Salcini Group
We are interested in mechanisms regulating signal transduction and we are focusing our attention on endocytosis, the crucial starting event of a signal cascade.
Endocytosis, by which eukaryotic cells communicate with the external environment, is a fine regulated process coordinating signaling, cell migration, polarity and development. Several endocytic mechanisms are identified at the plasma membrane and our aim is to identify new genes regulating these processes.
Endocytic pathways (Conner, Nature, 2003)
We are particularly interested in dynamin-independent endocytosis, a poorly characterized internalization process in which dynamin, a large GTPase responsible for the fission of the coated vesicles, is not required.
We are also interested to identify novel genes regulating endocytosis, using a family of proteins, the EH-containing proteins. The EH-containing proteins are involved in a variety of processes, including endocytosis, actin reorganization and mitogenic signaling. A number of different technologies and systems have been used to this end, including the identification of proteins by yeast two-hybrid assays, generation of C.elegans knockout animals1,2 and high-throughput RNAi screening to determine the physiological function of the newly identified genes.
The aim is to identify new proteins and mechanisms related to endocytosis that ultimately regulate cell homeostasis and cell proliferation in mammals. We are specifically interested in proteins with relevance in human diseases and our final goal is to investigate their roles in mechanisms related to malignancy human. In this optic we are investigating the role of endocytic proteins in internalization and intracellular trafficking of Amyloid Precursor Protein (APP), a key molecule in Alzheimer Disease.
Our model system: C. Elegans
C.elegans is our favorite model system for several reasons. Firstly, the organism has the advantage of a multicellular organism without the complexity of higher eukaryotes. The fact that C.elegans is a multicellular organism provides an opportunity to study tissue- or organ-specific proteins. Secondly, it is an appropriate system for genetic studies. Thirdly, the C.elegans genome has been completely sequenced and other additional information such as the phenotypes of RNA interference (RNAi) of each gene (even with some limitation of the analysis) or that of some mutants is publicly available.
Finally, in C. elegans gene families existing in higher eukaryotes are often represented by single genes, allowing a more suitable study in absence of functional redundancy.
Chromatin remodelling factors in C. Elegans development
In the nuclei of all eukaryotic cells, genomic DNA is tightly wrapped around histone complexes into a compacted structure called chromatin. Each histone complex is an octamer consisting of two copies of each of the histone core proteins H2A, H2B, H3 and H4. The N-terminal tails of these histones protrude from the octameric structure and are subject to a vast variety of post-translational modifications. These modifications remodel the structure of chromatin and represent a combinatorial “histone code” specifying the function of genomic regions in terms of chromosome segregation, DNA repair and transcriptional activity.
Methylation of lysines (K) on histone tails
Trimethylated K4, K36 and K79 on histone H3 generally mark actively transcribed regions, whereas trimethylated K9 and K27 on histone H3 and K20 on histone H4 mark regions of transcriptionally silenced gene. To date, more than 20 Histone Methyl Transferases (HMTs) have been characterised and shown to play key roles in the regulation of development, differentiation, cell fates and in cancer. HOX genes, whose expression is established during body formation by early signalling events, represent a good example of this regulation.
Until recently, methylation was considered to constitute a permanent and irreversible histone modification . However, the recent discovery of histone demethylases has challenged this view. The largest group of histone demethylases contains a Jumonji (Jmj)C-domain, which catalyzes demethylation of specific methyl-lysine residues on histone H3.
Since methylation state of histone is an important clue during development, C.elegans is a suitable model system to study the in vivo role of these demethylases, largely conserved in nematode. Characterization of mutant alleles carrying mutations in demethylases genes and microarray analysis identifying target genes will provide important contribution in understanding the roles of histone demethylases in a variety of processes such as embryonic and larval development, cell-fate decisions and organogenesis.
Defects in gonad migration (top) and gonad development (bottom) in JMJD3 mutant, lacking a H3K27me3 demethylase activity
Selected recent publications
Rose S., Malabarba M.G., Krag C., Schultz A., Tsushima H., Di Fiore P.P., Salcini A.E. (2007). C.elegans intersectin: a synaptic protein regulating neurotransmission. Mol Biol Cell 18(12):5091-9
Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rappsilber J, Issaeva I, Canaani E, Salcini AE, Helin K. (2007). UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature. 449(7163):731-4.
Christensen J, Agger K, Cloos PA, Pasini D, Rose S, Sennels L, Rappsilber J, Hansen KH, Salcini AE, Helin K. (2007). RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell. 128(6):1063-76.
Tosoni D, Puri C, Confalonieri S, Salcini AE, De Camilli P, Tacchetti C, Di Fiore PP. (2005). TTP specifically regulates the internalization of the transferrin receptor. Cell 123:875-88.
Polo S, Confalonieri S, Salcini AE, Di Fiore PP. (2003). EH and UIM: endocytosis and more. Sci STKE 2003(213):re17.
Salcini AE, Hilliard MA, Croce A, Arbucci S, Luzzi P, Tacchetti C, Daniell L, De Camilli P, Pelicci PG, Di Fiore PP, Bazzicalupo P. (2001). The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling. Nat Cell Biol 3:755-60.
Salcini AE, Confalonieri S, Doria M, Santolini E, Tassi E, Minenkova O, Cesareni G, Pelicci PG, Di Fiore PP. (1997). Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev 11:2239-49.
