Long-term objective: My long-term career goal is a commitment to basic investigations on chromatin structure and function. I am interested in studying reversible histone modification as epigenetic markers controlling basic chromatin processes and to understand how deregulation of chromatin dynamics lead to cancer development. ? ? Specific aims (health connection): My immediate aims are to understand how deregulation of histone modifications, e.g. phosphorylation, is linked to cancer development. Specifically: (1) Identify the phosphatase(s) leading to H3 dephosphorylation. (2) Unveil the signaling cascade(s) and the mechanism by which signaling molecules induce H3 dephosphorylation: investigate mTOR as the potential pathway controlling such events. (3) To achieve, by inducing histone dephosphorylation, a permanent phenotypic reversal of malignant cells leading to their elimination. Understand how deregulation of histone modifications affects gene expression, cell differentiation and division, and leads to cancer development. Research designs and methods: I will pursue my aims by biochemical, cell and molecular biology means. Rationale and techniques: Our preliminary results allow me to test the hypothesis that growing (cancer) cells sense changes in their environment through changes in histone modification, e.g. phosphorylation (chromatin environmental sensor). Thus, I am focusing on the mechanism and on the identification of the factors and signal transduction pathways involved in histone dephosphorylation (phosphatosome). The techniques to be used are: Isolation of large protein complexes (phosphatosome) using a variety of chromatographic techniques. Identification, by mass spectrometry or protein micro sequencing of their components. In vivo (dominant negative mutants and iRNA to the phosphatosome, its overexpression) and in vitro (using histones and their peptides and reconstituted polynucleosomes) characterization of phosphatosome enzymatic properties. Use specific agonist/antagonist to unveil the involved signal pathway(s). Use cell immunofluorescence, Western blot, gel electrophoresis, chromatin immuno- precipitation, co-immuno-precipitation to elucidate phosphatosome physiological role. Use HPLC technique for histone isolation and analysis of in vivo phosphatase activity. Analysis of histones by TAU and AU-gels. ? ? Knowing the mechanisms of how a variety of drugs provoke the lost of phosphate from histones which in turn leads to cancer cell death, will allow us to design more powerful and selective drugs inducing this stripping process to destroy tumors. ? ? ?
