate in the transcriptional upregulation of florigen genes? which is the site of ABA action in flowering? Two main avenues of research will be pursued. First, we will study the distribution of plant ABA by analysing available transgenic plants harbouring an ABA–responsive promoter fused to a reporter gene. We will next examine the spatial regulation of ABA signalling in flowering by expressing dominant ABA signalling components under the control of tissue–specific promoters. Alterations in flowering time of these transgenic plants will inform about where ABA signalling is required to stimulate or inhibit flowering. We will then address the physiological significance of the reported interaction between the ABA –downstream transcription factor ABI3 and master regulator of the photoperiodic response CONSTANS. Knowledge obtained through these experiments will provide concepts that help to understand how enormously variable water–dependent signals are translated into developmental information in plants. The project will also elaborate novel mechanisms underlying gene expression regulation by addressing the mechanistic basis of the interaction between ABA and photoperiod.


Project leader: CHIARA ZUCCATO ([email protected])



An in vivo study of the impact of ADAM10 dysfunction in Huntington’s Disease

Huntington's disease (HD) is a genetically dominant, neurodegenerative disorder caused by anelongated polyglutamine (polyQ) segment in the huntingtin (Htt) protein. Mounting evidenceindicates that mutant Htt disrupts normal synaptic function (contributing to HD behavioral,cognitive, and motor symptoms), and that alteration of the cortico-striatal excitatory circuit occursearly in HD progression (Zuccato and Cattaneo, Progress in Neurobiology 2007; Milnerwood andRaymond, TINS 2010; Zuccato et al., Physiological Review 2010). We believe that the search foreffective strategies to help restore the activity of the cortico-striatal synapse (regarding structure,function, and plasticity) is an important area in HD research that requires further study.This project focuses on the role of metalloprotease disintegrin ADAM10 in HD. ADAM10 iscritical both for the developing and adult brain. ADAM10 has only recently emerged in the HDfield on the basis of our demonstration that normal Htt inhibits its activity during brain development(Lo Sardo and Zuccato et al., Nature Neuroscience 2012). Recent works show that ADAM10 exertsa critical role in the adult brain by controlling the structure and function of excitatory synapticcircuitries (Malinverno et al., The Journal of Neuroscience 2010; Marcello et al., The Journal ofClinical Investigation 2013; Saftig and Reiss, Eur J Cell Biol. 2011). Therefore, ADAM10represents a good and novel target of investigation in HD due to its inherent structural andfunctional role in excitatory synaptic circuitries.Our preliminary data show that ADAM10 activity is significantly increased in the brain of HD miceand in post-mortem HD caudate. We also show increased proteolytic processing of ADAM10synaptic substrates in the brain of HD mice. Notably, administration of GI254023X, a compoundthat specifically targets the catalytic domain of ADAM10 with no effects on other ADAMs, rescuesthe severe brain defects observed in zebrafish embryos expressing human mutant Htt. Finally, wefound that treatment of organotypic slices from symptomatic HD mice with ADAM10 blockersrestores pro-survival pathways in the HD brain.The goal of this project is to evaluate whether the increased activity of ADAM10 is a relevantcomponent in the dysfunction of the cortico-striatal glutamatergic circuitry in HD. We willnormalize ADAM10 activity in the brain of HD mice by crossing HD mice with the new line ofCAMKIIalpha-cre Adam10Fl/+ heterozygous knock-out mice produced by P. Saftig at the Universityof Kiel (Prox et al., The Journal of Neuroscience 2013). By combining biochemical and behaviouralstudies we will test whether the inhibition of ADAM10 normalizes synaptic defects and is beneficial to HD mice.

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