Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. We show here that components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets.

Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

Maintenance of cell identity is a complex task that involves multiple layers of regulation, acting at all levels of chromatin packaging, from nucleosomes to folding of chromosomal domains in the cell nucleus. Polycomb-group (PcG) and trithorax-group (trxG) proteins maintain memory of chromatin states through binding at cis-regulatory elements named PcG response elements or cellular memory modules. Fab-7 is a well-defined cellular memory module involved in regulation of the homeotic gene Abdominal-B (Abd-B). In addition to its action in cis, we show here by three-dimensional FISH that the Fab-7 element leads to association of transgenes with each other or with the endogenous Fab-7, even when inserted in different chromosomes. These long-distance interactions enhance PcG-mediated silencing. They depend on PcG proteins, on DNA sequence homology, and on developmental progression. Once long-distance pairing is abolished by removal of the endogenous Fab-7, the derepressed chromatin state induced at the transgene locus can be transmitted through meiosis into a large fraction of the progeny, even after reintroduction of the endogenous Fab-7. Strikingly, meiotic inheritance of the derepressed state involves loss of pairing between endogenous and transgenic Fab-7. This suggests that transmission of nuclear architecture through cell division might contribute to inheritance of chromatin states in eukaryotes.[Keywords: Polycomb-group; nuclear architecture; chromosome pairing; chromatin/meiosis] Supplemental material is available at http://www.genesdev.org. Packaging of eukaryotic DNA into chromatin is achieved through its folding in a hierarchy of structures, such as nucleosomes, 30-nm chromatin fibers, higher order chromatin structures, and chromosomal domains. Chromatin folding organizes individual chromosomes in specific chromosomal territories in the cell nucleus (Cremer and Cremer 2001). Individual chromosomal territories are separated by an interchromatin compartment that contains diffusible components, protein complexes, and larger structures or nuclear bodies. All of these compartments are dynamic and continuously exchange components, although the motion of large bodies or chromosomal territories is constrained (Chubb and Bickmore 2003).It has been suggested that nuclear architecture might play a regulatory role in gene expression and cellular differentiation. In this view, genes would be segregated in specific nuclear domains and regulatory changes might move these genes into different compartments. Evidence has accumulated for precise gene localization and for changes in their nuclear position upon regulatory transitions (Gasser 2001). In vertebrate cell types, transitions between active and silent transcriptional states were shown to be accompanied by shuttling between euchromatic and heterochromatic nuclear compartments (Brown et al. 1997;Francastel et al. 1999;Lundgren et al. 2000). Similarly, silencing of the Drosophila euchromatic brown gene by insertion of a 2-Mb block of heterochromatin (bw D allele) has bee...

The Polycomb gene was discovered 60 years ago as a mutation inducing a particular homeotic phenotype. Subsequent work showed that Polycomb is a general repressor of homeotic genes. Other genes with similar function were identified and named Polycomb group (PcG) genes, while trithorax group (trxG) genes were shown to counteract PcG-mediated repression of homeotic genes. We now know that PcG and trxG proteins are conserved factors that regulate hundreds of different genomic loci. A sophisticated pathway is responsible for recruitment of these proteins at regulatory regions that were named PcG and trxG response elements (PRE and TRE). Once recruited to their targets, multimeric PcG and trxG protein complexes regulate transcription by modulating chromatin structure, in particular via deposition of specific post-translational histone modification marks and control of chromatin accessibility, as well as regulation of the three-dimensional nuclear organization of PRE and TRE. Here, we recapitulate the history of PcG and trxG gene discovery, we review the current evidence on their molecular function and, based on this evidence, we propose a revised classification of genes involved in PcG and trxG regulatory pathways.