Project Summary/Abstract Enhancer, promoters, and insulators are regulatory sequences essential for the establishment of specific patterns of gene expression during cell differentiation It is becoming increasingly evident that these sequences, often located far from each other in the genome, interact in the nuclear space over long distances to regulate the establishment of distinct transcriptional profiles characteristic of specific cellular fates. The requirement for loal interactions at specific loci, for example between enhancers and promoters, has been studied in detail. Nevertheless, the extent and function of long-range inter- and intra-chromosomal interactions mediated by these sequences at the level of the whole genome and its role in establishing and/or maintaining specific gene expression profiles is largely unexplored. During the last funding period we have characterized the protein composition of Drosophila insulators and determined the genome-wide localization patterns of known insulator proteins. We have also used 5C/HiC technology to gather data that give us a glimpse into the complexity of the intra-chromosomal interactions mediated by insulators. We are now in an ideal position to carry out the experiments required to understand the role of the three-dimensional arrangement of the chromatin fiber in the nuclear space in the regulation of gene expression. Here we propose to use HiC and ChIA-PET to establish detailed maps of intra- and inter-chromosomal interactions in the Drosophila genome. Preliminary experiments suggest that transcription and insulators collaborate in the establishment of the 3D organization of the genome and we will carry out functional experiments to test this hypothesis. Point to point interactions between enhancer, promoters and insulators will be analyzed by ChIA-PET in order to understand the mechanisms by which insulators control gene expression. The 3D arrangement of the chromatin during S and M phases will be analyzed by HiC and results will be compared to those obtained with interphase cells to understand the possible role of insulators in the control of genome architecture throughout the cell cycle. Finally, covalent modifications responsible for the regulation of insulator activity will be analyzed and their role in mediating specific inter- and intra-chromosomal interactions will be determined. In the end, we hope to define the role of insulators in the regulation of the three- dimensional organization of the genome and its role in different aspects of nuclear biology. This three-dimensional organization may serve as a fingerprint of cell identity that synthesizes the epigenetic status of the cell, and may help to identify the state of differentiated, trans- differentiated or reprogrammed cells for use in human cell therapy.