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TEMATICHE DI RICERCA DEL CORSO DI DOTTORATO IN BIOLOGIA MOLECOLARE E CELLULARE

(XXXII CICLO)

1. Aliverti Alessandro Targeting mitochondrial proteins involved in apoptosis and cellular metabolism regulation to counteract cancer progression/Proteine mitocondriali coinvolte nell’apoptosi come bersaglio di farmaci attivi contro la progressione tumorale

2. Bolognesi Martino sclerosis/Anti-viral drug discovery strategies: structure-based development of inhibitors of proteins involved in flaviviral replication/Strategie per l'identificazione di anti-virali: sviluppo di inibitori basato sulla struttura tridimensionale delle proteine coinvolte nella replicazione dei flavivirus

3. Cappelletti Graziella Protein-protein interaction in regulating microtubule dynamics: the role in neurogeneration/Interazione proteina-proteina nella regolazione della dinamica microtubulare: ruolo nella neurodegenerazione

4. Cattaneo Elena Stem cells for Huntington’s Disease/ Cellule Staminali per la Malattia di Huntington

5. Colombo Lucia Environmental and genetic control of seed number/Controllo ambientale e genetico del numero di semi

6. Fornara Fabio Rice adaptation to higher latitudes by modification of photoperiod sensitivity/ Adattamento del riso alle latitudini settentrionali attraverso la modifica della sensibilità fotoperiodica

7. Gnesutta Nerina NF-Y partners in the regulation of CCAAT promoters/I partner di NF-Y nella regolazione dei promotori CCAAT

8. Gregis Veronica Study of transcription factors involved in reproductive development in the model species Arabidospis thaliana/Studio di fattori di trascrizione coinvolti nello sviluppo riproduttivo nella specie modello Arabidopsis thaliana

9. Guerrini Luisa Analysis of the molecular mechanisms at the basis of thalidomide induced fin defects in zebrafish embryos/Analisi dei meccanismi molecolari alla base dei difetti indotti dalla talidomide in embrioni di Zebra fsish

10. Kater Martin Understanding the molecular control of inflorescence development for crop improvement/Controllo molecolare dello sviluppo dell’infiorescenza per il miglioramento delle specie coltivate

11. Landini Paolo The bacterial signal molecule c-di-GMP as main trigger for chronic inflammatory bowel disease: a biological study leading to a therapeutic approach/La molecola segnale batterica c-di-GMP come attivatore delle malattie infiammatorie intestinali corniche: uno studio biologico con sviluppi terapeutici

12. Lazzaro Federico Role of Translesion Synthesis (TLS) polymerases in rNMPs incorporation and bypass during DNA replication/Ruolo delle polimerasi translesione (TLS) nell'incorporazione e il bypass di rNMPs durante la replicazione del DNA

13. Mantovani Roberto Role of NF-YA in mouse Embryonic Stem cells/Ruolo di NF-YA nelle cellule embrionali Staminali di topo

14. Marini Federica Role of the SLX4 network in genome integrity maintenance and cancer prevention/ Ruolo di SLX4 nel mantenimento dell’integrità del genoma e nel prevenire l’insorgenza di tumori

15. Messina Graziella Development of genetic and cellular approaches to cure Muscular Distrophies and Cystic Fibrosis/Sviluppo di strategie genetiche e cellular nelal cura delal Distrofia Muscolare e della Fibrosi Cistica

16. Moroni Anna Chimeric ion channel as a tool to study conformational changes/ Produzione di canali chimerici per lo studio dei cambiamenti conformazionali

17. Moroni Anna Engineering of synthetic ion channels activated by remote stimuli/ Crezione di canali ionici sintetici attivati a distanza da stimoli fisici

18. Muzi Falconi Marco From molecular mechanisms to human pathologies linked to genome instability/ Dai meccanismi molecolari alle patologie legate ad instabilità del genoma

19. Muzi Falconi Marco Development of novel technologies for studying the interactions between physiological/pathological nucleic acid structures and proteins/ Sviluppo di nuove tecnologie per lo studio di interazioni tra acidi nucleici con strutture fisiologiche/patologiche e proteine

20.Nardini Marco Structural analysis of transcription factor/DNA complexes /Analisi strutturale di complessi fra fattori di trascrizione e DNA

21. Pellicioli Achille Polo kinases and genome instability: implication for cancer development and treatment/Polo chinasi e instabilità genomica: implicazioni per lo studio e la cura del cancro

22. Pesaresi Paolo Analysis of the molecular mechanisms responsible of chloroplast biogenesis/Studio dei meccanismi molecolari alla base della biogenesi del cloroplasto

23. Petroni Katia Role of anthocyanin-enriched diet on cardioprotection/Ruolo di una dieta ricca di antocianine nella cardioprotezione

24. Polissi Alessandra Envelope biogenesis in Gram-negative bacteria as molecular target for the development of next generation antibacterial drugs/Studio della biogenesi dell’envelope nei batteri Gram-negativi come bersaglio per lo sviluppo di antibatterici di prossima generazione

25. Ricagno Stefano Structural characterization of the receptor EP1 from Arabidopsis thaliana/Caratterizzazione strutturale del recettore EP1 di Arabidopsis thaliana

26. Tonelli Chiara Role of plant hormone signalling in the natural variation of flowering and adaptation in response to water scarcity through QTL and eQTL dissection/Ruolo dei segnali ormonali vegetali nella variabilità naturale della fioritura e delle risposte adattative a stress idrico tramite un approccio di analisi QTLs e eQTLs

27. Zuccato Chiara Huntington’s Disease: biological aspects and molecular pathogenesis/ Aspetti biologici e patogenesi molecolare della Malattia di Huntington

Project leader: Alessandro ALIVERTI ([email protected])

Location: Department of Biosciences, Dipartimento di Bioscienze

RESEARCH PROJECT SUMMARY

Targeting mitochondrial proteins involved in apoptosis and cellular metabolism regulation to counteract cancer progression.

The apoptosis inducing factor (AIF) is a mitochondrial flavoprotein endowed with NADH-dependent oxidoreductase activity that plays a dual role within eukaryotic cells (1). While its release into the cytosol and translocation to the nucleus are central steps in caspase-independent apoptosis, its redox activity is required to maintain mitochondrial structure and function. Several point mutations resulting in amino acid changes are known to cause oxidative phosphorylation defect, resulting in a variety of neurological diseases (2-4). In addition to its established role in the etiology of neurodegeneration, a prominent role for AIF in development, progression and invasiveness of various forms of tumors is emerging from recent studies (5-7). AIF is overexpressed in cancer cells and its downregulation or disruption of catalytic activity result in cell proliferation inhibition. It has been proposed that AIF acts as a redox sensor and metabolic regulator and that such function is hijacked by cancer cells to favor their proliferation and survival, particularly in advanced stages of the disease. Very recently, the putative molecular mechanism of the mitochondrial AIF action has been identified in its redox-dependent interaction with CHCHD4/MIA40, a protein involved in the import and folding of nuclear-encoded respiratory complex subunits (8).

The general aim of this research project is to provide further insight into the molecular mechanism of AIF function within mitochondria, with a particular emphasis on the mode of interaction with its protein partner CHCHD4/MIA40, and to exploit the resulting knowledge to design and characterize small-molecule ligands for their possible inhibitory activity towards this new target of anti-cancer therapies.

