Riassunti dei progetti del corso di dottorato di ricerca in biologia molecolare e cellulare
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Project leader: NERINA GNESUTTA (nerina.gnesutta@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYNF-Y partners in the regulation of CCAAT promoters Transcriptional regulation is at the heart of all biological process, and it is governed by transcription factors -TFs- which bind to discrete genomic regions. Many protooncogenes and tumor suppressors are TFs and their disregulation leads to changes in gene expression patterns that result in uncoltrolled cell growth and cancer. The CCAAT box is a DNA element which is found enriched in promoters of growth controlling genes, and is specifically bound by the trimeric transcription factor NF-Y. Recently, the locations of several TF binding sites has been mapped in vivo at the genomic level by the ENCODE consortium project. Bioinformatic analyses of these data, performed by our group and others’, have shown that NF-Y locations significantly overlap, within short distances, with a few set of other TFs, among which the protooncogenes Myc and Fos. Such analyses suggest that specific configurations of the relative binding sites in promoters, can underlie the rules of biochemical and functional interactions of NF-Y with its partners to control gene expression. Such information, together with the knowledge of the crystal structure of NF-Y bound to DNA, recently solved by our group in collaboration with proff Nardini and Bolognesi, is the foundation of the proposed project. The research project aims at understanding, at the biochemical, structural and functional levels, the interactions of NF-Y with TFs Myc (Myc/Max), and other E-box binding TFs (Max and USF1), and with Fos (Fos/Jun), based on “prototypical” CCAAT promoters bound by NF-Y and its genomic partners. Such informations will allow us to analyse larger sets of promoters, and could provide useful information to understand and predict TFs interactions in the regulation of CCAAT promoter genes. Specific aims of the project will include: in vitro biochemical analyses of purified proteins to evaluate TFs cooperativity in DNA binding by EMSA; isolation of TFs ternary complexes with DNA for SAXS structural analyses, to visualise surfaces involved in protein interactions; in vivo studies of promoter occupancy by ChIP, following TFs inactivation, to understand possible hierarchy in DNA binding; in vivo studies by transient expression of wt and mutant proteins with gene promoter-reporter assays to evaluate functional interactions in gene expression regulation.
-Dolfini D, Gatta R, Mantovani R. NF-Y and the transcriptional activation of CCAAT promoters. Crit. Rev. Biochem. Mol. Biol. 2012; 47: 29-49. -Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R. Struhl R. NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. Genome Res 2013; 23:1195-209 -Nardini M, Gnesutta N, Donati G, Gatta R, Forni C, Fossati A, Vonrhein C, Moras D, Romier C, Bolognesi M, Mantovani R. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination. Cell 2013; 152: 132-143. Project leader: LUISA GUERRINI (luisa.guerrini@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYRole of the p300 acetylase in regulating the activities of the p63 transcription factorThe transcription factor p63 is a key regulator of ectodermal, orofacial and limb development. In particular, it plays a critical role in epithelial biology, contributing to development and maintenance of the stratified epidermis (1). Dominant mutations in the p63 gene are causative of several human hereditary syndromes, such as AEC, EEC, LMS and SHFM-IV (2). Unequivocal establishment of the role of p63 in the pathogenesis of these human hereditary syndromes is complicated by the fact that this protein exists in multiple isoforms with different, often contradictory, biological activities. Despite increasing knowledge about the biological function of p63 in the tissues in which is expressed, relatively little is known about the mechanisms governing the expression levels of the p63 proteins. The function played by p63 in ectodermal differentiation and stratified epithelial progenitor-cell maintenance is well assessed, however, further studies are required to determine the interplay of p63 with other signalling pathways including those regulating the other p53 family members. It is likely that interrelationships between p53 and specific p63 isoforms play an essential role into the proliferation and differentiation program of developing cells. A current opinion is that p63 and/or p73 reside with p53 in larger transcriptional complex in which each sibling may regulate the activity of the others (3). To this respect, mutations affecting the relative stability of specific isoforms might grossly alter the fine tuning of p53 and p63 activity on specific promoters. It has already been demonstrated that one component of the principal p53 regulatory pathway, the p300 acetylase, is clearly involved also in the control of p73 isoforms (4, 5) with very little being known about its role on p63 isoforms (6). We are now interested in unravelling the role of the p300 acetylase on p63 regulation during keratynocites differentiation and during mouse limb development. The p300 acetylase has a critical role in regulating p53 and p73 protein stability and transcriptional activity. Recently, a new p300 site has been described in p53, lysine 164, which integrity is essential for p53 activities (7) that is perfectly conserved in p63 and is found mutated to glutamic acid (K193E) in patient affected by the SHFM-IV syndrome. Interestingly, we have made preliminary observations suggesting that the effects of p300 on p63 stability could be affected by this natural mutation. We intend now to better investigate the mechanisms through which p300 regulates p63 expression since, depending on their relative expression levels, p300 can mediate either p63 protein stabilization or p63 protein degradation. This dual role of p300 has already been described for the p53 protein (8). Our studies should contribute to the identification of the molecular mechanisms and the cellular players involved in the control of epithelial and limb development and homeostasis. References 1) Barbieri CE, et al (2006) p63 and epithelial biology. Exp Cell Res 312, 695-706 2) Brunner HG, et al (2002) P63 gene mutations and human developmental syndromes. Am J Med Genet 112, 284-290 3) King KE, Weinberg WC.(2007) p63: defining roles in morphogenesis, homeostasis, and neoplasia of the epidermis. Mol Carcinog 46,716-24
5) Mantovani F, et al Pin1 Links the Activities of c-Abl and p300 in regulating p73 Function. Mol Cell 2004; 14:625-636 6) MacPartlin M, et al. p300 regulates p63 Trasc-riptional Activity. Jour Biol Chem 280: 30604-30610. 7) Tang Y, et al. Acetylation Is Indispensable for p53 Activation. Cell 2008; 133:612-626 8) Grossman SR, et al.. Polyubiquitination of p53 by a ubiquitin Ligase activity of p300. Science 2008; 300:342-344.
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