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Chip-On-Chip Gene Regulation APPLICATION FOCUS: ChIP-on-chip Gene Regulation Chromatin immunoprecipitation (ChIP) is an effective tech- TABLE OF CONTENTS nique for enriching and identifying genomic DNA ChIP Applications 2 sequences bound by regulatory proteins, as well as iden- The ChIP-on-chip Toolbox 5 tifying sites of histone and DNA modification. The full Data Analysis Solutions 7 power of the application, however, is only realized when References 8 combined with very high-density, array-based detection. This allows interrogation of entire genomes at very high CRITICAL FEATURES resolution and has already revealed a wealth of essential Why are researchers choosing Affymetrix products for whole- information about regulatory activity in non-coding and genome ChIP-on-chip analysis? intergenic regions. ■ Lowest number of arrays possi- ble to cover the genome of It is no longer sufficient, or necessary, to limit regulatory studies to promoter re- interest gions or defined genomic loci. An unbiased, whole-genome approach reveals the full regulatory network activity of transcription factors and epigenetic modifications. ■ High-resolution probe design to Affymetrix' unique high-density tiling arrays accommodate 6.4 million features per pinpoint multiple data points array, allowing whole-genome (human or mouse) coverage on just a few arrays, de- per binding region, resulting in livering high-performance, cost-effective whole-genome ChIP-on-chip analysis. high accuracy and sensitivity ■ Easy-to-use tools for prelimi- nary and advanced data analysis Enhancer DNA methylation Activator Transcription Factor RNA Polymerase DNA Coding Region TATA Box Figure 1: The total genome activity of transcription factors and epigenetic modifications are far more complex than previously predicted. APPLICATION FOCUS: ChIP-on-chip Only Affymetrix high-density arrays Previous ChIP-on-chip experiments make whole-genome ChIP-on-chip ex- using focused arrays (as opposed to periments affordable and practical to whole- genome arrays) have traditionally Affymetrix whole-genome ar- conduct today—using just a single array analyzed candidate genes, annotated rays have already been used to for model organisms, and seven-array promoter regions or customized loci. sets for human and mouse. These These approaches have revealed only a map sites for transcription fac- whole-genome products allow you to partial picture of the total genome activ- tor binding, and DNA and his- analyze all coding and non-coding re- ity of key transcription factors and epi- tone modification, revealing gions for an unbiased view of the genetic modifications. genome, which is especially important key information about the func- With 25-mer probes spaced every 35 as non-coding regions contain several base pairs (bp) across an entire tions of various transcription enhancer/repressor binding sequences. genome, Affymetrix Tiling Arrays offer Single arrays from mouse and human regulators (including estrogen complete and unbiased genome cov- whole-genome sets are also available receptor [ER] and p53), and erage on a very small number of ar- for your initial assay development and rays. They offer the only practical, helping identify target genes optimization. Your initial experiments cost-effective and scalable approach give a realistic chromosome-wide view involved in disease progression to gene regulation analysis available of activity, and can be easily scaled up to date. and development—insights that for whole-genome analysis. Start your analysis with a single array could not have been made Recent reports have demonstrated that from a set, and confidently scale up to using focused arrays. epigenetic and transcription regulatory the entire genome, using the seven-array networks are far more complex than pre- set. No need to repeat large amounts of viously predicted, with individual tran- work—whole-genome analysis is afford- scription factors binding to thousands of Affymetrix Offers Unique Off- able and practical now. sites across the genome, and with much the-shelf Tools for Advanced of the regulatory activity located at distal Gene Regulation Analysis, sites far from promoter regions 5, 6. Which Provide Unrivalled Genome Coverage ■ Content: Over 6.4 million fea- tures per array for human and APPLICATIONS FOR IP-BASED ANALYSIS OF GENE REGULATION mouse sets The basics of Chromatin IP make it applicable to the study ■ Simplicity: Low number of arrays for human and mouse of any protein interaction with genomic DNA, or DNA or whole genomes, single whole- histone modification, provided an antibody exists that is genome arrays for other organ- isms specific for that protein or modification, and is suitable for ■ Resolution: 35 base pairs from immunoprecipitation. midpoint to midpoint of adja- cent