The workplan will build on previous work carried out in the laboratory (9), which resulted in a significant growth of knowledge of mammalian AIF structural and functional molecular properties, as well as in the development of a set of novel experimental tools for its in vitro study. A system for the overexpression and purification of recombinant mammalian CHCHD4/MIA40 will be developed. The interaction between this protein and AIF will be studied by a variety of in vitro techniques, including protein engineering and X-ray crystallography, in collaboration with Dr. Eloise Mastrangelo (CNR-IBF). The definition of the interacting regions of the two protein molecules and the definition of role of the AIF redox state in protein-protein interaction will be the main specific aims of this part of the program. Furthermore, already available and newly acquired structural information on the above proteins and their complex will be used to design by in silico docking low-molecular-weight interactors of AIF, aimed at disrupting its redox activity and complex-formation capability.

1. Sevriukova IF (2011) Antioxid Redox Signal 14, 2545-79.2. Zong L et al (2015) J Med Genet 52, 523-31.3. Diodato D et al (2015) Eur J Hum Genet 24, 463-6.4. Sevriukova IF (2016) J Mol Biol [Epub ahead of print] doi: 10.1016/j.jmb.2016.05.004.5. Lewis EM et al (2012) J Biol Chem 287, 43862-75.6. Fan T et al (2014) Tumor Biol 35, 519-27.7. Scott AJ et al (2016) BMC Cancer 16:286.8. Hangen E et al (2015) Mol Cell 58, 1001-14.9. Sorrentino et al (2015) Biochemistry 54, 6996-7009.

mailto:[email protected]

Project leader: MARTINO BOLOGNESI ([email protected])

Location: Department of Biosciences, Via Celoria 26, 20133 Milano


Anti-viral drug discovery strategies: structure-based development of inhibitors of enzymes involved in flaviviral replication

The total number of fatalities that occur worldwide as the result of infection by RNA viruses can be estimated in many millions every year. Newly emerging RNA viruses present an additional threat to mankind through their increasing impact on morbidity and mortality. Thus, there is an urgent need for advanced methods of medical intervention to support and improve the health perspectives for the world population.

In the context of new emerging viral threat, a primary role is played by flaviviruses. Flaviviruses have been recently “rediscovered” because of the increasing number of cases in a growing number of countries: e.g. dengue fever (300 million cases annually), encephalitis (several million cases annually), and Zika (recent outbreak in Brasil). Scientists need to look for new therapeutic and prophylactic treatments active against Flaviviruses, since those currently available are scarce and of poor potency. The common strategies used for the development of antiviral drugs are mainly based on knowledge accumulated through studies of virus genetics and structure. However, it is a paradox that genomic and structural characterization of RNA viruses have not been accepted as priorities until very recently. The lack of structural data on the virus replication machinery components, such as the RNA/NS3-NS5 complexes, is hampering not only structure-based drug design but also mechanism-of-action and drug-resistance studies.

The structural and functional study of flaviviral replication complex, proposed here as a PhD research project, will address many open issues on overall protein and protein-RNA interactions during RNA replication. The flavivirus replication complex carries out RNA synthesis, RNA capping and RNA

methylation steps, to produce the genome with a type 1 cap structure (m7GpppNm-RNA) at its 5’ end. Among the non-structural (NS) proteins, the functions of NS3 and NS5 in viral replication are well established. NS3 displays a serine protease domain (Pro) at its N-terminal end (NS3-N; 180 amino acids), which is functional only if associated to the central hydrophobic region of the NS2B protein, an ER resident integral membrane protein. The NS3 C-terminal domain (NS3-C, residue 180-620) is a multifunctional protein endowed with helicase, RNA triphosphatase and ATPase functions. NS5 contains at its N-terminal end a domain endowed with RNA guanylyltransferase and methyltransferase activities necessary for 5’-RNA capping and cap methylations (MTase; NS5-N, residue 1-270), and at its C-terminal end a RNA dependent RNA polymerase domain (RdRp; NS5-C, residue 280-900), involved in viral genome replication and carries out both (-) and (+) strand RNA synthesis.

The main aim of the PhD project is a comprehensive structural and functional characterization of the individual members of the replicative machinery (in particular of NS3 and NS5 proteins), of their mutual interactions within the core complex, and with RNA, during viral replication, through 3D structural analysis (X-ray crystallography), enzyme-activity assays, protein-mutants production, small angle X-ray scattering and cryo-electron microscopy analyses. This approach is part of a Europe-wide coordinated effort that can potentially disclose key features of the flaviviral RNA replication process, opening the way to new advances in virology and in drug design applications.


Project leader: Graziella Cappelletti ([email protected])

Department of Biosciences, University of Milan


Protein-protein interaction in regulating microtubule dynamics: the role in neurodegeneration

Neurons are a striking example of cells in which microtubules are essential to achieve a high degree of morphological and functional complexity. Neuronal microtubules display different orientation and dynamics in axons and dendrites, and interact with a plethora of specific associated proteins. In addition, the incorporation of tubulin isotypes and post-translational modifications of tubulin are selectively combined and distributed among different subcellular compartments, thus generating a tubulin code, that might regulate basic as well as higher-order neuronal functions.

The goal of the present project is to investigate if and how parkin, a well-known E3 ubiquitin ligase, interacts with tubulin and contributes to regulate the molecular architecture of neuronal microtubules in physiological and pathological context. Notably, parkin mutations are associated to autosomal recessive Parkinson’s disease. Although previous evidence links parkin deficiency to microtubule destabilization in neurons (Cappelletti G. et al., Biochem Soc Trans, 2015), the interaction of parkin with tubulin/microtubules remains a poorly investigated aspect of parkin’s biology. This project aims to fill this gap using a set of complementary in vitro and in cell strategies.

In the first part of the work, the PhD fellow will evaluate the impact of wild type and mutated parkin on microtubule assembly, structure, stability and dynamics in vitro. The second aim is to analyse the interplay between wt/mutated parkin and microtubule system in primary neuronal cultures from embryo midbrain of Parkin KO mice. Wt, truncated and point mutated parkin will be overexpressed in primary neurons and their impact on microtubule dynamics and subsets of post-translationally modified tubulin will be investigated as previously reported (Cartelli et al., J. Neurochem., 2010). Finally, advanced analyses of parkin/tubulin interaction in fixed and live cells will be performed by two single-molecule imaging techniques (FRET or dSTORM).If successful, the outcome of this research will lead to a mechanistic insight into the interaction of parkin with microtubules and to a better understanding of its physiological and pathogenic functions.

