Repressive Chromatin in Caenorhabditis elegans: Establishment, Composition, and Function.

Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in Caenorhabditis elegans, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in C. elegans, and should inform our understanding of repressed chromatin in other animals

In and out of the Replication Factory

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

How Broken DNA Finds Its Template for Repair: A Computational Approach

Homologous recombination (HR) is the process by which a double-strand break in DNA is repaired using an identical donor template. Despite rapid progress in identifying the functions of the proteins that mediate HR, little is known about how broken DNA finds its homologous template. This process, coined homology search, has been difficult to monitor experimentally. Therefore, we present here a computational approach to model the effect of subnuclear positioning and chromatin dynamics on homology search. We found that, in our model, homology search occurs more efficiently if both the cut site and its template are at the nuclear periphery, whereas restricting the movement of the template or the break alone to the periphery markedly increases the time of the search. Immobilization of either component at any position slows down the search. Based on these results, we propose a new model for homology search, the facilitated random search model, which predicts that the search is random, but that nuclear organization and dynamics strongly influence its speed and efficiency

The study of protein recruitment to laser-induced DNA lesions can be distorted by photoconversion of the DNA binding dye Hoechst [version 2; peer review: 1 approved, 2 approved with reservations]

A commonly used approach for assessing DNA repair factor recruitment in mammalian cells is to induce DNA damage with a laser in the UV or near UV range and follow the local increase of GFP-tagged proteins at the site of damage. Often these measurements are performed in the presence of the blue DNA dye Hoechst, which is used as a photosensitizer. However, a light-induced switch of Hoechst from a blue-light to a green-light emitter will give a false positive signal at the site of damage.  Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results

The study of protein recruitment to UV-induced DNA lesions can be distorted by photoconversion of DNA dyes like Hoechst or DAPI [version 1; referees: 1 approved, 2 approved with reservations]

A common approach used to assess DNA repair factor binding in mammalian cells is to induce DNA damage with a UV laser and follow the movement of GFP-tagged proteins to the site of damage. Often these measurements are performed in the presence of the blue DNA intercalating dye Hoechst or DAPI, which is used to label nuclear DNA. A UV-induced switch of Hoechst and DAPI from a blue-light to a green-light emitter will give a false positive signal at the site of damage.  Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results

Modulation of drug sensitivity in yeast cells by the ATP‐binding domain of human DNA topoisomerase IIα

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIα (htopoIIα), one in the catalytic core of the enzyme and one in the ATP‐binding N‐terminal domain. Here we have tested how N‐terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full‐length enzymes, mutations that lower the drug binding capacity of the N‐terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIα N‐terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N‐terminal ATP‐binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non‐equivalent drug‐binding site

Regulating repression : roles for the Sir4 N-terminus in linker DNA protection and stabilization of epigenetic states

The Gasser laboratory is supported by the Novartis Research Foundation and the EU training network Nucleosome 4D. SK was supported by an EMBO long-term fellowship, a Schrodinger fellowship from the FWF, and the Swiss SystemsX.ch initiative/C-CINA; HCF by an EMBO long-term fellowship.Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358) must be complemented with an N-terminal domain (Sir4N; residues 1-270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.Publisher PDFPeer reviewe

Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes

Temporal separation of replication and recombination requires the intra-S checkpoint

In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block