Proteomics and functional genomics for the analysis of the different level of gene expression regulation
Tiziana Bonaldi , PhD
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
T +39 02 94375123
The long-standing goal of my research is the elucidation of the molecular mechanisms that regulate gene expression using a combination of functional genomic approaches.
My interest is focused on the investigating how the establishment of gene expression patterns contributes to the definition of cell-fate during development and on understanding how this control is lost in progression of tumor states.
My unit will develop to lines of research
1st Project: The histone code revealed by Mass Spectrometry-based proteomics
Histone N-termini undergo diverse post-translational modifications that significantly extend the information potential of the genetic code. Moreover, they appear to "label" specific chromatin regions, modulating epigenetic control, lineage commitment and the overall function of chromosomes.
Recent work has shed a light onto how single histone modifications affect chromatin function, but to fully decipher the code of histone tails it is essential to understand how specific combinations of modifications are generated and how different modifications cross-talk one with the other. Hence it is of great interest to develop new strategies to "decode" the language of histone modifications. Such information is essential for cells to establish and memorize specific programs of gene expression, which are set during embryonic development and are prerequisite for differentiation of particular cell types in the adult organism.
Mass Spectrometry (MS) has already proven to be a successful tool to detect many histone PTMs and to reveal some interplay between neighboring ones.
State-of-the-art of the technology
I am currently setting up a panel of novel mass spectrometric technologies to evaluate the relative stoichiometry of combinations of PTMs, with a "bird-eye view" at the whole protein level. In addition, I am to perform quantitative analysis of modifications by employing Stable Isotope Labelling by Amino acids in Cell Culture (SILAC), based on a differential isotopic labelling of two cellular states that allows quantitative evaluation of proteomes by MS and their dynamics during different biological events.
Among the different modifications occurring on the core histones, methylation appears to be stable and transmitted across cell generations; therefore is the best characterized candidate for an epigenetic mark, and the major focus of my investigation. In my unit I will focus on the pure epigenetic aspects of this code, therefore I will concentrate on murine embryonic stem (ES) cells. Given the possibility to tightly regulate their differentiation in vitro into multiple cell types, and to manipulated them with a variety of technologies, like RNA interference (RNAi) and SILAC, this model system will allow to analyze:
1) The establishment of PTMs patterns in the move from the undifferentiated/pluripotent state thorough the first steps of differentiation;
2) The dynamics of such PTMs patterns during subsequent stages, up to the fully differentiated one.
As such, I plan to clarify the functional correlation between the onset of specific epigenetic marks on histones and the definition of specific cell identity.
2nd Project: Quantitative Proteomics for Functional Analysis of miRNAs targets
miRNAs are small non coding RNAs that regulate gene expression. The primary regulation of miRNAs is believed to occur at the translational level, even though cross talks with the RNA interference (RNAi) processes imply also a contribution at the level of transcription of mRNA. Great effort has been recently put in miRNA targets prediction, by computational studies, and in their identification by DNA micro arrays. Yet, the mechanism responsible for gene expression regulation by mRNA remains unclear.
Screening techniques to measure changes at the protein level in a comprehensive fashion are more difficult than profiling mRNA changes by micro-array approaches. However, recent works have demonstrated that proteomics can now quantify a very comprehensive fraction of the total proteome. When stable isotope labeling by amino acids in cell culture (SILAC) is employed, quantitation can be very accurate in these large-scale experiments. Thus, quantitative proteomics can now compete for large-scale analysis of gene expression with strategies based on detection of mRNAs, with the advantage of measuring actual protein levels, in this case a pre-requisite for the understanding of miRNA action.
By employing SILAC approach, my unit will investigate the effects of different miRNAs on the cells proteome. The plan is to transfect cell culture with different miRNAs and quantitatively profile changes in proteins. This should provide global information about target genes, to be compared to the data produced by DNA array technology and by computational prediction.
Ideally, proteomic measurements will be performed in parallel with microarray analysis that allow the evaluation of changes in transcript levels; the analysis of these data synergistically will be a gateway for understanding the mechanism of miRNA action at all levels of gene expression.