SUMMARY How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear “compartments”. Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

PC4-and SF2-interacting protein 1 (Psip1)-also known as lens epithelium-derived growth factor (Ledgf)-is a chromatin-associated protein that has been implicated in transcriptional regulation, mRNA splicing, and cell survival in vitro, but its biological function in vivo is unknown. We identified an embryonic stem cell clone with disrupted Psip1 in a gene trap screen. The resulting Psip1-␤geo fusion protein retains chromatin-binding activity and the PWWP and AT hook domains of the wild-type protein but is missing the highly conserved C terminus. The majority of mice homozygous for the disrupted Psip1 gene died perinatally, but some survived to adulthood and displayed a range of phenotypic abnormalities, including low fertility, an absence of epididymal fat pads, and a tendency to develop blepharitis. However, contrary to expectations, the lens epithelium was normal. The mutant mice also exhibited motor and/or behavioral defects such as hind limb clenching, reduced grip strength, and reduced locomotor activity. Finally, both Psip1 ؊/؊ neonates and surviving adults had craniofacial and skeletal abnormalities. They had brachycephaly, small rib cages, and homeotic skeletal transformations with incomplete penetrance. The latter phenotypes suggest a role for Psip1 in the control of Hox expression and may also explain why PSIP1 (LEDGF) is found as a fusion partner with NUP98 in myeloid leukemias.PC4-and SF2-interacting protein 1 (Psip1) has been isolated in a number of independent experimental settings and has been assigned a variety of names and putative functions. Psip1 encodes two protein isoforms with molecular masses of 52 and 75 kDa. p52 and p75 were identified as interacting with transcription factor PC4 and shown to act as transcriptional coactivators (8). p75 is also referred to as lens epithelium-derived growth factor (LEDGF) since it was identified as a cell survival factor under a variety of conditions of environmental stress, and it has been implicated as a transcriptional regulator of stress-related genes (32). PSIP1 (LEDGF) is also a nuclear autoantigen targeted by autoantibodies in some patients with atopic dermatitis and other inflammatory conditions involving dysregulated apoptosis. It is thought to exert an antiapoptotic effect by transcriptional activation of stress-related genes (7), and it is cleaved by caspases (38).Psip1 has been reported to localize to chromatin in both interphase and mitotic chromosomes (27,28,35,37). The N-terminal domain of Psip1 contains a PWWP domain (Fig. 1B), a member of the Tudor domain "royal family" that includes chromo domains-some of which are known to bind to chromatin (24). There are conflicting data on the ability of the Psip1 PWWP domain to bind to free DNA in vitro (33,36). The PWWP domain of the DNA methyltransferase Dnmt3b is required for targeting the protein to chromatin and chromosomes (2, 10), and the Psip1 PWWP domain also affects the interaction of the protein with chromatin in vivo (36). Psip1 also contains AT hook-like motifs (Fig. 1B), and some of the...

In vertebrates, cytosine methylation is an epigenetic DNA modification that participates in genome stability and gene repression. Methylation patterns are either maintained throughout cell division, or modified by global or local de novo methylation and demethylation. Site-specific demethylation is a rather elusive process that occurs mainly in parallel to gene activation during development. In light of our studies of the glucocorticoid-dependent DNA demethylation of the tyrosine aminotransferase gene, we discuss the potential biochemical mechanisms allowing DNA demethylation and its targeting to specific sequences by transcription factors as well as possible links to DNA replication and chromatin remodelling.

Cytosine methylation at CpG dinucleotides contributes to the epigenetic maintenance of gene silencing. Dynamic reprogramming of DNA methylation patterns is believed to play a key role during development and differentiation in vertebrates. The mechanisms of DNA demethylation remain unclear and controversial. Here, we present a detailed characterization of the demethylation of an endogenous gene in cultured cells. This demethylation is triggered in a regulatory region by a transcriptional activator, the glucocorticoid receptor. We show that DNA demethylation is an active process, occurring independently of DNA replication, and in a distributive manner without concerted demethylation of cytosines on both strands. We demonstrate that the DNA backbone is cleaved 3 to the methyl cytidine during demethylation, and we suggest that a DNA repair pathway may therefore be involved in this demethylation.

Position within chromosome territories and localization at transcription factories are two facets of nuclear organization that have been associated with active gene expression. However, there is still debate about whether this organization is a cause or consequence of transcription. Here we induced looping out from chromosome territories (CTs), by the activation of Hox loci during differentiation, to investigate consequences on neighboring loci. We show that, even though flanking genes are caught up in the wave of nuclear reorganization, there is no effect on their expression. However, there is a differential organization of active and inactive alleles of these genes. Inactive alleles are preferentially retained within the CT, whereas actively transcribing alleles, and those associated with transcription factories, are found both inside and outside of the territory. We suggest that the alleles relocated further to the exterior of the CT are those that were already active and already associated with transcription factories before the induction of differentiation. Hence active gene regions may loop out from CTs because they are able to, and not because they need to in order to facilitate gene expression.

To gain a better understanding of the nature of active chromatin in mammals, we have characterized in living cells the various chromatin modification events triggered by the glucocorticoid receptor (GR) at the rat tyrosine aminotransferase gene. GR promotes a local remodeling at a glucocorticoid-responsive unit (GRU) located 2.5 kb upstream of the transcription start site, creating nuclease hypersensitivity that encompasses 450 bp of DNA. Nucleosomes at the GRU occupy multiple frames that are remodeled without nucleosome repositioning, showing that nucleosome positioning is not the key determinant of chromatin accessibility at this locus. Remodeling affects nucleosomes and adjacent linker sequences, enhancing accessibility at both regions. This is associated with decreased interaction of both the linker histone H1 and the core histone H3 with DNA. Thus, our results indicate that nucleosome and linker histone removal rather than nucleosome repositioning is associated with GR-triggered accessibility. Interestingly, GR induces hyperacetylation of histones H3 and H4, but this is not sufficient either for remodeling or for transcriptional activation. Finally, our data favor the coexistence of several chromatin states within the population, which may account for the previously encountered difficulties in characterizing unambiguously the active chromatin structure in living cells.

Whey acidic protein (WAP) and casein (CSN) genes are among the most highly expressed milk protein genes in the mammary gland of the lactating mouse. Their tissue-specific regulation depends on the activation and recruitment of transcription factors, and chromatin modifications in response to hormonal stimulation. We have investigated if another mechanism, such as specific positioning of the genes in the nucleus, could be involved in their functional regulation. Fluorescent in situ hybridization was used to study the nuclear localization of WAP and CSN genes in mouse mammary epithelial cells (HC11) cultured in the absence and presence of lactogenic hormones. Automatic 3D image processing and analysis tools were developed to score gene positions. In the absence of lactogenic hormones, both genes are distributed non-uniformly within the nucleus: the CSN locus was located close to the nuclear periphery and the WAP gene tended to be central. Stimulation by lactogenic hormones induced a statistically significant change to their distance from the periphery, which has been described as a repressive compartment. The detection of genes in combination with the corresponding chromosome-specific probe revealed that the CSN locus is relocated outside its chromosome territory following hormonal stimulation, whereas the WAP gene, which is already sited more frequently outside its chromosome territory in the absence of hormones, is not affected. We conclude that milk protein genes are subject to nuclear repositioning when activated, in agreement with a role for nuclear architecture in gene regulation, but that they behave differently as a function of their chromosomal context.