Vernalization, the acceleration of flowering by winter, involves cold-induced epigenetic silencing of Arabidopsis FLC. This process has been shown to require conserved Polycomb Repressive Complex 2 (PRC2) components including the Su(z)12 homologue, VRN2, and two plant homeodomain (PHD) finger proteins, VRN5 and VIN3. However, the sequence of events leading to FLC repression was unclear. Here we show that, contrary to expectations, VRN2 associates throughout the FLC locus independently of cold. The vernalization-induced silencing is triggered by the cold-dependent association of the PHD finger protein VRN5 to a specific domain in FLC intron 1, and this association is dependent on the cold-induced PHD protein VIN3. In plants returned to warm conditions, VRN5 distribution changes, and it associates more broadly over FLC, coincident with significant increases in H3K27me3. Biochemical purification of a VRN5 complex showed that during prolonged cold a PHD-PRC2 complex forms composed of core PRC2 components (VRN2, SWINGER [an E(Z) HMTase homologue], FIE [an ESC homologue], MSI1 [p55 homologue]), and three related PHD finger proteins, VRN5, VIN3, and VEL1. The PHD-PRC2 activity increases H3K27me3 throughout the locus to levels sufficient for stable silencing. Arabidopsis PHD-PRC2 thus seems to act similarly to Pcl-PRC2 of Drosophila and PHF1-PRC2 of mammals. These data show FLC silencing involves changed composition and dynamic redistribution of Polycomb complexes at different stages of the vernalization process, a mechanism with greater parallels to Polycomb silencing of certain mammalian loci than the classic Drosophila Polycomb targets.Arabidopsis ͉ PHD protein ͉ PRC2 ͉ chromatin modifications P olycomb-group (PcG) proteins were identified first in Drosophila melanogaster as factors necessary to maintain the repressed state of homeotic (Hox) genes (1). They also were identified as regulators of Hox genes in vertebrates and have been implicated in stem cell identity, cancer, imprinting, and chromosome X inactivation (1, 2). Recent genomic analysis has shown that up to 5% of genes in mice, humans, and Drosophila are PcG targets (3). Contrary to the original belief that PcG proteins maintain chromatin states, they now are thought to play a dynamic role in the transcriptional regulation of many genes (2).PcG proteins function in multiprotein complexes. Polycomb Repressive Complex 2 (PRC2) consists of core components, E(Z) (a histone methyltransferase), extra sex combs (ESC), p55, and Su(z)12 (4), and is widely conserved from plants to humans (4, 5). PRC2 maintains repressed chromatin states through posttranslational modification of histone tails (specifically histone 3 lysine 27 trimethylation, H3K27me3). In flies, H3K27me3 is recognized by the chromodomain of Pc, one of the components of the PRC1 complex. However, components of PRC1 seem to be absent from plant genomes (4).Polycomb silencing is involved in many aspects of plant development (5, 6) and was found to be the mechanistic basis of vernalization, the accelerati...

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two 113s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a leftto a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame Nucleosome dynamics is a necessary requirement of DNA function in chromatin, including transcription, replication, or repair. It has long been thought to be mediated by the tripartite organization of the histone octamer, made of an (H3-H4)2 tetramer bound with two H2A-H2B dimers (1). Thermodynamic studies of octamer assembly and disassembly showed that the forces holding the tetramer and dimers together are of a different nature and much stronger than the forces binding the dimers to the tetramer (1, 2), inspite of an extensive dimer-tetramer interface. This interface is disrupted in the first step of octamer disassembly.The crystal structure of the nucleosome core particle (3), and even more so, of the histone octamer (4), subsequently confirmed this tripartite organization. Moreover, one of the H2A-H2B dimers in the crystal structure of the core particle appears significantly displaced from its original position (3), which probably reflects the effects of crystal-packing forces and the relative ease with which the dimers can move relative to the tetramer. Such dimer lability may also be important in vivo, as is emphasized by the observations of an H2A-H2B deficit in nucleosome cores originating from transcriptionally active chromatin (5), and of H2A-H2B exchange with the endogenous histone pool upon chromatin transcription (6-8).Here we provide evidence for a potential conformational flexibility of the (H3-H4)2 tetramer, a tribute to the role of this tripartite organization of the histone octamer in nucleosome dynamics. This conformational flexibility is suggested by the observation of a similar affinity of the tetramer for negatively and positively supercoiled DNA minicircles, which apparently arises from the ability of the wrapped DNA to switch from a left-to a right-handed superhelix. This transition is found to require only modest positive torsional stress in the DNA, and can be triggered by DN...

Heterochromatin protein 1 (HP1) was originally described as a non-histone chromosomal protein and is required for transcriptional gene silencing and the formation of heterochromatin. Although it is localized primarily at pericentric heterochromatin, a scattered distribution over a large number of euchromatic loci is also evident. Here, we provide evidence that Drosophila HP1 is essential for the maintenance of active transcription of euchromatic genes functionally involved in cell-cycle progression, including those required for DNA replication and mitosis. Depletion of HP1 in proliferating embryonic cells caused aberrant progression of the cell cycle at S phase and G2/M phase, linked to aberrant chromosome segregation, cytokinesis, and an increase in apoptosis. The chromosomal distribution of Aurora B, and the level of phosphorylation of histone H3 serine 10 were also altered in the absence of HP1. Using chromatin immunoprecipitation analysis, we further demonstrate that the promoters of a number of cell-cycle regulator genes are bound to HP1, supporting a direct role for HP1 in their active transcription. Overall, our data suggest that HP1 is essential for the maintenance of cell-cycle progression and the transcription of cell-cycle regulatory genes. The results also support the view that HP1 is a positive regulator of transcription in euchromatin.

Vernalization, the promotion of flowering by cold, involves Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC). Cold progressively promotes cell-autonomous switching to a silenced state. Here, we used live-cell imaging of FLC-lacO to monitor changes in nuclear organization during vernalization. FLC-lacO alleles physically cluster during the cold and generally remain so after plants are returned to warm. Clustering is dependent on the Polycomb trans-factors necessary for establishment of the FLC silenced state but not on LIKE HETEROCHROMATIN PROTEIN 1, which functions to maintain silencing. These data support the view that physical clustering may be a common feature of Polycomb-mediated epigenetic switching mechanisms.