Project leader: Elena Cattaneo ([email protected])

Location: Department of Biosciences and INGM, Via Sforza 35 Milano


Stem cells for studies of Huntington’s Disease

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. Current treatments fail to produce significant relief to the patients therefore HD results as 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. MSN are inhibitory projection neurons and are the primary source of striatal projections.The laboratory is actively involved in international research programs aiming at deriving specific and robust differentiation protocols for the generation of MSNs. 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 et al., Development 2013). Moreover, we are using stem cells to study the normal fuction of huntingtin along evolution ( Lo Sardo et al., Nat Neurosci 2012).In this project, by the combination of multiple approaches we aim to develop strategies to further improve the recovery and quality of fully functional human MSNs from hES/hiPS cells with the goal of using them to study huntingtin function in human cells and for future transplantation studies in HD. We will further explore the use of additional morphogenes/active molecules to increase frequency of authentic MSNs during differentiation and apply these to doxycycline-inducible hES lines that over-express critical combinations of transcription factors (TFs) known to be important for striatal specification and differentiation. As the optimization of MSN differentiation protocols depends critically on gene expression profiles during the development of these cells in vivo, we have developed a project to quantifying RNA expression in the regions of the embryonic brain that give rise to these cells. We plan to perform bulk and single cell analysis at different developmental time points of the developing human brain in order to reconstruct the differentiation trajectory that goes from neural progenitor cells to MSNs.In order to better recapitulate in vitro the events that are occurring in vivo, we generated a protocol to differentiate hES and iPS cells in a 3D fashion (“organoids”): with this procedure we are able to mimic the first 12weeks of human fetal development. This protocol will be used to model HD in vitro.Moreover, we plan to use recently developed genome editing tools to modify ad hoc endogenous gene expression by either insertion of fluorescent reporters to use as hallmarks for differentiation or to modulate and/or knock out gene of interest for gain/loss-of-function studies. 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. We will also use Cas9-mediated knock-out strategies to inactivate the HD gene and produce new lines expressing huntingtin protein with mutation in critical aminoacidic sites.In conclusion this project aims at (i) developing new hES cell lines over-expressing critical striatal TFs; (ii) modelling HD in vitro by using a 3D protocol; (iii) performing single cell analysis of the human fetal striatal development; (iv) using the cells to study huntingtin biology and for transplantation studies.


Project leader: Lucia Colombo ([email protected])

Location: Dipartimento di Bioscienze


Environmental and genetic control of seed number

Morphogenesis is the remarkable process by which a developing plant acquires its shape and size. Underlying the architectural complexity of plants are diverse cell types that easily reveal relationships between cell/tissue structures and specialized functions. This project focuses on fruit development. Upon fertilization, the pistil develops into a fruit and the ovules are transformed into seeds. Pistil size is often associated to ovule number and therefore to the seed yield.

We propose an innovative investigation project using complementary approaches such as molecular, genetic and biochemistry to identified key regulators of pistil/fruit size in Arabidopsis.

Furthermore, we will investigated the impact that environmental changes have on the determination of fruit size and consequently on seed number in Arabidopsis.


Project leader: Fabio Fornara ([email protected])



Rice adaptation to higher latitudes by modification of photoperiod sensitivity

Rice is a tropical plant that has been artificially adapted to grow in several temperate areas of the world, including Mediterranean Europe. Flowering (also called “heading” in cereals) starts when specific proteins, encoded by Heading Date 3a (Hd3a) and Rice Flowering Locus T1 (RFT1) are produced in the leaves and then move to the shoot apical meristem. Transcription of Hd3a and RFT1 is regulated by several genes, most of them acting as transcriptional repressors. Varieties adapted to higher latitudes frequently harbour mutations in floral repressor genes, releasing expression of Hd3a and RFT1 and allowing flowering to occur early also under unfavourable conditions. Among the floral repressors that have been identified to date, Heading date 1 (Hd1), Ghd8 (OsNF-YB11) and PRR37 encode the strongest repressors. Unpublished data from our laboratory indicate that heterodimers formed by Ghd8 and OsNF-YC proteins can recruit Hd1 or PRR37 in a trimeric NF-Y complex in yeast. These data suggest the existence of a NF-Y complex in rice where interchangeable subunits act to repress flowering under LD.The PhD student will join this project and address the genetics of the complex in rice plants by assaying the interactions between putative components of the complex and generating mutants in specific sub domains of each protein using genome-editing technologies.We will address the following questions:- Could more CCT domain proteins interact with the Ghd8 - OsNF-YC dimer? Besides PRR37 and Hd1, other CCT domain proteins, including PRR73, PRR59 and PRR95 represent suitable candidates to test this hypothesis. - Is the effect of mutations in PRR genes additive on flowering? Higher order mutant plants will be generated, in which Hd1 and PRR proteins will be mutated simultaneously in different combinations.This work will greatly improve our understanding of the regulatory mechanisms of flowering in rice and possibly demonstrate that adaptation of rice to European environments has been enabled by artificial selection acting on NF-Y complex components.


Project leader: Dr. Nerina Gnesutta – ([email protected])

Location: Dipartimento di Bioscienze, Università degli Studi di Milano


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 altered gene expression that results in uncontrolled cell growth and cancer. The CCAAT box is a common DNA element found enriched in promoters of growth controlling genes, and is specifically bound by the trimeric transcription factor NF-Y. Recently, the genomic locations of several TF binding sites have been mapped in vivo by the ENCODE project. Bioinformatic analyses have shown that NF-Y sites significantly overlap, within short distances, with a specific set of other TFs, among which the protooncogenes Myc and Fos. These data suggest that specific binding sites configurations 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, solved by our group in collaboration with proff Nardini and Bolognesi, is the foundation of the proposed project. More recently, we have obtained higher order structural informations on NF-Y/DNA complexes also containing bHLH TFs bound to their Ebox target site at the preferred 10/12bp distance from CCAAT. These data highlighted that among Ebox TFs of the bHLH/cMyc family, USF1 directly contacts NF-Y when bound to DNA, providing clues on the cooperative nature of their interactions. This (DNA-dependent) interaction is highly relevant, also considering that USF1 was reported to act in concert with NF-Y to control the regenerative capacity of the hematopoietic progenitor cells population.

The research project aims at understanding, at the biochemical, structural and functional levels, the observed interactions of NF-Y with bHLH TF USF1 and to extend our approach to other TF families, in particular oncogenic b-ZIP (Fos/Jun, MAFs), based on “prototypical” promoters bound by NF-Y and its genomic partners. 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 structural analyses, to visualise surfaces involved in protein interactions; in vivo studies of promoter occupancy following TFs inactivation, by ChIP-qPCR, and at genomic level by ChIP-Seq, to understand possible hierarchy in DNA binding; in vivo studies by transient expression of wt and mutant proteins with gene promoter-reporter assays to validate and define the functional interactions mechanisms involved in gene expression regulation.

-Dolfini D, et al. (2012). NF-Y and the transcriptional activation of CCAAT promoters. Crit. Rev. Biochem. Mol. Biol. 47: 29-49.

-Fleming JD, et al. (2013). 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. 23:1195-209

-Nardini M, et al. (2013). Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination. Cell 152: 132-143.

-Zhu J, et al. (2003). NF-Y cooperates with USF1/2 to induce the hematopoietic expression of HOXB4. Blood.; 102: 2420-7.

- Luo X, Sawadogo M. (1996). Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains. Mol Cell Biol. 16:1367-75.


