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Assays and Knowledgebases for Genomic Analysis


By Robert M. Frederickson

Sept. 18, 2006 |  Gene transcription and chromosome dynamics are regulated by a complex interplay of DNA-binding factors that interact with specific control elements. Genome-wide analysis of where such factors bind can provide the basis for understanding these complex gene and chromosome regulatory networks. This information has many applications in drug discovery, from toxicity studies and development of drug resistance to the analysis of gene targets for new cancer drugs, for example.

In 2000, the ChIP-Chip assay was developed for determining the many genetic targets for a given transcription factor (TF). The assay involved chromatin immunoprecipitation using antibodies specific for a particular TF, followed by labeling of the immunoprecipitation-captured DNA as a probe for hybridization to a DNA array pre-spotted with various promoter sequences.

The ChIP-GLAS (Chromatin Immunoprecipitation-Guided Ligation And Selection) assay is a similar, and includes an amplification step to enhance the sensitivity and reduce background. ChIP-GLAS technology was invented in the laboratory of Xiangdong Fu, at UC San Diego, and is licensed exclusively by Aviva Systems Biology. ChIP-GLAS permits genome-wide detection of promoters and enhancers that interact with proteins or carry modifications, such as methylation, that affect gene expression.

In ChIP-GLAS, the ChIP sample is selected, amplified and labeled, and then hybridized to a microarray containing 20,000 unique regions of human promoters. The arrayed oligonucleotides represent unique 40-bp sequences identified by bioinformatics analysis of putative gene-control regions of 20,000 human genes. The assay also allows capture and analysis of hypermethylated DNA using Aviva’s methylation-specific anti-5-methylcytosine monoclonal antibody.

An important feature of the system is the GLAS step, whereby the DNA of interest is specifically selected by annealing to promoter oligonucleotides on the microarray. This step decreases the sample complexity, thereby increasing the technique’s sensitivity and specificity. ChIP-GLAS has been selected for use by the NIH ENCODE (ENCyclopedia Of DNA Elements) development project to identify functional DNA elements within the human genome (see www.genome.gov/10005107).

Aviva Systems Biology has recently launched a ChIP-GLAS custom service for the simultaneous detection of 20,000 human promoters from a single sample. Researchers can send in a sample — either a cell or tissue sample or chromatin immunoprecipitated DNA samples — and will receive a list of potentially enriched genes in return. Also new this year, according to Julian Yuan, CEO of Aviva, is the release of more than 1,000 new antibodies for human and mouse transcription factors.

Transcribing Knowledge
An important aspect of any genomic analysis — whether expression profiling or analysis of DNA-binding elements as above — is the integration of the data with existing knowledge. Genomatix is a privately held company located in Munich, Germany, that offers software, databases, and services aimed at understanding gene regulation at the molecular level. Genomatix’s suite of products aims to facilitate understanding of gene regulation by integrating data and knowledge into a regulatory encyclopedia to facilitate annotation-driven microarray analysis, regulatory network and pathway mining, and promoter analysis at the sequence level.

Genomatix has recently released a TF knowledge database called MatBase. MatBase contains information on genomic TF binding sites and binding domains and related literature. It includes information on more than 27,000 known TF-gene interactions, descriptions of experimentally verified complexes of specific factors with other TFs, and weight matrix descriptions for the DNA binding sites of TFs.

The current MatBase (Release 1.0) contains information on 6,620 transcription factor genes from more than 80 different species, including all organisms featured in Genomatix’s Eldorado module. Eldorado contains more than 100 gigabytes of condensed and vetted information on several genomes. It comprises both publicly available data and Genomatix’s proprietary annotation, including promoters, transcription factor binding sites, promoter modules, scaffold/matrix attachment regions, and single nucleotide polymorphisms. The 511 functional promoter modules in MatBase describe compositions of binding sites characterized by a proven regulatory function. The 705 matrices covering 3,277 of the transcription factors correspond to those used by Genomatix’s MatInspector to identify TF binding sites.

All data come with references to scientific publications that can easily be looked up via PubMed. MatBase is available standalone or in combination with MatInspector, either accessible via the Web or as a local in-house installation.

E-mail Robert M. Frederickson at rfreder@yahoo.com.

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