Distinct 'safe zones' at the nuclear envelope ensure robust replication of heterochromatic chromosome regions.

Chromosome replication and transcription occur within a complex nuclear milieu whose functional subdomains are beginning to be mapped out. Here we delineate distinct domains of the fission yeast nuclear envelope (NE), focusing on regions enriched for the inner NE protein, Bqt4, or the lamin interacting domain protein, Lem2. Bqt4 is relatively mobile around the NE and acts in two capacities. First, Bqt4 tethers chromosome termini and the mat locus to the NE specifically while these regions are replicating. This positioning is required for accurate heterochromatin replication. Second, Bqt4 mobilizes a subset of Lem2 molecules around the NE to promote pericentric heterochromatin maintenance. Opposing Bqt4-dependent Lem2 mobility are factors that stabilize Lem2 beneath the centrosome, where Lem2 plays a crucial role in kinetochore maintenance. Our data prompt a model in which Bqt4-rich nuclear subdomains are 'safe zones' in which collisions between transcription and replication are averted and heterochromatin is reassembled faithfully.

Finding a place in the SUN: telomere maintenance in a diverse nuclear landscape.

Telomeres function in the context of a complex nuclear milieu in which telomeres tend to occupy distinct subnuclear regions. Indeed, regulation of the subnuclear positioning of telomeres is conserved from yeast to human, raising the age-old question: to what extent is location important for function? In mitotically dividing cells, the positioning of telomeres affects their epigenetic state and influences telomere processing and synthesis. In meiotic cells, telomere location is important for homologue pairing, centromere assembly and spindle formation. Here we focus on recent insights into the functions of telomere positioning in maintaining genome integrity.

Closed mitosis: A timely move before separation.

Faithful chromosome segregation entails long-range chromosome movement into newly dividing cells. A recent study implicates CDK1 function in releasing mitotic telomeres from the nuclear envelope, thereby liberating chromosomes for mitotic segregation.

Casein kinase I delta controls centrosome positioning during T cell activation.

Although termed central body, the centrosome is located off-center in many polarized cells. T cell receptor (TCR) engagement by antigens induces a polarity switch in T cells. This leads to the recruitment of the centrosome to the immunological synapse (IS), a specialized cell-cell junction. Despite much recent progress, how TCR signaling triggers centrosome repositioning remains poorly understood. In this paper, we uncover a critical requirement for the centrosomal casein kinase I delta (CKIδ) in centrosome translocation to the IS. CKIδ binds and phosphorylates the microtubule plus-end-binding protein EB1. Moreover, a putative EB1-binding motif at the C terminus of CKIδ is required for centrosome translocation to the IS. We find that depletion of CKIδ in T lymphocytes and inhibition of CKI in epithelial cells reduce microtubule growth. Therefore, we propose that CKIδ-EB1 complexes contribute to the increase in microtubule growth speeds observed in polarized T cells, a mechanism that might serve to generate long-stable microtubules necessary for centrosome translocation.

Early initiation of a replication origin tethered at the nuclear periphery.

Peripheral nuclear localization of chromosomal loci correlates with late replication in yeast and metazoan cells. To test whether peripheral positioning can impose late replication, we examined whether artificial tethering of an early-initiating replication origin to the nuclear periphery delays its replication in budding yeast. We tested the effects of three different peripheral tethering constructs on the time of replication of the early replication origin ARS607. Using the dense-isotope transfer method to assess replication time, we found that ARS607 still replicates early when tethered to the nuclear periphery using the Yif1 protein or a fragment of Sir4, whereas tethering using a Yku80 construct produces only a very slight replication delay. Single-cell microscopic analysis revealed no correlation between peripheral positioning of ARS607 in individual cells and delayed replication. Overall, our results demonstrate that a replication origin can initiate replication early in S phase, even if artificially relocated to the nuclear periphery.

Release of yeast telomeres from the nuclear periphery is triggered by replication and maintained by suppression of Ku-mediated anchoring.

The perinuclear localization of Saccharomyces cerevisiae telomeres provides a useful model for studying mechanisms that control chromosome positioning. Telomeres tend to be localized at the nuclear periphery during early interphase, but following S phase they delocalize and remain randomly positioned within the nucleus. We investigated whether DNA replication causes telomere delocalization from the nuclear periphery. Using live-cell fluorescence microscopy, we show that delaying DNA replication causes a corresponding delay in the dislodgment of telomeres from the nuclear envelope, demonstrating that replication of individual telomeres causes their delocalization. Telomere delocalization is not simply the result of recruitment to a replication factory in the nuclear interior, since we found that telomeric DNA replication can occur either at the nuclear periphery or in the nuclear interior. The telomere-binding complex Ku is one of the factors that localizes telomeres to the nuclear envelope. Using a gene locus tethering assay, we show that Ku-mediated peripheral positioning is switched off after DNA replication. Based on these findings, we propose that DNA replication causes telomere delocalization by triggering stable repression of the Ku-mediated anchoring pathway. In addition to maintaining genetic information, DNA replication may therefore regulate subnuclear organization of chromatin.