Project leader: GREGIS VERONICA ([email protected])

Location: Department of Bioscience


Study of transcription factors involved in reproductive development in the model species Arabidospis thaliana

This research project will focus on the development of flower meristems and fruits, two extremely important agronomical traits. MADS-domain transcription factors (TFs) are key regulators of these processes and the project will especially focus on how these TFs and their interacting partners control the regulatory network leading to reproductive meristem formation and fruit development in the model plant Arabidopsis thaliana.An excellent starting point for this research project is the large amount of information and tools that we and other groups have already generated for one of the key players in both vegetative and reproductive meristem development: the MADS-domain TF SHORT VEGETATIVE PHASE (SVP). SVP acts as a repressor of the floral transition in the vegetative meristem and later as a key factor floral meristem identity factor (Gregis et al., 2013). Moreover, we have recently characterized the function of the complex composed of SVP and BASIC PENTACYSTEINE factors (BPCs) (Simonini et al., 2012). BPCs are transcription factors with affinity to GA-repeated sequences, involved in transcription regulation through their interaction with other TFs and chromatin modifiers. We showed that BPC binding sites are important for BPC recruitment to the DNA but also for binding of SVP to nearby MADS-domain binding sites (CArG boxes).This project will study two important interconnected research lines that are related to the regulatory mechanisms that underlay reproductive development: (i) the mechanistic molecular aspects of target gene regulation by SVP and its interacting partners BPCs and (ii) the analysis of bpc multiple mutants combinations phenotype and the characterization of their role in fruit development.The project will focus on the molecular mechanisms by which SVP and its interaction partners control their targets. MADS-domain factors have been studied deeply from a genetic point of view and are recognised as key controllers of development in plants. However, little is known about the molecular aspects (DNA binding mechanisms, recruitment of complex members etc.). Within the frame of this project we will analyse the importance of the interaction between these transcription factors in more detail and provide deep mechanistic insights about complex formation and recruitment of the complex to the DNA in a context of a changing histone-landscape.

Gregis et al., Genome Biol. 2013 Jun 11;14(6):R56. [Epub ahead of print]Simonini et al., The Plant Cell 2012 (10):4163-72.


Project leader: Luisa Guerrini ([email protected])



Analysis of the molecular mechanisms at the basis of thalidomide-induced limb defects in zebra fish embryos

Severe developmental malformations were detected in human feti in the 1950s, when women used the anti nausea and sedative drug thalidomide in the first trimester of pregnancy. The striking similarities between the phenotypic abnormalities of babies born from mothers exposed to thalidomide during pregnancy and patients affected by syndromes associated to mutations in the p63 gene, prompted us to verify whether p63 could be a molecular target for the thalidomide drug. Our results indicate that the Np63 and Np63 proteins, but not Np63 and p53, are degraded through the proteasome upon thalidomide exposure in several human cell lines expressing either the endogenous or the transfected p63 proteins. By serial deletions analysis, we identified serine 383 in p63 as necessary for thalidomide mediated degradation of p63, since mutation of S383 to alanine abolished thalidomide action on p63. GSK3 kinase is responsible for S383 phosphorylation since the use of a specific GSK3 inhibitor also abolished thalidomide action on p63. Thalidomide has been recently been shown to have teratogenic effects also in the zebra fish; we have evidences that thalidomide modulated p63 protein levels also in vivo in developing Zebra fish embryos, with concomitant teratogenic fin defects. The aim of the project will be to dissect the molecular pathways altered by thalidomide treatment at the basis of the observed limb defects, using zebrafish as animal model.


Project leader: Prof. Martin Kater ([email protected])

Location: Department of Pharmacological and Biomolecular Sciences and the Department of Biosciences


Rice Yield Increase by Altering Inflorescence Architecture

Rice is one of the most important crops for feeding the world. Rice yield increase over the next 25 years is of enormous importance to meet the demand of a rapidly growing world population. This rise in yield will have to be sustainable and without increase of cultivated land. This project focuses on improving rice yield through altering the inflorescence structure. The inflorescence or panicle has a main axis on which primary branches develop. From the primary branches the secondary branches arise from which the spikelets are born. The number of branches varies between rice varieties and by that the number of seeds that develop on a panicle. Panicle branching is therefore an important character for rice crop improvement.

Very little is known about the genetic control of panicle branching. Recently, Yoshida et al. (2013) identified a regulator of rice panicle branching called TAWAWA1, however the molecular mechanism is still far from understood. We have in the frame of the French-Italian EVOREPRICE project isolated by laser micro-dissection microscopy isolated the apical, primary and secondary branch meristems of rice inflorescences. This material has been used for RNA extraction and next generation sequencing analysis. The transcriptomes have been analysed and key genes putatively involved in regulating branching have been identified (Harrop et al., 2016). These will be subjected to functional analysis using molecular and genetic tools. For making mutants we will use the latest genome editing approaches.

Literature:

- Yoshida et al. (2013). TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition. Proc Natl Acad Sci U S A 110, 767-772.

- Harrop, T.W.R. , Ud Din, I., Gregis, V., Osnato, M., Jouannic, S., Adam, H. and Kater, M.M. (2016). Gene expression profiling of reproductive meristem types in early rice inflorescences by laser microdissection. Plant J. 86, 75-88.


Project leader: Paolo Landini ([email protected])

Location: Department of Biosciences


Inhibition of bacterial antigen production as a strategy to counteract chronic inflammation in Crohn’s disease.

In Crohn’s disease (CD) overly stimulation of gut-associated lymphoid tissue by components of the enteric flora leads to the chronic intestinal inflammation typical of the disease. In many bacteria, immunogenic structures are controlled by the bacterial signal molecule c-di-GMP, itself a powerful inducer of the human innate immune system. We hypothesize that inhibition of c-di-GMP biosynthesis could help prevent chronic inflammation in CD. Recently, we found that azathioprine, widely used as an anti-inflammatory drug in CD patients, is an inhibitor of c-di-GMP production, thus suggesting that c-di-GMP inhibition might contribute to azathioprine modulation of the immune response. To verify this hypothesis, we will test the effects of azathioprine on c-di-GMP biosynthesis, and on adhesion factor production, in the adherent-invasive E. coli (AIEC) strain LF82, a bacterium associated with CD. We will create mutant strains of LF82 impaired in c-di-GMP production by overexpression of c-di-GMP phosphodiesterases. We will test cytokine production and proliferation of dendritic cells, from either healthy subjects or individuals affected by Crohn’s disease, upon exposure either to LF82 or to its mutant derivatives impaired in c-di-GMP production. Likewise, we will pre-treat LF82 bacterial cells with azathioprine and test their ability to induce cytokine production in human T cells. The work proposed may lead to the identification of new bacterial targets to counteract inflammation and thus define a novel therapeutic approach for CD.


Project leader: Federico Lazzaro ([email protected])



Role of Translesion Synthesis (TLS) polymerases in rNMPs incorporation during DNA replication

Ribonuclease H (RNase H) are evolutionary conserved enzymes capable of removing the RNA moiety in RNA:DNA hybrid molecules. Mutations in RNase H2 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 can 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). Moreover our preliminary results show that TLS polymerases have an important role not only in bypassing rNMPs incorporated but can also incorporate rNMPs during DNA replication. In particular the evolutionary conserved TLS polymerase Pol-eta shows elevated predisposition to misincorporate rNMPs when the dNTPs pools became extremely limited. Here we will investigate in yeast and human cells how these particular events are regulated, extending the results to any processes where the DNA synthesis is essential even though dNTPs are deficient.Evidence in yeast indicates that the simultaneous deletion of all genes coding RNase H enzymes is tolerated by the cell, suggesting that rNMPs normally incorporated by DNA polymerases are maintained at sub-lethal levels by other repair pathways and these pathways may be new candidates involved in AGS. In order to identify these possible new pathways we applied the “synthetic genetic array” SGA approach to search for new genes functionally interacting with RNases H (Costanzo et al., 2010).An initial analysis identified promising candidate genes involved in recombination/repair 38%; replication/transcription 24%; chromatin 7%; meiosis/mitosis 7%; mitochondria 10%; RNA metabolism 3%; uncharacterized 10%. In this project we will validate and characterize these positives with particular attention to candidates conserved in human cells.