oligos Protein-DNA complexes are chemically comparison with non-enriched sam- ■ Specificity: 25-mer probes de- cross-linked, then purified from the cell ples for identification of all binding se- liver high sensitivity with a low and immunoprecipitated using an anti- quences—known and novel. incidence of false positives in body specific for the regulatory protein initial tests The ChIP-on-chip assay can be used to or modification under scrutiny. The examine gene regulation by both epige- DNA in the enriched fraction can be netic modifications, such as histone amplified using real-time PCR to con- and/or DNA modification, and by tran- firm the presence of expected se- scription machinery, such as transcrip- quences, and then applied to arrays for 2 Application Focus: ChIP-on-chip APPLICATION FOCUS: ChIP-on-chip tion factor binding. ChIP-on-chip has sites. Dr. Kevin Struhl, an author of the also been used to study other protein- paper, discussed this work in the Octo- mediated processes such as DNA repli- ber 2005 issue of the Affymetrix Mi- “The combination of this unique cation and repair. croarray Bulletin. resource [whole-genome ChIP- Using a human whole-genome set Mapping Transcription Factor from Affymetrix, Yang, et al. (2006) ex- on-chip analysis at 35-bp resolu- Binding Sites amined the global binding behavior of tion] with gene expression data Recent evidence shows that many p63 and the evolutionary conservation transcription protein-binding sites in of p63 binding sites. sets serves to elucidate the the genome are actually in non-coding mechanisms underlying estro- regions once considered “junk” DNA, Mapping Histone Modifications and can sometimes be up to 100 kilo- gen-regulated gene expression Bernstein, et al. (2005) used Affymetrix bases (kb) from the gene they regulate. Tiling Arrays to map the location of two In addition, transcription factors are in breast cancer.” Carroll, et al. (2006) methylated lysines in the histones of more active than previously thought, embryonic stem cells. This modifica- with a high number of binding sites, tion pattern appears to be required to not always predictable from either silence developmental genes while canonical promoter regions or keeping the cells poised to differenti- “consensus motif” searches. ate. An interview with Dr. Bernstein Carroll, et al. (2006) published a discussing this work can be found in genome-wide map of estrogen recep- the October 2006 issue of the tor and RNA Polymerase II binding Affymetrix Microarray Bulletin. sites in breast cancer cells. The study Bernstein, et al. previously used tiling identified cis-binding sites in previously arrays to map histone methylation and unexplored regions of the genome, acetylation across non-repetitive por- and investigated cooperation between tions of human chromosome 21 and 22 transcription factors in estrogen signal- in both the human and mouse ing associated with breast cancer. Q- genomes. Bernstein’s team deter- PCR validation of 15 randomly selected mined that sites of trimethylation cor- binding sites confirmed ER recruitment Dr. Myles Brown in the October 2005 edition related with transcription start sites to all of the ChIP-on-chip identified of the Affymetrix Microarray Bulletin. and that methylation sites are strongly sites and none of the negative con- conserved between the two species. trols. Dr. Myles Brown, an author of Distribution of All TFBS Regions the paper, discussed this work in the Mapping DNA Methylation Pseudogene/ 5’ to a Known October 2005 issue of the Affymetrix Ambiguous Gene Microarray Bulletin (AMB), a magazine Zhang, et al. (2006) recently reported that highlights key microarray-based re- the first genome-wide analysis of DNA search from around the world. methylation in Arabidopsis thaliana at 35-bp resolution, using Affymetrix Tiling Cawley, et al. (2004) used Affymetrix Arrays. Using an anti-methylcytosine an- Tiling Arrays to map binding sites for tibody to precipitate methylated frag- the transcription factors Sp1, cMyc ments of the genome, the study found and p53. Thousands of previously un- that 19 percent of the Arabidopsis known transcription factor-binding sites Within 3’ or Flanking genome is methylated. They unexpect- Novel were identified—only about 22 per- to a Known Gene edly found that 35 percent of expressed cent of which were located at the 5’ Figure 2: Cawley, et al. (2004) Unbiased genes contain some methylation within end of protein-coding genes—disprov- mapping of transcription factor binding coding regions, while around 5 percent ing common assumptions about the sites along human chromosomes 21 and show methylation in promoter regions. location of transcription factor-binding 22 points to widespread regulation of noncoding RNAs. Cell 116, 499-509. Application Focus:
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