Dr. Sadikovic and his coworkers have also performed epi-toxicogenomic studies in which antibodies against acetylated histones were used to immunoprecipitate chromatin extracted from cells exposed to benzo(a)pyrene. Then Affymetrix promoter tiling microarrays were probed to investigate the relationship between such environmental exposure and global gene-specific changes in histone acetylation.
“It’s important to carry out replicate studies in order to lower noise and the chance for erroneous signals,” Dr. Sadikovic notes. “Repeatability has bedeviled the discovery process in genomics, which may account for the failure of genome analysis to uncover new biomarkers.”
The group’s studies have turned up a number of genes altered in their expression by polycyclic hydrocarbons, including those affecting proliferation, apoptosis, and cell-cycle regulation that may serve as targets for future anticancer drugs.
“Aviva Systems Biology has developed a transcription network mapping system,” explains Jeff Falk, Ph.D., director of technology and business applications at Aviva. One of the functions of this platform is to provide transcriptional fingerprinting of hepatotoxicity regulatory networks.
For the last six years, Aviva has been building ChIP-based technologies for mapping genome-wide gene transcription regulation, and it has applied these technologies to profiling multiple tissues and key disease-related systems as well as mapping the response of liver specific genes to toxic substances.
“Now the initial pilot studies are generating a complete map of the human liver transcriptional networks,” Dr. Falk says. “This will provide a framework for identifying the critical pathways and biomarkers of toxicity.”
A major component of the company’s strategy is a multiepitope polyclonal antibody collection, which includes a complete set of antibodies for all human transcription factors. The company has developed a high-throughput, automated ChIP-on-chip screening capability that allows prevalidation of the antibodies directed against various regulatory proteins.
Dr. Falk argues that a stable of powerful polyclonal antibodies directed against putative marker proteins and peptides is absolutely essential for a successful ChIP- on-chip program, a view that he shares with other scientists.
Dr. Falk compares the Aviva platform with conventional toxicology testing in which compounds are initially screened and moved through preclinical and clinical evaluation, with painfully high attrition rates, resulting in a spectacular loss of time and great expense.
Aviva’s DNA/protein interaction mapping technologies focus on three variations of the traditional ChIP-on-chip platform, which uses antitranscription factor antibodies to identify genome-wide promoter/enhancer interactions, epigenetic modifications, and DNA-methylation sites. These include ChIP-DSL (DNA selection and ligation), a promoter array technology that is faster and much more sensitive; ChIP-Seq, known as sequencing-based profiling of transcriptional fingerprinting interactions; and ChIP-qPCR, for validation of the identified binding interactions at specific targets.
“DSL refers to the improvements made to the ChIP-on-chip procedure. In typical ChIP-on-chip reactions, the immunoprecipitated DNA products undergo general blunt-end ligation of linkers and subsequent PCR amplification,” Dr. Falk explains. “This is an inefficient process since blunt-end ligation only attaches linkers to a small percentage of the actual immunoprecipitated DNA, so the background is very high.”
With DSL, there is a specific amplification process. The immunoprecipitated DNA is first hybridized to a pool of 20,000 oligo pairs that correspond directly to the 20,000 arrayed promoter oligos. The hybridized oligo pair is then ligated into a 40 base template using Taq DNA Ligase, which only occurs if there is a 100% direct match. As a result, the specificity is dramatically increased, since only the immunoprecipitated sequences present in the promoter array are amplified. In addition, the reactions are more efficient so that DSL requires less starting material.
Among the many possible applications for products in the clinic, the Aviva chip technologies are being applied to a variety of cancer prognostics as well as hepatotoxicity biomarkers.