Project leader: ROBERTO MANTOVANI (mantor@unimi.it)

Project leader: ROBERTO MANTOVANI (mantor@unimi.it)

Location: Department of Biosciences, University of Milan, Italy

RESEARCH PROJECT SUMMARY

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

1. Nardini M., Gnesutta N., Donati G., Gatta R, Forni C., Fossati A., Vonrhein C., Moras D., Romier C., Bolognesi M., Mantovani R. NF-Y is a sequence-specific transcription factor displaying histone-like DNA binding and H2B-like ubiquitination. Cell, 152, 132-143 (2013).
2. Dolfini D. and Mantovani R. Targeting the Y/CCAAT box in cancer: YB-1 or NF-Y?

Cell Death and Differentiation, 20, 676-685 (2013).

Project leader: FEDERICA MARINI (federica.marini@unimi.it)

Location: Department of Biosciences, University of Milan, Italy

RESEARCH PROJECT SUMMARY

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

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

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

The PhD student will use both yeast and human cell lines as model systems to investigate the role of Slx4 in the maintenance of genomic stability. He/She will study the kinetic of a DSB resection and repair by Southern blotting, the recruitment of checkpoint and recombination factors by ChIP and indirect immunofluorescence. The functional interaction between Slx4 and 53BP1/Rad9 will be investigated through genetic and biochemical approaches. Furthermore, He/She will set up specific screening to identify novel genes and factors involved in DSB repair.

1) Jackson, S. P. & Bartek, J. (2009) Nature 461(7267), 1071--1078.

2) Lazzaro, F. et al. (2008) EMBO J 27(10), 1502--1512.

3) Zimmermann M, de Lange T. (2013) Trends Cell Biol. doi: 10.1016/j.tcb.2013.09.003.

4) Ohouo PY et al. (2013) Nature. 3;493(7430):120-4.

5) Kim Y, et al. (2013) Blood 3;121(1):54-63.