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

Costanzo M, Baryshnikova A, Bellay J, Kim Y, Spear ED, Sevier CS, et al. The Genetic Landscapeof a Cell. Science. 2010;327:425–31

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])



Role of NF-YA isoforms in Embryonic Stem cells

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). The CCAAT box is often present in promoters of genes overexpressed in different types of cancer, and NF-Y plays an important role in mediating high levels of expression (2). We showed that one of the splicing isoform of NF-YA plays a crucial role in maintaining the mouse ES stemness potential (2, 4). 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 precisely regulated. Surprisingly, it has recently emerged that the two isoforms have different, often opposing roles in important cellular processes.

The aim of the project is to investigate the mechanistic role of NF-YA isoforms in mouse ES cells. The expansion of the stem cells compartment(s) has been associated to NF-YAs (NF-YA-short), and we have preliminary data indicating that NF-YAl (NF-YA-long) is involved in early differentiation. The exact role of NF-Ys in stemness is likely related to synergy with important ES regulators (Sox2, Nanog, KLF4) to their genomic sites. In general, we have a wealth of data on the major NF-Y transcription factor partners, derived from ENCODE (5, 6). We will analyze profilings of mES cells in which the isoforms are overexpressed, or inactivated in mES and human iPS cells. In addition, we will specifically delete with CRISPR/Cas9 technology the extra 28 aminoacids of NF-YAl coded by Exon 3 in mES cells, and examine the biological behavior of cells only expressing NF-YAs. We expect to gain a full understanding of the NF-Y-driven molecular mechanisms that lead to differentiation or to the expansion of the stem cells pools.

1) Nardini M., Gnesutta N., Donati G., Gatta R, Forni C., Fossati A., Vonrhein C., Moras D., Romier C., Bolognesi M., Mantovani R. 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).

3) Dolfini D, Minuzzo M, Pavesi G, Mantovani R. Stem Cells. 30, 2450-9 (2012). 4) Oldfield AJ, Yang P, Conway AE, Cinghu S, Freudenberg JM, Yellaboina S, Jothi R. Mol Cell. 55:708-22

(2014).5) Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R, Struhl K. Genome Res. 23:1195-209.

(2013).6) Dolfini D., Zambelli F., Pedrazzoli M., Mantovani R., Pavesi G. Nucleic Acids Res., in press (2016).


Project leader: Federica Marini ([email protected])

Location: Dipartimento di Bioscienze, Via Celoria 26 20133 Milano

RESEARCH PROJECT ABSTRACT

Role of the SLX4 network in genome integrity maintenance and cancer prevention

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 (ssDNA) 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 MRE11, CtIP/Sae2 and BLM/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 (2,3). 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 (4). Therefore, the studying of the regulation of the DSB resection is fundamental to understand why defects in this process lead to chromosome rearrangements and cancer. Importantly, we recently found that the Slx4 protein counteracts Rad9 binding near a DSB in yeast, promoting both the inactivation of the DNA damage checkpoint and the DSB resection and repair (5).

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 is 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 (6).

The PhD student will investigate the role of the Slx4 pathway in DBS repair and in the maintenance of genomic stability in different human cell lines, already available in our laboratory. Particularly useful to characterize SLX4 role in checkpoint activation and inactivation and DSB repair will be cell lines derived from FANCP patients. He/She will also study the involvement of SLX4 and 53BP1 in the repair of DNA damages caused by camptothecin, a topoisomerase-aborting agent, which is highly used in chemotherapy. Furthermore, He/She will set up specific screening to identify novel genes involved in DSB repair.

REFERENCES1. Jackson, S.P. and Bartek, J. (2009) The DNA-damage response in human biology and disease.

Nature, 461, 1071-1078.2. Ferrari, M., Dibitetto, D., De Gregorio, G., Eapen, V.V., Rawal, C.C., Lazzaro, F., Tsabar, M., Marini,

F., Haber, J.E. and Pellicioli, A. (2015) Functional interplay between the 53BP1-ortholog Rad9 and


the Mre11 complex regulates resection, end-tethering and repair of a double-strand break. PLoS Genet, 11, e1004928.

3. Lazzaro, F., Sapountzi, V., Granata, M., Pellicioli, A., Vaze, M., Haber, J.E., Plevani, P., Lydall, D. and Muzi-Falconi, M. (2008) Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres. Embo J, 27, 1502-1512.

4. Zimmermann, M., Lottersberger, F., Buonomo, S.B., Sfeir, A. and de Lange, T. (2013) 53BP1 regulates DSB repair using Rif1 to control 5' end resection. Science, 339, 700-704.

5. Dibitetto D., Ferrari M., Rawal C. C., Balint A., Kim T., Zhang Z., Smolka M. B., Brown G. W., Marini F. and Pellicioli A. (2016) Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks. Nucleic Acids Research 44(2): 669-82.

6. Kim, Y. (2014) Nuclease delivery: versatile functions of SLX4/FANCP in genome maintenance. Molecules and cells, 37, 569-574.

Project leader: Prof. Graziella Messina ([email protected])



Development of genetic and cellular approaches to cure Muscular Dystrophies and Cystic Fibrosis

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. 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, through the expression of different genes2,3. A genome wide gene expression analysis carried on purified embryonic and fetal myoblasts 4 identified many differentially expressed genes, clearly revealing that embryonic and fetal myoblasts are intrinsically different populations of myoblasts with distinct genetic programs. In 2010, we demonstrated the pivotal role of the transcription factor Nuclear Factor IX, Nfix, in driving the transcriptional switch from embryonic to fetal myogenesis5. Interestingly, satellite cells express high levels of Nfix and we have recently observed that Nfix has also a crucial role in post-natal life during muscle regeneration upon injury6.

In light of this evidence, this project has as first aim the identification of all the targets of Nfix in skeletal muscle through a genome-wide ChiP-seq analysis to discover other and new pathways in which Nfix may play new functions during muscle development and regeneration.

Muscular dystrophies (MDs) are clinically and molecularly heterogeneous diseases, characterized by primary wasting of skeletal muscle that compromises patient mobility and, in the most severe cases, respiratory and cardiac functions, leading to wheelchair dependency, respiratory failure and premature death7. In many cases, the mutation affects proteins that form a link between the cytoskeleton and the basal lamina. Absence of one protein often causes the disassembly of the whole multiprotein complex associated with dystrophin, leading to increased fragility of the sarcolemma, especially during intense contractile activity. Muscular dystrophies are among the most difficult diseases to treat, although the underlying pathogenesis is well understood. Skeletal muscle is indeed the most abundant tissue of the body and is composed of large multinucleated fibers, whose nuclei cannot divide. Consequently, any cell or gene replacement strategy must restore proper gene expression in hundreds of millions of post-mitotic nuclei, which are embedded in a highly structured cytoplasm and surrounded by a thick basal lamina. Damaged or dead fibers can be repaired or replaced by allogenic cell therapy approach by using the satellite cells (SCs), the adult stem cells of skeletal muscle. Unfortunately, many evidence demonstrated the inability for SCs to cross the blood vessel wall upon systemic delivery, making these cells not eligible for a cell based therapy of MDs. In our lab we performed a comparative analysis of transcriptomes from SCs and a well-known population of adult progenitor cells (named Mesoangioblasts, MABs), that we demonstrated to be able to cross the blood vessel wall and differentiate in skeletal muscle8,9. This analysis revealed important differences in the expression of genes involved in cell migration.Therefore, the second aim of this project will be developed by the identification and the validation of possible molecular players that would activate and mobilize SCs for an efficient skeletal muscle tissue repair in MD mouse models.


