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The 2019 Gairdner Prize will be given to John F.X. Diffley and Bruce Stillman for their groundbreaking work on the mechanisms and control of the initiation of eukaryotic DNA replication. No two people have contributed more extensively, or over a longer period of time, to enlighten us on how our genomes replicate themselves once and only once per cell cycle.

In eukaryotic organisms, gene regulation occurs in the context of chromatin. In the interphase nucleus, euchromatin and heterochromatin occupy distinct space during cell differentiation, with heterochromatin becoming enriched at the nuclear and nucleolar peripheries. This organization is thought to fine-tune gene expression. To elucidate the mechanisms that govern this level of genome organization, screens were carried out in C. elegans which monitored the loss of heterochromatin sequestration at the nuclear periphery. This led to the identification of a novel chromodomain protein, CEC-4 (Caenorhabditis elegans chromodomain protein 4) that mediates the anchoring of H3K9 methylation-bearing chromatin at the nuclear periphery in early to mid-stage embryos. Surprisingly, the loss of CEC-4 does not derepress genes found in heterochromatic domains, nor does it affect differentiation under standard laboratory conditions. On the other hand, CEC-4 contributes to the efficiency with which muscle differentiation is induced following ectopic expression of the master regulator, HLH-1. This is one of the first phenotypes specifically attributed to the ablation of heterochromatin anchoring.

Mechanistic analyses based on improved imaging techniques have begun to explore the biological implications of chromatin movement within the nucleus. Studies in both prokaryotes and eukaryotes have shed light on what regulates the mobility of DNA over long distances. Interestingly, in eukaryotes, genomic loci increase their movement in response to double-strand break induction. Break mobility, in turn, correlates with the efficiency of repair by homologous recombination. We review here the source and regulation of DNA mobility and discuss how it can both contribute to and jeopardize genome stability.

SummaryHigh-fidelity chromosomal segregation requires the properly timed establishment of sister-chromatid cohesion mediated by the Cohesin complex, and its resolution at the metaphase-to-anaphase transition. We have examined cell-cycle progression in a yeast strain from which the origin recognition complex protein Orc2 was depleted after the assembly of prereplication complexes. We find that Orc2 depletion causes a delay in progression through mitosis, reflecting activation of both the DNA-damage and Mad2-spindle checkpoints. Surprisingly, sister-chromatid cohesion is impaired in Orc2-depleted cells, although Cohesin subunits are properly associated with chromatin. Reexpression of Orc2 in late G2/M phase restores chromatid cohesion. Finally, the targeting of Orc2 to a specific chromosomal locus suppresses premature sister-chromatid separation locally in a temperature-sensitive cohesin mutant. We conclude that ORC mediates sister-chromatid interaction on a pathway that is additive with Cohesin-mediated pairing.

The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8. Strains lacking INO80, ARP5, or ARP8 have defects in transcription. Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease. The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations. Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates. Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129. Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage. These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

An elegant study in Drosophila has expanded the evidence that different types of heterochromatin repeats have different characteristics by analyzing a number of randomly inserted transgenes that landed either in telomeric associated sequences on chromosomes 2 and 3, or in the repetitious transposable elements found at the telomere of chromosome 4 (Cryderman et al., 1999xCryderman, D.E., Morris, E.J., Biessmann, H., Elgin, S.C., and Wallrath, L.L. EMBO J. 1999; 18: 3724–3735Crossref | PubMed | Scopus (111)See all References)(Cryderman et al., 1999). As expected, these telomeric transgenes were subject to variegated repression, but they showed different degrees of repression and responded differently to mutations in genes known to affect PEV, namely, suppressor 2 of zeste (Su(z)2) and Su(var)2-5, which encodes HP1, a component of pericentric heterochromatin. Localization of the transgenes within the nucleus by in situ hybridization, showed that the transgenes that are sensitive to HP1 mutations were spatially juxtaposed to centromeric heterochromatin, while those insensitive to HP1 were not. Reciprocal translocations allowed the authors to correlate changes in nuclear position with changes in the strength of the repression. However, the local “heterochromatin” context of the chromosome 2 transgene was dominant over its nuclear position, i.e., the transgene did not respond to changes in HP1 concentrations even when spatially associated with centromeric domains (Cryderman et al., 1999xCryderman, D.E., Morris, E.J., Biessmann, H., Elgin, S.C., and Wallrath, L.L. EMBO J. 1999; 18: 3724–3735Crossref | PubMed | Scopus (111)See all References)(Cryderman et al., 1999). The fact that both local repeat context and position within the nucleus influence the character of gene repression, hints at the diversity that may be possible in mammalian cells, which seem to have numerous variants of repetitive elements. Due to the inherent “stickiness” of repetitive DNA, and its capacity for self-recognition, it is possible that different satellites' repeats will form distinct clusters, each able to offer a unique set of transcription factor recruitment sites. Cell-type specific patterns of gene regulation could then be mediated, at least in part, by a selective sequestering of genes into distinct heterochromatin compartments, which may respond differentially to modifiers of chromatin structure, although they use the same general mechanism of repression (Figure 2)(Figure 2). In view of the data suggesting that it is largely chromatin structure and not active transcription that correlates with a position in the nucleus, it is perhaps more appropriate to propose that relocated genes assume positions of potential, reflecting their potential for repression or expression, due to a position near heterochromatin or distant from it.Figure 2Cell Type–Specific Juxtaposition of Genes to Satellite DNAA hypothetical situation in two differentiated higher eukaryotic cells, each containing three different types of repetitive elements (here arbitrarily labeled SatA, SatB, and SatC). Different promoter-bound complexes (in blue or red, which may contain cell-type specific factors) recruit target genes (X or Z) to the zones of heterochromatin in which they have binding sites, depending on the cell type (compare A and B). Repression is likely to be achieved by a common mechanism involving histone modification and long-range chromatin compaction in both cases. Variations in spatial organization permit many different patterns of repression, making use of both cell type–specific transcription factors and general repressors of active chromatin (indicated as blue, red, or green spots within the heterochromatin)View Large Image | View Hi-Res Image | Download PowerPoint SlideNaturally it is the inheritance of differentiated gene expression patterns that is of primary importance in the development of a complex organism. Thus, there may be need for a system for inheriting three-dimensional order. Since both repetitive DNAs and nuclear localization tend to influence the timing of replication, these pockets of potential repression or expression will tend to be replicated at specific times in S phase. One can speculate that the late replication of genes associated with heterochromatin propagates not only a local chromatin organization, but a tag that maintains its localization within a subnuclear zone (see Cimbora and Groudine [this issue of Cell]). We should remember, however, that the nucleus is not hard wired, and that there is a great deal of movement of both DNA and proteins throughout the cell cycle. Perhaps in addition to the zones of heritable repression established by the Polycomb group genes, the eukaryotic nucleus profits from the tendancy of repetitive, heterochromatic domains to cluster, to set up networks of interactions within the nucleus. It is tempting to speculate that these compartments and their associated genes embody the molecular memory of genetic expression.Cimbora Groudine 2001xCimbora, D.M. and Groudine, M. Cell. 2001; 104: 643–646PubMedSee all ReferencesCimbora Groudine 2001

