MADISON, Wis., June 13, 2007 — The Encyclopedia of DNA Elements (ENCODE) consortium today published a set of papers in Nature and Genome Research that sum the work of more than four years of effort dedicated to understanding the functional elements of the human genome – a step toward the goal of using such information to diagnose, treat and prevent disease. NimbleGen Systems, Inc. played a primary role in this international discovery effort, both as the chosen chromatin immunoprecipitation microarray (ChIP-chip) platform for a majority of investigators who participated in this study and as the recipient of $2.5 million in ENCODE research grants. At least 15 papers have been published to date by ENCODE participants using the NimbleGen platform.¹

"With the innovative microarray technologies that NimbleGen provides, we have been able to identify the transcription factor binding sites and chromatin modifications along the human genome in multiple cell types," stated Dr. Bing Ren, assistant professor at the University of California, San Diego School of Medicine and Ludwig Institute for Cancer Research (LICR), and participant in the ENCODE consortium. "The high quality data from such experiments have allowed us to annotate promoters, enhancers, insulators and other transcriptional regulatory elements in the human genome and examine the mechanisms that drive cell type-specific gene expression programs in human cells. We anticipate that this information will provide a valuable resource for study of human development, disease process and evolution."

Begun in September 2003 by the National Human Genome Research Institute (NHGRI), the ENCODE project (www.genome.gov/ENCODE) picks up the search for understanding the human genome where the Human Genome Project (www.genome.gov/10001772) left off. Composed of scientists from 35 groups within 80 organizations in government, industry, and academia across the world, ENCODE is dedicated to producing a comprehensive catalog of elements crucial to biological function in the roughly 98 percent of the human genome that does not code for proteins. Approximately 1% of the human genome, or 30 million DNA base pairs, was selected for in-depth investigation; the targets were strategically selected to provide a representative cross section of the entire human genome.

The results have led researchers to rethink some long-held views about what genes are and what they do, as well as how the genome's functional elements have evolved.

The ENCODE consortium's major findings include the discovery that the majority of DNA in the human genome is transcribed into functional molecules, called RNA, and that these transcripts extensively overlap one another. This broad pattern of transcription challenges the long-standing view that the human genome consists of a relatively small set of discrete genes, along with a vast amount of so-called "junk" DNA that is not biologically active. The new data indicate the genome contains very little unused sequences and is a complex, interwoven network.

Other highlights of the ENCODE work include:

• Identification of numerous previously unrecognized start sites for DNA transcription.
• Evidence that, contrary to traditional views, regulatory sequences are just as likely to be located downstream of a transcription start site on a DNA strand as upstream.
• Identification of specific signatures of change in histones, which are the proteins that organize DNA, and correlation of these signatures with different genomic functions.
• Deeper understanding of how DNA replication is coordinated by modifications in histones.

All data generated by ENCODE participants was rapidly released into public databases. The main portal for ENCODE data is the University of California, Santa Cruz's ENCODE Genome Browser.

NimbleGen's ChIP-chip technology was the primary ChIP-chip platform for ENCODE research on regulation of gene expression. "We chose NimbleGen because of its ability to produce arrays with long oligonucleotide probes, which allows for higher sensitivity and specificity. The capacity for flexible design and high probe density also made the NimbleGen platform a good choice," stated Dr. Jason Lieb, assistant professor in the Department of Biology at University of North Carolina.

Lieb and researchers at UNC developed a method for rapidly mapping regions of a wide range of genomes that are depleted of nucleosomes, a hallmark of gene activity. Using a method termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements), the group was able to distinguish active and inactive regions of the genome and compare these patterns between different cell types. The method utilizes NimbleGen high-density DNA arrays to map regions of chromatin activity at high resolution, genome wide. Distinguishing patterns of chromatin activity provides insight on cellular regulatory processes in both normal and diseased cells.

"Our experiments proved that FAIRE, a new technology developed under the ENCODE project, is a simple and effective method for high-throughput mapping of open chromatin throughout the human genome," said Lieb. "The high quality of the NimbleGen data allowed us to create the open chromatin maps quickly and at high confidence with relatively few experiments."