References:

1. Davis, R.L., Weintraub, H. & Lassar, A.B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987-1000 (1987).

2. 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).

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. 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).

5. Messina, G., et al. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell 140, 554-566 (2010).

6. Rossi, G., et al. Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression. Cell reports (2016).

7. Emery, A.E. The muscular dystrophies. Lancet 359, 687-695 (2002).8. Bonfanti, C., et al. PW1/Peg3 expression regulates key properties that determine mesoangioblast

stem cell competence. Nature communications 6, 6364 (2015).9. Minasi, M.G., et al. The meso-angioblast: a multipotent, self-renewing cell that originates from the

dorsal aorta and differentiates into most mesodermal tissues. Development 129, 2773-2783 (2002).

Project leader: ANNA MORONI [email protected],

Location: Dept. of Biosciences


Engineering of synthetic ion channels activated by remote stimuli

Optogenetics uses visible light to regulate the activity of ion channels and pumps. Light-gated proteins can be expressed by standard genetic tools in a cell specific manner in any animal tissues, opening the possibility to control cellular functions by light with unprecedented high temporal and spatial precision. In the last few years the progress in this field went as far as controlling neuronal activity in vivo and even the behavior of animal models with light.

The current optogenetic toolbox has one serious limitation in that the available opsins are confined to action spectra that typically peak at 450-545 nm (blue-green). In mammalian systems, this light however does not penetrate deep into tissue since it is strongly absorbed by endogenous chromophores and scattered more strongly than yellow-red wavelengths. Moreover the energy rich light bears the hazard of photo oxidative damage. Because of the very low penetration of blue light it is necessary to introduce thin optical fibers into the target tissue e.g. the brain. However this invasive procedure requires precise positioning that may not always be possible. As a negative side effect the fibers may also damage tissue or cellular structures during insertion. Moreover, the implantation of fibers requires difficult surgery, which limits experiments with free moving animals.

Our goal is to engineer ion channel proteins for genuine remote manipulation of cells. These channels will respond to signals which are not harmful to human health but still penetrate deep into tissue and even through the skeleton. In this project we intend to exploit the possibility of engineering a channel that responds to near infrared radiation. The natural receptors of NIR in the spectral window of 680-880 nm (NIRW) are bacteriophytochromes. Their chromophore, biliverdin IXa, is naturally made in animal tissue as the first intermediate in the heme degradation pathway, which obviates the need to supply the chromophore for application in animals. Bacteriophytochromes possess a common photoreceptor module, PAS-GAF-PHY, usually linked to a histidine kinase output. This modality perfectly fits the architecture of the Kcv pore that assembles correctly as dimer of dimers. The NIR channel will be engineered by fusing a bacterial phytochrome moiety to the N terminus of the Kcv dimer. The mechanical movement induced in the bacteriophytochrome dimer in response to light absorption will be used to gate the pore.


Project leader: ANNA MORONI [email protected],

Location: Dept. of Biosciences


Chimeric ion channel as a tool to study conformational changes during pore gating

Hyperpolarization activated cyclic nucleotide gated (HCN) channels open in response to voltage and cAMP binding. Our goal is to reconstruct the allosteric pathway of the cAMP signal within the C-terminal portion of the channel that includes a cyclic nucleotide binding domain (CNBD) connected to the pore via the C-linker. By comparing the structures of the CNBD solved in the cAMP bound and unbound form, we have reconstructed the conformational changes leading to the upward displacement of the C-linker. In this project we will continue this investigation acquiring information on the C-linker movement initiated by the CNBD and terminating into pore opening. To this end we will engineer constructs in which the C-linker CNBD soluble domain will be anchored to a rather simple ion channel pore, derived from the bacterial channel KcsA. This pore offers the possibility to produce the chimeric construct in E.coli by means of an expression protocol previously optimized (ref). The protein, purified from the membranes in the presence of detergents, will be functionally characterized in planar lipid bilayer and further studied by NMR and x-ray crystallography.


Project leader: Marco Muzi Falconi ([email protected])

Location: Dipartiemnto di Bioscienze


From Molecular Mechanisms to Human Pathologies Linked to Genome Instability

Carcinogenesis is associated to alterations in the sequence and organization of the genome. Maintenance of genome integrity is essential for all living organisms and genomic instability is a hallmark of cancer cells. DNA metabolism is integrated with cell cycle progression through checkpoint mechanisms. Understanding the mechanistic details of the cellular response to DNA damage is thus critical for the comprehension of tumor development and for the design of better therapeutic approaches. In general, this proposal aims at understanding how cells maintain genome integrity and what happens when these controls are lost. The project is based on integrated approaches to identify genes and pathways, define the molecular mechanisms of these pathways, determine what happens when they are misfunctional and, in the long term, connect such defects to human pathologies. In particular we plan to study the regulation of Exo1 nuclease, which acts in several pathways (e.g. mismatch repair, DSB repair, telomere maintenance, replication, UV-induced checkpoint activation). Indeed, a tight control of nucleolytic activities is fundamental to prevent excessive processing of DNA intermediates and despite the importance of Exo1 in DNA metabolism, very little detail of its regulation is known. Through a genetic screening we identified mutants (called DSL) that are likely to increase the levels of endogenous DNA damage. We plan to characterize the DSL2 gene.

Preliminary data show that dsl2∆ cells are sensitive to DNA damaging agents, increase rDNA instability and exhibit chromosome alterations. More detailed investigations are required to define the mechanistic details and to determine other functions of DSL2.

Project leader: Marco Muzi Falconi ([email protected])

Location: Dipartimento di Bioscienze/ Dipartimento di Biotecnologie Mediche e Medicina Traslazionale


Development of novel technologies for studying the interactions between physiological/pathological nucleic acid structures and proteins

DNA binding proteins play a key role in the physiology and pathology of cell, and thus the measurement of the specificity, strength and kinetics of such interaction is a must of modern biology. In particular, the growing awareness of the importance atipical DNA structural motifs such as ribonucleotide inclusions, Hoogsteen interactions, G-quadruplexes, make the study of DNA-protein interaction a topic of current interest.

Previous results showed that accumulation of ribonucleotides in genomic DNA leads to genome instability and that inactivation of RNase H2, the enzyme responsible for recognizing and removing genomic ribonucleotides, is responsible for the Aicardi-Goutières Syndrome (AGS). AGS is an inflammatory encephalopathy presenting during infancy, and cells from AGS children accumulate genomic ribonucleotides. Understanding how RNase H2 recognises and is recruited to ribonucleotide-containing DNA will be relevant to further characterize the molecular details of the pathology.

G-quadruplexes (G4) are endogenous DNA secondary structures that have an important impact on chromosome stability and maintenance. We have identified a protein that, when mutated, results in chromosome rearrangements at G4 sites. It will be important to verify and characterize the interaction between this factor and G4- containing sequences.

A research activity is proposed in which this topic will be tackled by exploiting new approaches which involve the use of Reflective Phantom Interface (RPI)

a technology for biosensing recently developed within the University of Milano and of the so-called structural DNA nanotechnology.

