Positional analyses of BRCA1-dependent expression in Saccharomyces cerevisiae
Mutations in BRCA1 account for a significant proportion of hereditary breast and ovarian cancers, but analysis of BRCA1 function is complicated by pleiotropic effects and binding partners (Pol II holoenzyme and transcription factors, chromatin remodelers, recombination complexes and E3 ligases). In vertebrate cells, efforts to elucidate BRCA1 transcriptional effects have focused on specific genes or restricted portions of the genome – limiting analyses of BRCA1 effects on adjoining DNA sequences and along chromosome lengths. Here, we use microarray analyses on the genetically tractable yeast cell system to elucidate BRCA1-dependent genome-wide positional effects on both gene induction and repression. Yeast responses may be of clinical relevance based on findings that BRCA1 severely diminishes yeast growth kinetics but that BRCA1 mutated at sites identified from breast tumors is no longer able to retard yeast. Our analysis reveals that BRCA1 acts through both transcription factors to up-regulate specific loci and chromatin remodeling complexes to effect global changes in gene expression. BRCA1 also exhibits gene repression activities. Cluster-functional analysis reveals that these repressed factors are required for mitotic stability and provide a novel molecular explanation for the conditional lethality observed between BRCA1 and chromosome segregation genes.
RVS support from the Susan G. Komen for the Cure Foundation (Award BCTR0707708)
Elg1p, an alternative replication factor C complex, functions in sister chromatid cohesion
Replication factor C (RFC) complexes catalyze the loading of PCNA-like sliding clamp complexes onto primed DNA. RFC complexes consist of four small subunits plus one of the four interchangeable large subunits. Rfc1-RFC is the only essential RFC complex and is utilized for processive DNA replication. Ctf18-RFC and Rad24-RFC function in various DNA repair processes. Elg1-RFC functions in homologous recombination, replication fork restart, S phase checkpoint pathways and Okazaki fragment maturation (Bellaoui et al 2003, Ben-Aroya et al 2003, Kanellis et al 2003, Banerjee and Myung 2004, Ayora and Kupiec 2005).
Of the four RFC complexes, only Ctf18-RFC has been identified as promoting sister chromatid pairing – or cohesion (Skibbens et al 1999, Mayer et al 2001, Hanna et al 2001). However, physical interaction between all RFC subunits and the cohesion establishment factor Ctf7p/ Eco1p suggests that additional RFC complexes may be involved in chromatid cohesion (Kenna and Skibbens 2003, Satish and Skibbens, unpublished data). Here, we demonstrate a new pathway for the Elg1-RFC complex in chromatid cohesion. In contrast to the synthetic lethal effect ctf18 has on ctf7 mutants, deletion of elg1 rescues both temperature sensitivity of ctf7eco1-1 and cohesion loss. While elg1 rescues ctf7eco1-1 phenotypes, elg1 and mcd1-1 are synthetically sick. This evidence suggests that Elg1p and Ctf18p play opposing roles in cohesion establishment.
The Role of Cohesion Establishment in Genomic Maintenance
Genome fidelity and cell fitness relies on a system that ensures that each daughter cell obtain a single copy and only a single copy of every chromosome while maintaining genomic integrity through repair processes. Previously, cohesion establishment was shown to act during S phase through Ctf7p/Eco1p (Skibbens et al 1999, Toth et al 1999) and more recently during DNA damage repair (Sjogren and Nasmyth 2001, Strom et al2007, Unal et al 2007). Although little is known about the process of cohesion establishment research has linked Ctf7p to replication factors PCNA (Skibbens et al 1999, Moldovan et al 2006) and RFC large subunits Rfc1p, Ctf18p and Rad24p (Skibbens et al 1999, Kenna and Skibbens 2003). This information lead us to hypothesize that Elg1p, the least characterized of large RFC alternative subunits, may also be implicated in cohesion establishment. Genetic screens revealed a role for Elg1p in homologous recombination, replication fork restart, S phase checkpoint pathways and Okazaki fragment maturation (Bellaoui et al 2003, Ben-Aroya et al 2003, Kanellis et al 2003, Banerjee and Myung 2004, Ayora and Kupiec 2005).
Here we show a novel physical and genetic interaction between cohesion establishment factor Ctf7p and alternative RFC large subunit Elg1p. Pull down experiments involving Ctf7p-Gst (Bait) and Elg1p-Myc13 (target) showed a novel physical interaction between these two proteins. Furthermore, ctf7-203 temperature sensitivity is partially suppressed by deletion of ELG1. These results suggest a role for cohesion establishment factor Ctf7p in genome integrity and maintenance.