Alessandra Agresti
Alessandra Agresti
affiliation: San Raffaele Scientific Institute
research area(s): Molecular Biology, Cell Biology
  • Cell and Molecular Biology
  • Basic and Applied Immunology
University/Istitution: Università Vita-Salute San Raffaele
1993: Ph.D; Biotechnology, School of Veterinary Medicine, University of Milan
1986: National Board Exam
1984: degree in Biological Sciences, University of Milan

2009-present Group leader, In vivo chromatin and trascription, S.Raffaele Scientific Institute, Milan
2000-2009 Staff Scientist, Chromatin Dynamics Unit, S.Raffaele Scientific Institute, Milan
1996-2000 Researcher, Molecular Immunology Unit, S.Raffaele Scientific Institute, Milan
1993-1996 Researcher, Molecular Immunology of HIV Unit, S.Raffaele Scientific Institute, Milan
1986-1993 Postdoctoral fellow, Dept. Biology and Genetics, School of Medicine, University of Milan
1985-1986: Research fellow, "Istituto Nazionale per lo studio e la cura dei tumori", Milan

2009-2012: European Community Collaborative Research Project FP-7:
"Genomics determinants of inflammation: from physical measurements to system perturbation and mathematical modeling".

2001-present Tutorship, "Genetics and Developmental Biology"; Università Vita-Salute San Raffaele Milan Italy
1995-2001 Professor, Biology Course for Nurses and Neurophysiopathology Technicians
Transcription factor nuclear dynamics in living cells (NF-kB, Glucocorticoid Receptor and HMGB1). The non-histone nuclear protein HMGB1 in chromatin architecture, control of transcription and inflammation. Early transcriptional events regulated by NF-kB in isotype switching in allergy. Molecular tools to diagnose viral infections in mammals. Identification of new families of repetitive sequences in mammals, chromatin structure and genome complexity in evolution.

Current research in details
Our focus is on the quantitative analysis of NF-κB transcriptional dynamics in living cells, at single cell level. Single cell analysis provides a sharper description of dynamic events and bypass the inherent limitation of kinetic studies in cell populations due to averaged results from standard biochemical approaches.
In resting cells, NF-kB is mainly cytoplasmic and translocates to the nucleus upon inflammatory stimuli. Activated gene transcription leads to the resynthesis of several negative feedback genes that cause the inactivation of NF-kB and its re-localization to the cytoplasm. In the past, NF-kB nuclear cytoplasmic translocation has been reported to have a biphasic pattern with a sharp first nuclear translocation peak followed by a prolonged nuclear persistence with minor fluctuations. We demonstrated multiple consistent peaks of p65 nuclear localization (Sung et al, 2009) in single living fibroblasts from GFP-p65 knock-in mice that allow the detection of physiological levels of endogenous p65. Fourier analysis demonstrated that oscillations are sustained in the majority of the cells (79%) with 2.2 hours period (median value). Furthermore, p65 in late translocations is capable of diffusing on and interacting with the genome as effectively as the first p65 molecules activated after TNF-α by FRAP.
Mathematical modeling and computational simulations predicted that two different system perturbations would abolish oscillations, constrain p65 in the nucleus and result in opposite functional consequences of p65 transcriptional activity. Leptomicyn and Cycloheximide (LMB and CHX) used to mimic high IKK inactivation rate and low IκBα synthesis rate, respectively showed constitutive nuclear localization while p65 mobility increased with LMB and decreased with CHX suggesting different transcriptional activities. Indeed, expression of NF-kB target genes was either inhibited (LMB) or profoundly enhanced (CHX) suggesting that NF-kB oscillations encode specific cellular signaling information.
These results suggest that the oscillatory mode of NF-kB action may be a cellular trade-off between efficient pulses of expression and the need for NF-kB to monitor the signaling status for several hours to tune optimal transcriptional responses

1. Celona B, Weiner A, Felice FD, Mancuso FM, Cesarini E, Rossi RL, Gregory L, Baban D, Rossetti G, Grianti P, et al.: Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output. PLoS Biology 2011,in press.
2. Sung MH, Salvatore L, De Lorenzi R, Indrawan A, Pasparakis M, Hager GL, Bianchi ME, Agresti A: Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles. PLoS One 2009, 4:e7163
3. Bosisio D, Marazzi I, Agresti A, Shimizu N, Bianchi ME, Natoli G: A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NF-kappaB-dependent gene activity. Embo J 2006, 25:798-810.
4. Bianchi ME, Agresti A: HMG proteins: dynamic players in gene regulation and differentiation. Curr Opin Genet Dev 2005, 15:496-506.
5. Agresti A, Scaffidi P, Riva A, Caiolfa VR, Bianchi ME: GR and HMGB1 Interact Only within Chromatin and Influence Each Other's Residence Time. Mol Cell 2005, 18:109-121.
6. Agresti A, Bianchi ME: HMGB proteins and gene expression. Curr Opin Genet Dev 2003, 13:170-178.
7. Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, Rubartelli A, Agresti A, Bianchi ME: Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. Embo J 2003, 22:5551-5560.

