Forrest Spencer

A Model Based Background Adjustment for Oligonucleotide Expression Arrays

High-density oligonucleotide expression arrays are widely used in many areas of biomedical research. Affymetrix GeneChip arrays are the most popular. In the Affymetrix system, a fair amount of further preprocessing and data reduction occurs after the image-processing step. Statistical procedures developed by academic groups have been successful in improving the default algorithms provided by the Affymetrix system. In this article we present a solution to one of the preprocessing steps—background adjustment—based on a formal statistical framework. Our solution greatly improves the performance of the technology in various practical applications. These arrays use short oligonucleotides to probe for genes in an RNA sample. Typically, each gene is represented by 11–20 pairs of oligonucleotide probes. The first component of these pairs is referred to as a perfect match probe and is designed to hybridize only with transcripts from the intended gene (i. e., specific hybridization). However, hybridization by other sequences (i. e., nonspecific hybridization) is unavoidable. Furthermore, hybridization strengths are measured by a scanner that introduces optical noise. Therefore, the observed intensities need to be adjusted to give accurate measurements of specific hybridization. We have found that the default ad hoc adjustment, provided as part of the Affymetrix system, can be improved through the use of estimators derived from a statistical model that uses probe sequence information. A final step in preprocessing is to summarize the probe-level data for each gene to define a measure of expression that represents the amount of the corresponding mRNA species. In this article we illustrate the practical consequences of not adjusting appropriately for the presence of nonspecific hybridization and provide a solution based on our background adjustment procedure. Software that computes our adjustment is available as part of the Bioconductor Project (http://www.bioconductor.org).

Multiple-laboratory comparison of microarray platforms

Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC–Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.

Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers.

Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few genes that might cause this phenotype have been identified and no general mechanism underlying their function has emerged. To systematically identify somatic mutations in potential CIN genes in colorectal cancers, we determined the sequence of 102 human homologues of 96 yeast CIN genes known to function in various aspects of chromosome transmission fidelity. We identified 11 somatic mutations distributed among five genes in a panel that included 132 colorectal cancers. Remarkably, all but one of these 11 mutations were in the homologs of yeast genes that regulate sister chromatid cohesion. We then demonstrated that down-regulation of such homologs resulted in chromosomal instability and chromatid cohesion defects in human cells. Finally, we showed that down-regulation or genetic disruption of the two major candidate CIN genes identified in previous studies (MRE11A and CDC4) also resulted in abnormal sister chromatid cohesion in human cells. These results suggest that defective sister chromatid cohesion as a result of somatic mutations may represent a major cause of chromosome instability in human cancers.

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion.

ABSTRACT CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of eitherCTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, anRFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase κ, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-CCTF18 may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.

Gene function prediction from congruent synthetic lethal interactions in yeast

We predicted gene function using synthetic lethal genetic interactions between null alleles in Saccharomyces cerevisiae. Phenotypic and protein interaction data indicate that synthetic lethal gene pairs function in parallel or compensating pathways. Congruent gene pairs, defined as sharing synthetic lethal partners, are in single pathway branches. We predicted benomyl sensitivity and nuclear migration defects using congruence; these phenotypes were uncorrelated with direct synthetic lethality. We also predicted YLL049W as a new member of the dynein–dynactin pathway and provided new supporting experimental evidence. We performed synthetic lethal screens of the parallel mitotic exit network (MEN) and Cdc14 early anaphase release pathways required for late cell cycle. Synthetic lethal interactions bridged genes in these pathways, and high congruence linked genes within each pathway. Synthetic lethal interactions between MEN and all components of the Sin3/Rpd3 histone deacetylase revealed a novel function for Sin3/Rpd3 in promoting mitotic exit in parallel to MEN. These in silico methods can predict phenotypes and gene functions and are applicable to genomic synthetic lethality screens in yeast and analogous RNA interference screens in metazoans.

