Archive for December, 2008
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)
Gene Expression Profiles of HCT116-p53-/- cells Treated with Paclitaxel or Stathmin-siRNA
Among the most difficult cancers to treat are those having mutated or non-functional p53, with concurrent overexpression of stathmin (stmn1), amicrotubule (MT) destabilizer (e.g. Yuan et al. 2006. J. Pathol. 209: 549-8). Alli et al. (2007. Oncogene. 26:1003-12) demonstrated that this type of cancer cell undergoes apoptosis in response to stmn1 knock down by siRNA. The mechanism by which stmn1 siRNA promotes apoptosis in cancerous cell lines is not understood, while efficient delivery of targeted siRNA therapies remains an elusive challenge. In this work, we aim to understand the apoptotic processes activated by stmn1 siRNA in a p53-null human colon cancer cell line, HCT116-p53-null, and specifically what is unique about stmn1 siRNA induced apoptosis compared to that induced by paclitaxel treatment, a MT stabilizing drug. We find that both stmn1 depletion and paclitaxel addition slowed cell growth and caused ~25% cell death beginning ~48 h after initial treatment. Microarray analyses of gene expression profiles were then examined to provide an unbiased screen for genes up or down regulated by either treatment. Time courses (~55-72 h) were analyzed for annotated genes showing expression fold changes of greater than or equal to 2. In stmn1 siRNA treated cells, we found 215 down-regulated and 157 up-regulated genes. Paclitaxel treatment resulted in 162 down-regulated and 642 up-regulated genes. Of the differentially regulated genes from each treatment, the number of shared genes included 27 of 350 (8%) down-regulated genes and 89 of 710 (13%) up-regulated genes. These data indicate that most expression changes were unique to each treatment. Comparing those genes whose expression is oppositely changed between the two treatments also demonstrated a low level of overlap (95 of 1081 genes, or ~9%) between treatments. To date our results indicate that stmn1 siRNA and paclitaxel induce apoptosis through unique upstream signals, although each treatment acts to stabilize MTs. Continued analysis and microarray verification are ongoing to confirm our conclusions. Understanding how stmn1 siRNA induces apoptosis could lead to identification of novel chemotherapy targets.
Partially funded by an HHMI grant to Lehigh and NIH (LC).
Analysis of Yeast Chimeric rRNAs Harboring Fly D7a Expansion Segment Substitutions Within the Binding Site for RPL25
Although a conserved rRNA core structure and ribosomal protein families are the hallmark of the ribosome, it is clear that prominent structural differences within rRNA and ribosomal protein components exist between lineages that may contribute to interspecific differences in ribosome assembly, ribosome function, or the regulation of protein synthesis. Our interests are focused on determining how structural variation in L25/L23a ribosomal protein family and its 28S rRNA binding site impacts ribosome assembly and function. The L23a binding site is bisected by the presence of the highly divergent D7a rRNA expansion segment, which in some lineages (including Drosophila) is subject to a maturation event called “gap processing”, creating 28Sα and 28Sβ rRNAs. Within some insect lineages, there has been mutual expansion of both RpL23a and the D7a segment, suggesting co-evolution of these components. The factors that limit D7a size expansion nwithin a given lineage are unknown; however, it is presumed that a D7a expansion threshold must exist above which rRNA-protein binding and/or ribosome assembly and function would be disrupted. As a test of the structural limits of the D7a expansion segment within yeast rRNA, we have designed a series of chimeric yeast rDNA constructs (using a high copy number plasmid carrying a GAL7-35S rRNA gene) for expression within different yeast strains to determine the impact of D7a structural complexity on ribosome biogenesis and function. In each construct the yeast D7a region was replaced with Drosophila D7a sequences, effectively introducing a fly-specific “tag”. Following galactose induction of transfected wildtype yeast strains, chimeric yeast/fly construct expression was determined by RT-PCR. Preliminary results show differences in chimeric rDNA expression, likely due to differences in rRNA stability. D7a structure may have an effect rRNA interactions with yeast L25, leading to rRNA degradation. Expression of constructs within a yeast L25 chromosomal knock-out strain that is dependent on fly L23a for ribosome function and viability will determine if the presence of fly L23a alters chimeric rRNA maturation and stability.
Supported by research funds from Lehigh University.
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.