Specifically, the PhD student will develop new protocols and schemes in which the interactions between protein and variously structured and modified DNA

will be detected on the RPI surface both directly and through the amplification provided by the switching and growth of multistrand DNA nanoconstructs.

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. 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 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: Achille Pellicioli ([email protected])

Location: Dipartimento di Bioscienze, Via Celoria 26 20133 Milano


Polo kinases and genome instability: implication for cancer development and treatmentBackgroundPolo kinases (PLK1-4, in human cells) are fundamental regulators of mitosis and cell cycleprogression. Remarkably, PLK1 is up regulated in many cancers and its overexpression isconsidered as prognostic marker. In response to DNA damage, PLK1 is temporarily inactivated but,after a prolonged cell cycle block, it is required to switch the checkpoint off, leading to cellproliferation. Several experimental evidences show that PLK1 overexpression overrides the DNAdamage checkpoint and further leads to genome instability. Importantly, even reduced levels ofPLK1 have been found to promote tumor formation. This urges to carefully study the regulation ofPLK1 activity and its effect on genome stability.HypothesisBased on the variations in PLK1 levels observed in different types of cancers, the hypothesis is thatPLK1 activity may be important to preserve genome stability, even in response to induced DNAdamage. In particular, the student will take advantage of budding yeast as a model organism tocharacterize the functional role of Cdc5, the only PLK1 expressed in yeast, in maintaining genomeintegrity. Then, depending upon the results in yeast, we will transfer the obtained information toPLK1 in human cell lines.Aims and experimental approachesThe hosting laboratory has already produced novel cdc5 alleles and reagents that promise to beuseful to further characterize the Cdc5 regulatory network. Preliminary results have shown thatCdc5 kinase activity is required for spontaneous and induced recombination. Furthermore, some ofthe novel cdc5 alleles are sensitive to agents that cause DNA damage and are defective incheckpoint adaptation after persistent double strand DNA break (DSB).To further characterized the cdc5 alleles, during the Ph.D. project the student will study criticalaspects of DSB repair and recombination (such as DNA ends tethering and resection, binding ofrecombination and checkpoint factors on to DNA lesions, processing of recombinationintermediates, and others). One main part of the project will be focused on a special mechanism ofrecombination, called break-induced replication, that is responsible of severe genomerearrangements found in cancer cells. The student will also use yeast backgrounds designed to studychromosome loss and rearrangements, which are tracts of genome instability. Using these systems,the student will set up genetic screens to define functional interplay between Cdc5 and DNArecombination processes. She/He will also perform analysis in human cell lines to study the role ofPLK1 in DSB repair and recombination pathways. We will take advantage of specific PLK1inhibitors that are already used in clinical trials for cancer treatments.Altogether, the obtained results may be of interest to optimize the use of PLK1 inhibitors, as well asto develop novel and more specific inhibitors to modulate PLK1 activity for cancer treatment, alsoaiming to reduce unwanted side effects and genome instability.


Project leader: Paolo Pesaresi, [email protected]



BarPLUS: modifying photosynthesis to maximize barley biomass and yield for biofuel production

Barley is a major crop worldwide, with Europe producing the greatest share (~60 MT/yr). Beside grains barley plants produce an almost equivalent amount of straw that in the past was considered as a secondary product of minimal value. Indeed, most of the genetic progress to increase yield was obtained through a change in biomass partitioning from straw to grains and the current plant architecture has been mainly driven by the necessity of increasing the harvest index. Nevertheless, the increasing demand for renewable materials makes straw, and especially barley straw characterized by the largest content of carbohydrates among cereals, a valuable product for its potential conversion into biofuels and other products. Indeed, barley crop residues are desirable feedstocks because of their low cost, immediate availability, no competition with food, and relatively concentrated location in the major grain growing regions. Given this perspective, we believe that the current barley photosynthesis performance should be revised to maximise the farmer income (grain value plus straw value). To this aim, BarPLUS will identify genes, alleles and lines needed to increase barley plant biomass, without penalty on grain yield, in the agro-climatic and management scenarios of Italy. This goal will be achieved through improved efficiency of the photosynthetic process and field trial evaluation. We will deploy mutagenized lines and diverse accessions carrying natural allelic variants in candidate genes (CGs) for components of the light absorption and photo-protection mechanisms of the photosynthetic apparatus to evaluate their usefulness to increase barley biomass production. Taking advantage of the unique resources of mutants and exome-resequencing data available for barley,

BarPLUS will deliver knowledge and tools to develop a new barley variety, which will provide farmers with 5-to-10 % more biomass per hectare without compromising grain yield.

Project leader: KATIA PETRONI ([email protected])

Location: Department of BioSciences


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 chemotherapeutic agents, ii), to investigate the effects of dietary anthocyanins on specific microRNAs involved in drug cardiotoxicity, 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 leader: Alessandra Polissi ([email protected])



Envelope biogenesis in Gram-negative bacteria as molecular target for the development of next generation antibacterial drugs

The OM is the hallmark of Gram-negative bacteria. This membrane is unique in its composition and asymmetrical distribution of lipids, with the inner leaflet containing phospholipids and the outer leaflet mainly composed of lipopolysaccharide (LPS). Besides representing an essential structural component of the cell envelope of most Gram-negative bacteria, LPS also largely contributes to the impermeability of the OM, as the highly-negative charged LPS molecules are tightly packed and form an extremely efficient barrier against anionic and hydrophobic molecules, including several antibiotics. Accordingly, genetic mutations affecting the assembly of LPS into the OM have a profound impact on cell envelope integrity and tolerance to harsh conditions, and are generally incompatible with growth. Targeting the mechanisms responsible for LPS assembly and, more in general, OM biogenesis therefore represents a promising strategy to reduce the in vivo fitness of Gram-negative bacteria as well as to increase their susceptibility to currently-available antibiotics. LPS precursors are synthesized in the cytoplasm, assembled into mature LPS on the periplasmic side of the IM, and then translocated into the OM by a multi-protein complex named the lipopolysaccharide transport (Lpt) machinery. Most of what is known about the LPS transport pathway derives from studies in the model Gram-negative bacterium Escherichia coli, where the Lpt complex has been found to consist of seven essential proteins (LptABCDEFG), structurally conserved among different species, which likely form a continuous bridge across the IM, the periplasm and the OM. At the IM, the LptBFG complex constitutes an ABC transporter that provides the energy for LPS transport. LptC is a small bitopic protein, which interacts with the LptBFG complex and with the periplasmic protein LptA. LptA is thought to oligomerize to form a protein bridge across the periplasm, which delivers LPS to the LptDE protein complex of the OM, which is ultimately responsible for the assembly of LPS at the cell surface. While the LPS transport machinery is generally thought to be crucial for growth, pathogenicity and drug resistance of the great majority of Gram-negative pathogens, thus implicating it as a promising target for the generation of novel antimicrobials, recent findings from our laboratory strongly suggest that the functioning of this protein apparatus may diverge among bacterial species, and that also in E. coli the Lpt complex may accept alterations to its typical architecture. In particular, in the opportunistic human pathogen Pseudomonas aeruginosa, the LptC and LptE proteins appeared to be dispensable for growth under laboratory conditions, in contrast to what observed for other components of the Lpt complex that, as expected, were found to be strictly essential for cell viability. Moreover, through a purpose-developed genetic selection approach aimed at clarifying the functional role of E. coli LptC, viable E. coli mutants deleted in lptC have recently been obtained. Whole genome sequencing of different ΔlptC mutants revealed single amino acid substitutions at a unique position in the periplasmic domain of LptF, strongly suggestive of a gain-of-function mutation which allows the E. coli Lpt machinery to work as an LptC-independent six-component protein complex. Based on the rationale that understanding the mechanisms by which LPS is assembled into the OM could lead to the development of better strategies to target Gram-negative infections, the functional and molecular characterization of these “atypical” Lpt machineries, and the evaluation of their impact on bacterial fitness and ability to cause infection, are mandatory to further characterize this essential cellular pathway of Gram-negative bacteria and to evaluate its protein components as potential molecular targets for novel antibacterial drugs.


