Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. In budding yeast and mice, Exo1 creates single-stranded DNA (ssDNA) at uncapped telomeres. This ssDNA accumulation activates the checkpoint response resulting in cell cycle arrest. Here, we demonstrate that Exo1 is phosphorylated when telomeres are uncapped in cdc13-1 and yku70D yeast cells, and in response to the induction of DNA damage. After telomere uncapping, Exo1 phosphorylation depends on components of the checkpoint machinery such as Rad24, Rad17, Rad9, Rad53 and Mec1, but is largely independent of Chk1, Tel1 and Dun1. Serines S372, S567, S587 and S692 of Exo1 were identified as targets for phosphorylation. Furthermore, mutation of these Exo1 residues altered the DNA damage response to uncapped telomeres and camptothecin treatment, in a manner that suggests Exo1 phosphorylation inhibits its activity. We propose that Rad53-dependent Exo1 phosphorylation is involved in a negative feedback loop to limit ssDNA accumulation and DNA damage checkpoint activation.

HIRA-like (Hir) proteins are evolutionarily conserved and are implicated in the assembly of repressive chromatin. In Saccharomyces cerevisiae, Hir proteins contribute to the function of centromeres. However, S. cerevisiae has point centromeres that are structurally different from the complex centromeres of metazoans. In contrast, Schizosaccharomyces pombe has complex centromeres whose domain structure is conserved with that of human centromeres. Therefore, we examined the functions of the fission yeast Hir proteins Slm9 and the previously uncharacterised protein Hip1. Deletion of hip1 ؉ resulted in phenotypes that were similar to those described previously for slm9⌬ cells: a cell cycle delay, synthetic lethality with cdc25-22, and poor recovery from nitrogen starvation. However, while it has previously been shown that Slm9 is not required for the periodic expression of histone H2A, we found that loss of Hip1 led to derepression of core histone genes expression outside of S phase. Importantly, we found that deletion of either hip1 ؉ or slm9 ؉ resulted in increased rates of chromosome loss, increased sensitivity to spindle damage, and reduced transcriptional silencing in the outer centromeric repeats. Thus, S. pombe Hir proteins contribute to pericentromeric heterochromatin, and our data thus suggest that Hir proteins may be required for the function of metazoan centromeres.

The fission yeast HIRA proteins Hip1 and Slm9 are members of an evolutionarily conserved family of histone chaperones that are implicated in nucleosome assembly. Here we have used single-step affinity purification and mass spectrometry to identify factors that interact with both Hip1 and Slm9. This analysis identified Hip3, a previously uncharacterized 187-kDa protein, with similarity to S. cerevisiae Hir3. Consistent with this, cells disrupted for hip3 ؉ exhibit a range of growth defects that are similar to those associated with loss of Hip1 and Slm9. These include temperature sensitivity, a cell cycle delay, and synthetic lethality with cdc25-22. Furthermore, genetic analysis also indicates that disruption of hip3 ؉ is epistatic with mutation of hip1 ؉ and slm9 ؉ . Mutation of hip3 ؉ alleviates transcriptional silencing at several heterochromatic loci, including in the outer (otr) centromeric repeats, indicating that Hip3 is required for the integrity of pericentric heterochromatin. As a result, loss of Hip3 function leads to high levels of minichromosome loss and an increased frequency of lagging chromosomes during mitosis. Importantly, the function of Hip1, Slm9, and Hip3 is not restricted to constitutive heterochromatic loci, since these proteins also repress the expression of a number of genes, including the Tf2 retrotransposons.Centromeres play a critical role in the precise segregation of chromosomes, and as a result, defects in centromere function lead to aneuploidy (1). The fission yeast Schizosaccharomyces pombe provides an excellent system for the study of centromeres (2). In contrast to the budding yeast Saccharomyces cerevisiae, which has simple "point" centromeres (3), S. pombe has large complex centromeres that occupy between 35 and 110 kb and are arranged as a central core (cnt) flanked by arrays of repeated (imr and otr) elements (2). In this respect, S. pombe centromeres are reminiscent of the complex regional centromeres of metazoans. Furthermore, ultrastructural studies have revealed that the overall architectural organization of fission yeast centromeres is conserved with their human counterparts (4). Fission yeast centromeres are organized into distinct chromatin domains (2, 5). An inner domain is assembled into specialized chromatin in which core histone H3 is replaced by Cnp1, the fission yeast homologue of CENP-A (6), whereas the outer regions are associated with chromatin that resembles the pericentric heterochromatin of higher cells (2). Marker genes inserted into these outer regions are subject to heritable inactivation (7,8), which is dependent upon the RNA interference machinery, the methylation of histone H3 on lysine 9, and the association of a number of proteins, including Swi6, a homologue of mammalian HP1 (heterochromatin protein 1) (2, 5, 9 -12). In addition, the integrity of pericentric heterochromatin in fission yeast is dependent upon the two HIRA proteins Hip1 and Slm9 (13,14), since loss of either protein alleviates silencing in the otr centromeric repeats and results in...

PEA3 is a member of a subfamily of ETS domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression. PEA3, ER81, and ERM comprise the PEA3 subfamily of ETS domain transcription factors (reviewed in references 9 and 10). These proteins show a high degree of sequence conservation within their ETS DNA-binding domains and also in an N-terminal acidic domain and the region C-terminal to the ETS domain. These proteins also exhibit a high level of evolutionary conservation with homologues of human PEA3 and ERM, having been identified in other vertebrates such as zebra fish (4, 26). Biologically, PEA3 subfamily members have been shown to be involved in a number of processes including neuronal pathfinding (1, 24) and to play an important role in HER2/Neu-mediated mammary oncogenesis (37). Developmentally, members of the PEA3 subfamily are important recipients of fibroblast growth factor signaling (11,27,33,34). Fibroblast growth factor signaling acts via activating the extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase pathway and leads to the upregulation of the expression of PEA3 subfamily members. In addition, the transcriptional activity of several family members is enhanced in response to ERK pathway activation (18,19,28). A number of target genes have been identified (reviewed in reference 9 and 10), including genes with important roles in tumor growth and metastasis such as COX-2 (16, 42) and MMP-1 (3).The LPP (lipoma-preferred partner) protein and zyxin, ajuba, LIMD1, and TRIP6 form a subfamily of LIM domain proteins that are characterized by the presence of three tandem C-terminal LIM domains (44). LPP was first isolated as part of a fusion protein created by chromosomal translocations, in which the C-terminal part of LPP is fused to the N terminus of HMGA2/HMGIC (32). This suggests an important role for LPP in tumorigenesis and, in particular, the C-terminal region containing the LIM domains. LPP is usually localized at the cell periphery in focal adhesions and cell-cell contacts, where it associates with proteins such as ␣-actinin (23) and Scrib (31). However, in common with...