Systematic genome instability screens in yeast and their potential relevance to cancer

To systematically identify genes that maintain genome structure, yeast knockout mutants were examined by using three assays that followed marker inheritance in different chromosomal contexts. These screens identified 130 null mutant strains exhibiting chromosome instability (CIN) phenotypes. Differences in both phenotype severity and assay specificity were observed. The results demonstrate the advantages of using complementary assays to comprehensively identify genome maintenance determinants. Genome structure was important in determining the spectrum of gene and pathway mutations causing a chromosome instability phenotype. Protein similarity identified homologues in other species, including human genes with relevance to cancer. This extensive genome instability catalog can be combined with emerging genetic interaction data from yeast to support the identification of candidate targets for therapeutic elimination of chromosomally unstable cancer cells by selective cell killing.

The CHL 1 (CTF 1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M.

We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome transmission fidelity and shown to correspond to the previously identified chl1 mutation. Chl1 null mutants exhibited a 200‐fold increase in the rate of chromosome III missegregation per cell division, and near wild‐type rates of marker homozygosis on this chromosome by mitotic recombination. Analysis of the segregation of a marker chromosome indicated that sister chromatid loss (1:0 segregation) and sister chromatid non‐disjunction (2:0 segregation) contributed equally to chromosome missegregation. A genomic clone of CHL1 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL1 revealed a 2.6 kb open reading frame with a 99 kd predicted protein sequence that contained two PEST sequences and was 23% identical to the coding region of a nucleotide excision repair gene, RAD3. Domains of homology between these two predicted protein sequences included a helix‐turn‐helix motif and an ATP binding site containing a helicase consensus. Mutants lacking the CHL1 gene product are viable and display two striking, and perhaps interrelated, phenotypes: extreme chromosome instability and a delay in cell cycle progression in G2/M. This delay is independent of the cell cycle checkpoint that requires the function of the RAD9 gene.

Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae.

A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function.

Analysis of chromosome segregation in Saccharomyces cerevisiae.

Publisher Summary The yeast Saccharomyces cerevisiae is an excellent organism for the study of mitotic and meiotic chromosome segregation because it is possible to isolate mutations that effect the fidelity of this process. This chapter presents two methods for analyzing chromosome segregation in these mutants. In the first method, digital imaging microscopy is used on individual live cells to analyze the segregation of fluorescently labeled chromosomes. In this method, changes in nuclear DNA staining pattern are observed using a microscope equipped with epifluorescence optics suitable for DAPI-stained sampies. A low-light level imaging device, such as a silicon-intensified tube camera or a multichannel plate intensifier, is coupled to the microscope and used to obtain video images. In the second method, the construction of artificial chromosomes is described that have been specifically designed for analysis of the fidelity of chromosome segregation in mitosis and meiosis.

Performance assessment of copy number microarray platforms using a spike-in experiment

MOTIVATION Changes in the copy number of chromosomal DNA segments [copy number variants (CNVs)] have been implicated in human variation, heritable diseases and cancers. Microarray-based platforms are the current established technology of choice for studies reporting these discoveries and constitute the benchmark against which emergent sequence-based approaches will be evaluated. Research that depends on CNV analysis is rapidly increasing, and systematic platform assessments that distinguish strengths and weaknesses are needed to guide informed choice. RESULTS We evaluated the sensitivity and specificity of six platforms, provided by four leading vendors, using a spike-in experiment. NimbleGen and Agilent platforms outperformed Illumina and Affymetrix in accuracy and precision of copy number dosage estimates. However, Illumina and Affymetrix algorithms that leverage single nucleotide polymorphism (SNP) information make up for this disadvantage and perform well at variant detection. Overall, the NimbleGen 2.1M platform outperformed others, but only with the use of an alternative data analysis pipeline to the one offered by the manufacturer. AVAILABILITY The data is available from http://rafalab.jhsph.edu/cnvcomp/. CONTACT pevsner@jhmi.edu; fspencer@jhmi.edu; rafa@jhu.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.