The Rome Oncogenomic Center (ROC), together with AIRC, has organized the ‘First ROC international workshop and practical course on chromatin immunoprecipitation related techniques’. The workshop has involved a practical course focused on current methods for the analysis, on a genome-wide scale, of chromatin remodeling and molecular complexes identification in vivo. During the course, the students (36) have learned how to address these topics from a standpoint and how this field has progressed in the last years from a scientific, technological and bioinformatic point of view.
As documented in several talks, a great effort has been devoted in the last 2 years to develop microarray technologies for understanding protein–DNA binding events (ChIP-on-chip). Different technologies are used by different groups. By analyzing genome-wide target site profiling of several transcription factors through the whole-genome tiling array NimbleGen platform, Henk Stunnenberg (University of Radboud, Nijmegen, The Netherlands) has shown how TFs regulate their target genes mostly from distant DNA regions rather than proximal promoters. Motif searches revealed that a rather large number of the in vivo target sites did not contain the expected cis-acting sequence, indicating that transcriptional regulation frequently involves crosstalk with other factors. Similar results have been reached by Myles Brown (DFCI/HMS/HSPH, Boston, USA). To identify the complete set of steroid receptor regulatory sites, he has undertaken an unbiased approach combining ChIP with Affimetrix oligonucleotide microarrays tiled at high density across the entire human genome. He coined the term CISTROME to define the set of cis-regulatory targets of trans-acting factors across the entire genome. With this technology he has identified 3365 ER target sites, most of them in regions previously unrecognized as cis-regulatory elements, suggesting that studies focusing only on promoter regions are insufficient. In contrast, he showed that RNA PolII is significantly biased to promoter-proximal regions and concluded that the communication between the transcription factors and the basal transcription machinery is often mediated at long distances. To gain an unbiased view of in vivo NF-Y binding, Roberto Mantovani (ROC, Milan, Italy) performed ChIP-on-chip experiments on the chromosome 21 tiling array Nimblegen platform and found 400-600 NF-Y binding sites. Only 20% of the identified sites reside in the promoters. The vast majority of the sites are contained within transcribed regions (50%), with the remaining 30% located far from annotated genes. One type of array suitable for Chip-on-chip takes advantage of the strong association between CpG islands (CGIs) and gene regulatory regions. Linda Penn (University of Toronto, Canada) has obtained 20736 clones from a CGI Library and used these to construct CGI arrays containing 9595 distinct genomic loci. Using these slides to study Myc target genes, she has optimized key parameters for the ChIP-on-chip assay. She observed that the dye-swap, as well as the combination of sample versus input rather than sample versus negative control, does not play an important role in the result outcomes. Conversely, antibody quality, array batch, amplification and hybridization need to be tightly controlled. She conducted a large-scale comparison of different algorithms for ChIP-on-chip data analysis, reporting that part of the data interpretation depends on the in silico analysis. The contribution of Shirley Liu (DFCI/HSPH, Boston, USA) was specifically focused on the in silico analysis of ChIP-on-chip results. She developed a series of algorithms for the analysis of ChIP-on-chip on genome tiling microarrays, including (a) microarray blob remover (MBR) to filter probes in blob defects on the array, (b) an algorithm for eXtreme fast MApping of Nucleotide (XMAN) probes to the genome, (c) a model-based analysis of tiling arrays (MAT) that models probe baseline behavior from probe sequence and genome copy numbers, and (d) a web cis-element annotation system (CEAS) for a comprehensive annotation of TF-bound ChIP regions in the genome. Using ChIP-on-chip technology, Xavier Gidrol (CEA, Evry, France) demonstrated that E2F transcription factors are able to regulate various cellular processes and are subjected to a functional plasticity during differentiation of human keratinocytes. He used a DNA microarray containing promoter regions of 12000 human genes (−800/+200 bp relative to the start site). In order to map the replication time zones of the mouse, Itamar Simon (University of Hebrew, Jerusalem, Israel) isolated newly replicated DNA at distinct time points during S phase using a synchronization methodology that he called ‘baby machine’. DNA replication at each time point was detected by hybridization to high-density genomic microarrays. He observed that housekeeping genes as well as genes involved in apoptosis and stress responses are replicated early, while tissue-specific genes are replicated late. In order to identify potential correlation between the stress–response and the nuclear location of ERp57/GRP58, Silvia Chicchiarelli (University of Rome, Italy), one of the three selected speakers in this section, cloned the immunoprecipitated DNA fragments and localized them in the human genome. To study how Stat2 (the IFNα-specific component of ISGF3) modulates transcription, Barbara Testoni (ROC, Rome, Italy) built an oligo-based array containing 113 putative IFNα target promoter regions and performed ChIP-on-chip experiments with anti-Stat2 and the activated form of Stat2 antibodies before and after IFNα treatment in hepatoma cells. This allowed her to describe a scenario of Stat2 recruitment to ISG promoters, which greatly extends previous knowledge. To validate the ChIP-on-chip data, she utilized the Applied Biosystems real-time PCR cards to customize an expression assay containing 47 ISGs. With the aim of identifying a specific transcriptional signature for gain-of-function of mutant p53 proteins, Giulia Fontemaggi (ROC, Rome, Italy) described an oligonucleotide array for ChIP-on-chip, enclosing 152 putative mutant p53 targets.
