Progetto EPIGEN

  • MIUR - EPIGENOMICS  FLAGSHIP  PROJECT - (Progetto Bandiera Epigenomica) - EPIGEN
  • CALL FOR PROPOSALS 2012
  • Chronic disruption of the circadian rhythmicity and chromatin epigenetic modifications in the model organism Drosophila melanogaster

Chromatin remodeling is fundamental for the control of a large number of nuclear processes. Recent findings have linked cellular metabolism, epigenetic status, and the circadian clock (1-3). The circadian clock machinery, evolutionarily conserved among animals, consists of a complex transcriptional regulatory network that controls the expression of a significant portion of the genome (4, 5). The circadian oscillation of gene expression is paralleled by chromatin remodeling that confers specificity to circadian regulation (6, 7).
Chronic disruption of the circadian rhythmicity in humans has been linked to cardiovascular diseases,metabolic syndromes, sleep disorders, and tumorigenesis (8-10). In industrialized countries about 30% ofpeople is a shift-worker, consequently the chronic disturbance of the circadian clock has important social and economic implications. The classical view that the epigenetic control of circadian clock is primarily regulated by transcription factors capable to activate or repress specific genes must be certainly expanded today, in light of the recent discovery that many additional regulatory layers exist in cells, based on the presence of multiple RNA molecules devoid of coding potential. For instance, it is well known that the expression of miRNAs can strongly condition the translational output of the mRNA transcriptome. In addition, it has recently been appreciated that mRNAs can feed back on the activity of miRNAs through ‘sponging’ mechanisms (11-13). Finally, it has been recently shown that so far neglected RNA classes such as lncRNAs (14) and pseudogene-derived RNAs (15) can significantly affect the complex interplay between mRNAs,,miRNAs and chromatin status. Systems biology offers the potential to obtain new insights into our understanding of such complexity, providing quantitative models of how the single molecular players and events are integrated (16). Obtaining such kind of models would be of the greatest relevance to devise efficient strategies that could conveniently modify the end-points. To reach this goal, it is essential on one hand to characterize which molecules are present and at which levels they are expressed in the particular experimental setting. On the other hand, it is also essential to study how the different regulatory layers are integrated, by considering at a global level the interactions between the involved players. We plan to work along this line combining experimental and computational strategies with the aim to characterize the epigenomic modifications produced by chronic perturbations (shift-working) of the circadian machinery in the model organism Drosophila melanogaster. In particular, we will deeply characterize DNA methylation, hystone modifications, transposon and transcriptional activity. We will then integrate these new datasets with a previously generated mRNA and miRNA  transcriptome profiling to generate a network-based model of the transcriptional and post-transcriptional modifications induced by shift-working. In order to reach our aims we will use next generation sequencing (NGS)-based methods, which have been shown to provide a comprehensive and unbiased view of the epigenome, avoiding content-limited microarray platforms. Indeed, DNA methylation, histone modifications, the transcriptome, and DNA–protein interactions are now routinely being investigated genomewide using NGS (17-20). The combination of chromatin immuno-precipitation with NGS technology (ChIP-seq) is a powerful method for mapping protein–DNA interactions in a genome-wide fashion allowing us to detect histone modifications (i.e. acetylation and methylation) (21) and to investigate DNA methylation at a single nucleotide level (22). Finally, the total RNA extracted from the same individuals will be subjected to RNA-Seq analysis in order to define the long non coding RNAs (lncRNA) expression profiling. Inferential analysis on gene expression data (RNA-seq) will identify transcripts significantly modified in the shift-work model with respect to the control.
Drosophila are a useful and cost-effective model for shiftwork and recapitulate some of the damaging health effects observed in humans (see next Section 2.2 “Preliminary Results”). The results of the project outlined above will illuminate the intimate molecular nature of the many impaired physiological functions, which may characterize chronic disturbances of the circadian clock also in our species.

Principal Investigator

Partnership

  • Università degli studi di Torino - Dipartimento di Biotecnologie Molecolari e Scienze per la Salute