The silent mating loci and chromosomal regions adjacent to telomeres of S. cerevisiae have features similar to heterochromatin of more complex eukaryotes. Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4. We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro. Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro. Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres. Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.

A prior genetic study indicated that activity of Sir silencing proteins at a hypothetical AGE locus is essential for long life span. In this model, the SIR4-42 mutation would direct the Sir protein complex to the AGE locus, giving rise to a long life span. We show by indirect immunofluorescence that Sir3p and Sir4p are redirected to the nucleolus in the SIR4-42 mutant. Furthermore, this relocalization is dependent on both UTH4 a novel yeast gene that extends life span, and its homologue YGL023. Strikingly, the Sir complex is relocalized from telomeres to the nucleolus in old wild-type cells. We propose that the rDNA is the AGE locus and that nucleolar function is compromised in old yeast cells in a way that may be mitigated by targeting of Sir proteins to the nucleolus.

We have identified a yeast protein that binds specifically to the ARS consensus sequence. By two-step chromatography we have purified the factor to apparent homogeneity as a single polypeptide of 67 kd. The purified ARS consensus-binding protein (ACBP) recognizes the ARS consensus of the four genomic ARS elements tested, binding preferentially to the T-rich single strand. Point mutations in the consensus significantly reduce the affinity of the single-strand binding. At the histone H4 ARS, ACBP recognizes both the perfect ARS consensus and a 911 match 3′ of it. These two binding sites correlate with the boundaries of the minimal functional H4 ARS element. A similar configuration of binding sites is found at ARS1. We propose a model implicating this factor in an early step of the initiation of DNA replication.

DNA fragments containing the silencers that flank the mating type genes at HML alpha are shown to bind specifically to the nuclear scaffold of yeast. The scaffold proteins are solubilized with urea and then renatured to form a soluble extract which allows reconstitution of sequence-specific DNA loops. At the silent mating type locus HML alpha, loops are formed by either silencer-silencer (E-I) interaction or silencer-promoter interactions (E-P and I-P). The nuclear protein RAP-1 fractionates efficiently with the nuclear scaffold, and binds to the E, I, and promoter regions. Affinity purification of RAP-1 and oligonucleotide competition show that RAP-1 is necessary for reconstitution of loops in vitro. These results are consistent with a model in which silencers define a chromatin loop within which occur modifications that maintain the promoter in an inactive state.

Epigenetic mechanisms silence the HM mating-type loci in budding yeast. These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin. Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku. Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage. HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy. Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus. Silent chromatin anchorage requires the action of either Ku or Esc1. In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent. Thus, transcriptional repression can be sustained without perinuclear anchoring.

In this issue of Cell, Kitamura et al. (2006) use live-fluorescence microscopy to monitor individual genomic loci as they replicate in budding yeast. They confirm that DNA is recruited to replication factories and show that sister replication forks initiated from the same origin are held together within a single replication factory.

We find DNA fragments attached to the nuclear scaffold (SARs) both 5′ and 3′ of three Drosophila genes, defining looped domains ranging from 4.5 to 13 kb. For the two-promoter-containing gene Adh (alcohol dehydrogenase), we find two upstream and two downstream SARs. For Sgs-4, the 5′ SAR covers 866 bp immediately upstream of the transcript, and in the case of fushi tarazu, the 5′ SAR is found on a small fragment 4.8 kb upstream of the start of transcription. These four upstream scaffold-attached fragments comap with enhancer-like regulatory sequences. Sequence analysis of five upstream SARs reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II, several copies of a specific 10 bp A-rich sequence (AATAAATCAAA), and another 10 bp T-rich stretch.