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Polycomb in development, genome regulation and cancer

Final Report Summary - FLYINGPOLYCOMB (Polycomb in development, genome regulation and cancer)

The ERC project FlyingPolycomb was aimed at studying the role of epigenetic regulatory factors, particularly those named Polycomb group proteins, in regulation of gene expression, both during normal development and in cancer. Polycomb group (PcG) proteins are key regulators of the expression of major developmental genes and they coordinate the processes of cell differentiation and cell proliferation. PcG proteins are able to silence gene expression in the appropriate cells. They are able to maintain the memory of silent regulatory states through mitotic divisions of the different cell lineages. They act by binding to specific regulatory elements called Polycomb response elements (PREs) and by modifying chromatin flanking them. Importantly, PcG proteins regulate the organization of their target genes in the three-dimensional space of the nucleus, and this regulatory function is involved in the maintenance of cellular memory. In the framework of the FlyingPolycomb project, we proposed to study the mechanisms of PcG and trxG protein recruitment as well as of Polycomb mediated gene silencing at the genome-wide scale. Furthermore, we dissected the functional role of PcG proteins in normal development and cancer and we studied the role of PcG proteins in the regulation of the nuclear organization of their target genes. Finally, we planned to perform a new type of large scale screen in order to identify new genes involved in the function of PcG and trxG proteins.
The reseach we have accomplished allowed to reach most of our goals. This project had four aims. Aim 1 was to elucidate mechanisms of Polycomb protein targeting and of Polycomb-dependent chromatin silencing. Thanks to genome-wide mapping of Polycomb proteins in various Drosophila species, coupled with sequence analysis and to new technology to map chromatin domains (called Hi-C) we could derive important principles explaining recruitment of Polycomb proteins to chromatin thanks to a combination of cues, where specific DNA sequences attract binding of transcription factors, which recruit Polycomb proteins that, in turn, form large chromosome domains leading to spatial interactions of Polycomb binding sites. These interactions stabilize Polycomb domains and favor the binding of Polycomb recruiter proteins to chromatin, such that a mutual reinforcement between Polycomb proteins and transcription factors favors the maintenance of Polycomb domains through evolution even if individual consensus motifs in the DNA sequences mautate their sequence.
Aim 2 was to elucidate the role of Polycomb proteins in development and cancer. Our results showed that, in late developmental stages such as in larvae or adults, the function of Polycomb proteins is less to silence Hox genes than to modulate the expression of other genes, such as those involved in regulation of the cell cycle in the female germ line, and cell cycle, polarity and signaling in the larval epithelia, where Polycomb proteins prevent the occurrence of cancer.
Aim 3 was to understand the role of Polycomb proteins in the regulation of 3D organization of chromosomes. In this framework, we described the 3D dynamic organization of Polycomb proteins during embryogenesis. Furthermore, we extensively used chromosome conformation capture technology (3C, 4C, Hi-C...) in order to describe chromatin folding around a PRE in transgenic constructs. We expanded these studies to show, thanks to 4C and microscopy, that a network of 3D contacts among Polycomb target genes is formed in the cell nucleus, which can stabilize silencing of Polycomb target genes. We then established Hi-C, in order to show at the genome-wide level the 3D organization principles of a metazoan genome. This work provided the first evidence of the existence of 3D chromatin domains dubbed as TADs.
Aim 4, the final goal of the project, was to identify components that regulate the 3D organization of Polycomb proteins. Thanks to a breaktrhough technology combining high-throughput RNAi to high-end confocal microscopy, we were able to analyze the identify 130 genes involved in modulating the 3D organization of Polycomb in the cell nucleus and showed for two of them that they are important Polycomb cofactors involved in the SUMOylation of Polycomb proteins.