Project Title:
Nucleosome number modulation as a novel layer of transcriptional regulation in inflammation
We had known for some time that HMGB1 (High Mobility Group Box1) is a nuclear protein that interacts with nucleosomes and several transcription factors (1, 2), including NF-kB, the major player in inflammation (3). In addition we had shown HMGB1 is also a secreted signalling molecule that triggers responses to cell and tissue damage (4, 5). Importantly, LPS stimulation of monocytic cells leaves the nucleus almost completely devoid of HMGB1 (5).
As expected for a molecule that appears following tissue damage, HMGB1 then: 1. conveys the message of danger to other cells, 2. triggers inflammation and innate immunity, to stop the damage, 3. plays a role in cell-cell communication involved in adaptive immunity, to help establish immunological memory of the adverse event, and 4. helps orchestrate tissue repair and healing.

We have recently found that mammalian cells lacking HMGB1 (a proxy for cells that have removed HMGB1 from the nucleus as a response to inflammation) have a reduced amount of histones and nucleosomes (Figure 1 and (6)). Moreover, cells with fewer nucleosomes have a specific transcriptional profile, whereby genes associated with response to stress are highly activated.

We thus have a nuclear protein that, following cues of cell stress, can exit the nucleus and eventually the cell. We then asked how the two roles -nuclear factor and secreted protein- relate to each other, notably in relation to inflammatory processes.

More specifically, the general question we are trying to tackle is: does HMGB1 orchestrate chromatin re-organization and gene transcription following inflammatory and stress responses?
The student will characterize the chromatin structure of inflammatory proficient cells like macrophages exposed to LPS or TNF-α through a high throughput approach (deep sequencing). Transcription profiles will be correlated with nucleosomal position/occupancy on differentially expressed genes. Results will be compared with those obtained in primary macrophages from Hmgb1-/- embryos where we predict an inflammatory profile.
Finally we propose to characterize chromatin structure of promoters of genes that require the dynamic recruitment of NF-kB for expression in inflammatory and stress responses.
We expect to describe new genome-wide and gene-specific nucleosomal landscapes associated with inflammation obtained through nucleosome number modulation. This mechanism will represent a novel layer of epigenetic control of transcription, and of cell identity.
1. T. Bonaldi, G. Langst, R. Strohner, P. B. Becker, M. E. Bianchi, EMBO J. 21, 6865 (December 16, 2002, 2002).
2. A. Agresti, M. E. Bianchi, Curr Opin Genet Dev 13, 170 (Apr, 2003).
3. A. Agresti, R. Lupo, M. E. Bianchi, S. Muller, Biochem Biophys Res Commun 302, 421 (Mar, 2003).
4. P. Scaffidi, T. Misteli, M. E. Bianchi, Nature 418, 191 (2002).
5. T. Bonaldi et al., Embo J 22, 5551 (Oct 15, 2003).
6. B. Celona et al., PLoS Biol in press (2011).

Project Title:
Smart drugs to counteract NF-kB driven proliferation in Multiple Myeloma
NF-kB and tumors: Cancer is a disease characterized by self-sufficiency in growth signals, evasion from apoptosis, limitless replicative potential, tissue invasion and metastasis, and sustained angiogenesis (1). All these cellular processes are directly or indirectly controlled by Nuclear Factor (NF)-kB pathways(2) that activate the transcription of genes associated with cell cycle progression, proliferation, angiogenesis, and suppression of apoptosis as well as adhesion, migration, and invasion.
NF-kB and Multiple Myeloma (MM): MM is a mostly incurable plasma cell malignancy, characterized by the accumulation of a monoclonal plasma cell population in the bone marrow (BM). NF-kB pathways are activated in more then 80% of human cases (3) due to genetic mutations or aberrant signaling in the BM. Moreover, chemotherapeutics are genotoxic and activate the DNA damage response via NF-kB recruitment that, in turn, promotes evasion from cell cycle checkpoints. At present, most of the anti-NF-kB available drugs have broad aspecific effects and high toxicity (4).

Therefore, there is a clear need for new therapeutic strategies, either standing alone or acting as sensitizers to chemo- and radio-therapy, that target pathogenetic events in MM and represent powerful improvements to current therapeutic protocols in oncology.

Preliminary results: We have recently identified three molecules, "smart drugs", that show inhibitory activity on NF-kB driven transcription and cell proliferation. These molecules increase the concentration of inactive NF-kB that now acts as a Dominant Negative form of itself and inhibit transcriptional activity.
We plan to further improve the molecules tested so far to find specific weapons against MM growth, either alone or in cotreatment with chemotherapeutics. This project will also uncover new molecular targets to constrain the aggressive phenotype of MM.
The student will use standard and high throughput techniques to:
- analyse NF-kB regulated transcription and proliferation in MM cells upon the inhibitory treatments
- reduce MM proliferation in vivo with the identified treatments using a mouse model of human Multiple Myeloma (RAG2-/-, γchain-/-(5)).
- find first hints on the molecular mechanisms leading to NF-kB inhibition by SILAC-based expmts and MSquant/MawQuant analyses

1 D. Hanahan and R. A. Weinberg, Cell 100 (1), 57 (2000).
2 A. Mantovani, P. Allavena, A. Sica et al., Nature 454 (7203), 436 (2008).
3 C. M. Annunziata, R. E. Davis, Y. Demchenko et al., Cancer cell 12 (2), 115 (2007).
4 T. Hideshima, D. Chauhan, P. Richardson et al., J Biol Chem 277 (19), 16639 (2002).
5 C. S. Mitsiades, K. C. Anderson, and D. R. Carrasco, Hematology/oncology clinics of North America 21 (6), 1051 (2007).