Furthermore, the amount Everolimus of PcG proteins within PC foci correlates with the size of the genomic domains forming them. Large genomic domains such as the Hox complexes form intense PC foci, whereas narrow genomic domains are found in weak PC foci. When genes located in homologous chromosomes pair, the underlying PC foci are more intense than in nuclei where the same genes do not pair [ 14]. Taken together, these data indicate that PC foci are not structures onto which PcG target genes have to be directed for silencing. Instead, PcG proteins bound to chromatin marked with H3K27me3 form PC foci because their target chromatin fibres fold into small discrete nuclear volume parcels

( Figure 1). To study the Gemcitabine purchase folding of the chromatin fibre and explain how large genomic domains covered with histone H3K27me3 can form PC foci in the

cell nucleus, 3C technology was used in order to monitor interactions between chromatin segments. PREs located in the Drosophila bithorax complex can contact other PREs of repressed Hox genes. These multiple loops within a genomic domain describe a repressive chromatin hub which is dependent on Polycomb [ 13]. In addition, the Drosophila gypsy insulator can prohibit contacts between a PRE and a distal promoter. This insulator-dependent chromatin conformation confines H3K27me3 and PcG proteins within a specific domain, suggesting that endogenous insulators may confine chromatin loops within for Polycomb domains without affecting adjacent genomic regions [ 15]. In mammalian embryonic stem cells, the locus GATA-4 has a multi-loop conformation which depends on PcG proteins. Multiple internal long-range contacts rely on silencing because they

are completely lost after the differentiation signal inducing GATA-4 expression [ 16]. Taken together, these works suggest that multiple loops in chromatin regions repressed by PcG proteins might cluster PREs and explain the generation of chromatin structures giving rise to discrete PC foci in microscopy. Nevertheless, one should be cautious about the interpretation of 3C data. Indeed, even if 3C identifies numerous loops between discrete genomic elements such as PREs, promoters, enhancers, insulators [ 17, 18 and 19], the unknown frequency of these chromatin contacts, the ability to only detect bipartite and not multipartite chromatin interactions and the lack of simultaneous information about the neighboring regions prevent an understanding of the exact 3D folding path of the chromatin fibre. A modification of the 3C technology by using an unbiased approach to monitor all the contacts made by a genomic bait of interest (4C) has revealed a more complex conformation of PcG-bound chromatin. Two studies using 4C in Drosophila to map contacts established by PcG target loci revealed that most of the contacts made by the bait regions are precisely confined with the genomic region covered by H3K27me3 in which the bait is located.