Project leader: Stefano Ricagno ([email protected])



Structural characterization of the receptor EP1 from Arabidopsis thaliana

This project aims to understand the fundamental mechanisms of reproductive isolation in plant by investigating the genetic parameters controlling reproduction success. The evolutionary divergence of reproductive processes after ecological and geographic separation leads to reduce reproductive success and gene flow between populations or closely related species, and that reproductive divergence therefore constitutes an important step in speciation. Gametophytic interaction during fertilization is of fundamental importance to the successful reproduction process. In this contest ligand–receptor interactions are predicted to be involved in a complex network of extracellular signalling modules in gamete recognition and fusion.

Many receptors–ligand systems have been identified but their precise role in the interaction and recognition between male and female gametophyte is still unclear. Unpublished data points to EP1 as an important receptor involved in the release of male gametes inside the female gametophyte. This project aims to understand the molecular details of EP1 signalling.

The first step of this project consists in the expression and purification of EP1, which will be structurally and biophysically characterised. Through in vitro experimental approaches, the peptidic ligands of EP1 will be identified; then synthetic compounds, which could artificially activate this receptor, will be designed. The structural characterisation of the complexes between EP1 and its ligands will be also performed. The effects on such ligands on EP1 function will be tested in plants.

Inactive and hyperactive mutants of EP1 will be designed starting from structural considerations and their effects will be tested in vivo (transgenic plants).


Project leader: Chiara Tonelli ([email protected])

Location: Dept. Biosciences

Role of plant hormone signalling in the natural variation of flowering and adaptation in response to

water scarcity through QTL and eQTL dissection


Plant responses to varying water availability often involves phenotypic changes in flowering time and growth. The genetic basis of these adaptive traits is complex, although the florigen genes, encoding a class of hormone-like signaling proteins, may play a major role. Florigen genes have been implicated in the control of different aspects of plant vegetative development, including growth regulation and guard cell movements/transpiration 1-3. The question arises as to how florigen genes are regulated, temporally and spatially, under varying growth conditions, and whether variations in their transcript accumulation accounts for the diverse modes of developmental reprograming under drought stress observed in plants. This question has been addressed experimentally with an association genetics approach aimed at comprehensively relating different developmental adaptations (i.e. via putative effects on growth or on the duration of the vegetative phase) to patterns of florigen gene accumulation in maize germplasm. We have screened a panel of maize lines (about 320) for different developmental and physiological traits including flowering and growth under two watering conditions (i.e. normal watering and reduced watering). For these lines a dense genotyping is available to us in the form of Genotyping by Sequencing (GBS) dataset. Interrogation of phenotyping and genotyping data allowed the identification of QTLs of the studied phenotypic variables. We were also able to relate physiological QTLs with genetic variants affecting florigen expression (eQTLs) and identified putative co-locations 4.

The clearest trend that emerges from most GWA studies (including ours) is that the majority of genetic variants affecting complex traits (including eQTLs) resides outside of protein-coding regions. These trait-associated noncoding variants may contain transcriptional enhancers, but their molecular function remains largely unknown. The student will take advantage of a mix of tools and approaches to identify the source of the variation in florigen transcript accumulation (and its related phenotypic traits) across maize lines under different environments. Technically, the project will involve positional cloning followed by targeted gene replacement/editing, the setting-up of quantitative assays aimed at reconstruct regulatory chromatin landscapes 5 6 and target capture/resequencing using massive parallel sequencing. As a whole, the student will conceive follow-up functional studies to understand how genetic variants in genes or DNA functional elements identified from GWAS exert their effects on florigen gene regulation 7. This project balances elements of risk and more straightforward approaches that could yield important advances in our understanding of transcriptional regulation processes in plants and beyond.

1. Ando, E. et al. TWIN SISTER OF FT, GIGANTEA, and CONSTANS Have a Positive But Indirect Effect on Blue Light-Induced Stomatal Opening in Arabidopsis. Plant Physiol. 162, 1529–1538 (2013).

2. Danilevskaya, O. N., Meng, X., McGonigle, B. & Muszynski, M. G. Beyond flowering time: pleiotropic function of the maize flowering hormone florigen. Plant Signal Behav 6, 1267–1270 (2011).

3. Riboni, M., Galbiati, M., Tonelli, C. & Conti, L. GIGANTEA Enables Drought Escape Response via Abscisic Acid-Dependent Activation of the Florigens and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Plant Physiol. 162, 1706–1719 (2013).

4. Schadt, E. E. et al. Genetics of gene expression surveyed in maize, mouse and man. Nature 422, 297–


302 (2003).

5. Sullivan, A. M. et al. Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana. Cell Rep 8, 2015–2030 (2014).

6. Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).

7. Price, A. H. Believe it or not, QTLs are accurate! Trends Plant Sci. 11, 213–216 (2006).

Project leader: Chiara Zuccato ([email protected])

Location: Department of Biosciences and INGM, Via Sforza 35 Milano


Huntington’s Disease: biological aspects and pathogenesis

The Huntington's disease gene, encoding for huntingtin (HTT) protein, can be traced back through 800 million year of evolution to before the protostome-deuterostome divergence. HTT appears to have originally exerted non-neuronal functions and only later, during deuterostome evolution, acquired more specific activities that are important for central nervous system (CNS) development and function (Zuccato et al., Physiol Rev, 2010). The HD gene contains a polymorphic trinucleotide CAG repeat that is translated into polyglutamine amino acid (polyQ) residues in HTT.When this polyQ stretch, at the 18 aminoacid (aa) position of the protein, expands to over 39residues, HD occurs, a fatal, genetically dominant, neurodegenerative disease.We are currently investigating the biology of the HD gene and our aim is to re-construct, by using bioinformatic and sequencing approaches, the history of HTT along evolution. Here, we will also explore the relevance of different molecular and cellular dysfunctions previously identified in the lab in order to identify those that significantly contribute to HD pathogenesis. In particular we will focus on reduced transcription of genes controlled by REST/NRSF factor, including BDNF (Zuccato et al., The Journal of Neuroscience, 2007; Conforti et al., Gene Therapy, 2012) and reduced synthesis of cholesterol in the HD brain (Valenza et al., The Journal of Neuroscience, 2005; Leoni et al., Brain 2008; Valenza et al., The Journal of Neuroscience, 2010, Valenza et al., Cell death and differentiation 2015). More recently, we have started investigating a new target(ADAM10/NCadherin) that has emerged from our evolutionary study (Lo Sardo and Zuccato et al., Nat Neurosci. 2012), which is critical for excitatory synaptic circuitries affected in HD.


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