Another ENCODE grant recipient, Peggy Farnham and researchers at the University of California, Davis Genome Center, used NimbleGen ChIP-chip technology to analyze how human cells regulate gene expression by identifying protein binding sites in the genome at high resolution. In recent work, the group focused on identifying partners for a key regulatory protein (OCT4) that controls how stem cell develops into many different cell types. They found that in testicular embryonal carcinoma cells, the protein SRY works together with OCT4 to control gene expression in a cooperative manner. This is an important first step towards our understanding of the intricate network that controls how and when cells develop, and it could lead to new insights in stem cell therapy for many human diseases.

"We chose to use NimbleGen arrays for our ENCODE research due to the high sensitivity and reproducibility of the ChIP-chip data," Farnham stated. "Also, the high signal-to-noise ratio obtained using NimbleGen arrays enabled us to easily compare binding patterns of different transcription factors and allowed us to demonstrate a striking correspondence between binding of E2F family members and RNA polymerase II."

As the ENCODE project moves into the production phase, NimbleGen's ChIP-chip technology will be applied to analysing regulation of gene expression in the remaining 99% of the human genome. According to the ENCODE consortium, one of the goals of this project is to establish a reference base against which scientists will subsequently compare variation in regulation of gene expression involving thousands of different factors, in hundreds of target tissues, under numerous conditions.

In May 2007, the NHGRI expanded the scope of the ENCODE project to include identifying the functional elements in the genomes of the fruit fly (Drosophila melanogaster) and round worm (Caenorhabditis elegans) (www.genome.gov/25521166). The scientific community relies heavily on these model organisms to identify common genes, regulatory sequences and processes that underlie human conditions. Four out of the five groups performing ChIP research in this project, dubbed model organism ENCODE, or modENCODE, are using NimbleGen ChIP microarrays.

¹References:

The ENCODE Project Consortium. 2007. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 13 June [epub ahead of print]. (doi:10.1038)

Jin, V.X. et al. 2007. Identification of an OCT4 and SRY regulatory module using integrated computational and experimental genomics approaches. Genome Res. 17: 807-817. (doi:10.1101/gr.6006107)

Emanuelsson, O. et al. 2007. Assessing the performance of different high-density tiling microarray strategies for mapping transcribed regions of the human genome. Genome Res. 17: 886-897. (doi:10.1101/gr.5014607)

Euskirchen, G.M. et al. 2007. Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies. Genome Res. 17: 898-909. (doi:10.1101/gr.5583007)

Dennis, J.H. et al. 2007. Independent and complementary methods for large-scale structural analysis of mammalian chromatin. Genome Res. 17:928-939. (doi:10.1101/gr.5636607)

Giresi, P.G. et al. 2007. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. Genome Res. 17: 877-885. (doi:10.1101/gr.5533507)

King, D.C. et al. 2007. Finding cis-regulatory elements using comparative genomics: Some lessons from ENCODE data. Genome Res. 17: 775-786. (doi:10.1101/gr.5592107)

O'Geen, H, et al. 2006. Comparison of sample preparation methods for ChIP-chip assays. BioTechniques. 41(5): 577-580.

Jin, VX, et al. 2006. A computational genomics approach to identify cis-regulatory modules from chromatin immunoprecipitation microarray data – A case study using E2F1. Genome Res. 16(12):1585-95.

Sabo, PJ, et al. 2006. Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nature Methods. 3(7) 511-18.

Crawford, GE, et al. 2006. DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nature Methods, 3(7) 503-9.

Squazzo, SL, et al. 2006. Suz12 binds to silenced regions of the genome in a cell-type-specific manner. Genome Res. 16:890-900.

Bieda, M, et al. 2006. Unbiased location analysis of E2F1-binding sites suggests a widespread role for E2F1 in the human genome. Genome Res. 16:595-605.

Kirmizis A, et al. 2004. Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes Dev. 18(13):1592-605.

Feingold, EA, et al. 2004. The ENCODE (ENCyclopedia Of DNA Elements) Project. The ENCODE Project Consortium. Science. 306(5696):636-640.

About NimbleGen Systems, Inc.

For more information about NimbleGen, please visit the company's website at www.nimblegen.com