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The onset of random X inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. Here we show that RIF1 and KAP1 are two fundamental factors for the definition of the asymmetry. Our data show that at the onset of mESC differentiation, upregulation of the long non-coding RNA Tsix weakens the symmetric RIF1 association with the Xist promoter, and opens a window of opportunity for a more stable association of KAP1. KAP1 is required to sustain high levels of Tsix, thus reinforcing and propagating the asymmetry, and, as a result, marking the future active X chromosome. Furthermore, we show that RIF1 association with the future inactive X chromosome is essential for Xist upregulation. This double-bookmarking system, based on the mutually exclusive relationships of Tsix and RIF1, and RIF1 and KAP1, thus coordinates the identification of the inactive and active X chromosomes and initiates a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X chromosome state.

Eukaryotic genomes are replicated starting from multiple origins of replication. Their usage is tightly regulated, and not all the potential origins are activated during a single cell cycle. In addition, the ones that are activated are activated in a sequential order. Why don't origins of replication normally all fire together? Is this important? And if so, why? Would any order of firing do, or does the specific sequence matter? How is this process regulated? These questions concern all eukaryotes but have proven extremely hard to address because replication timing is a process intricately connected with multiple aspects of nuclear function.

The eukaryotic genome is replicated according to a specific spatio-temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S-phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re-organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication-timing programme in mammalian cells.

We report on the function of the human ortholog of Saccharomyces cerevisiae Rif1 (Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates their length. In contrast, human Rif1 did not accumulate at functional telomeres, but localized to dysfunctional telomeres and to telomeric DNA clusters in ALT cells, a pattern of telomere association typical of DNA-damage-response factors. After induction of double-strand breaks (DSBs), Rif1 formed foci that colocalized with other DNA-damage-response factors. This response was strictly dependent on ATM (ataxia telangiectasia mutated) and 53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition resulted in radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase checkpoint phenotype was independent of Nbs1 status, arguing that Rif1 and Nbs1 act in different pathways to inhibit DNA replication after DNA damage. These data reveal that human Rif1 contributes to the ATM-mediated protection against DNA damage and point to a remarkable difference in the primary function of this protein in yeast and mammals.

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12Δ and slk19Δ mutants.

The orderly reduction in chromosome number that occurs during meiosis depends on two aspects of chromosome behavior specific to the first meiotic division. These are the retention of cohesion between sister centromeres and their attachment to microtubules that extend to the same pole (monopolar attachment). By deleting genes that are upregulated during meiosis, we identified in Saccharomyces cerevisiae a kinetochore associated protein, Mam1 (Monopolin), which is essential for monopolar attachment. We also show that the meiosis-specific cohesin, Rec8, is essential for maintaining cohesion between sister centromeres but not for monopolar attachment. We conclude that monopolar attachment during meiosis I requires at least one meiosis-specific protein and is independent of the process that protects sister centromere cohesion.

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Scc1, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cell's differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3′ untranslated region (3′ UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1,2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3′ UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5′ to 67 nucleotides 3′ of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.

Fixing Broken DNA Some physiological processes, such as immunoglobulin class switching and telomere attrition, result in double-stranded DNA breaks. The DNA damage repair protein, 53BP1, prevents nucleolytic processing of these breaks, but the proteins it partners with to do this are unknown (see the Perspective by Lukas and Luka s ). Di Virgilio et al. (p. 711 , published online 10 January), using mass spectroscopy–based methods, and Zimmermann et al. (p. 700 , published online 10 January), using a telomere-based assay, identify Rif1 as a 53BP1 phosphorylation- and DNA damage–dependent interaction partner. Mice with a B cell–specific deletion in Rif1 showed impaired immunoglobulin class switching. Rif1-deficient cells exhibited extensive 5′-3′ resection at DNA ends, with enhanced genetic instability. Thus, Rif1 partners with 53BP1 to promote the proper repair of double-stranded DNA breaks.

Multifunctional protein Rif1 accumulates at stalled replication forks to facilitate DNA repair during S phase., Rif1, originally recognized for its role at telomeres in budding yeast, has been implicated in a wide variety of cellular processes in mammals, including pluripotency of stem cells, response to double-strand breaks, and breast cancer development. As the molecular function of Rif1 is not known, we examined the consequences of Rif1 deficiency in mouse cells. Rif1 deficiency leads to failure in embryonic development, and conditional deletion of Rif1 from mouse embryo fibroblasts affects S-phase progression, rendering cells hypersensitive to replication poisons. Rif1 deficiency does not alter the activation of the DNA replication checkpoint but rather affects the execution of repair. RNA interference to human Rif1 decreases the efficiency of homology-directed repair (HDR), and Rif1 deficiency results in aberrant aggregates of the HDR factor Rad51. Consistent with a role in S-phase progression, Rif1 accumulates at stalled replication forks, preferentially around pericentromeric heterochromatin. Collectively, these findings reveal a function for Rif1 in the repair of stalled forks by facilitating HDR.

A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis. Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II. The eukaryotic cell's cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process.