Genomewide identification of transcription factor binding sites by DNAseI footprinting
The complement of DNA-binding proteins and their occupancy of sites throughout the genome determine an organism’s programs of gene expression, DNA replication and other chromosome-based processes. A detailed picture of factor binding on a genome-wide basis exists for Saccharomyces cerevisiae, obtained by a combination of transcriptional profiles, chromatin immunoprecipitation of more than 200 transcription factors, computational analyses and other assays. In an alternative approach, we have used digestion of chromatin by DNase I followed by high throughput DNA sequencing to identify sites of increased nuclease accessibility throughout the yeast genome. The resulting set of more than 10 million sequence reads provides both a global view of chromatin architecture as well as a gene-by-gene view of regulatory sequences protected from digestion by the presence of bound proteins. Unlike the case with results from chromatin immunoprecipitation, these gene-by-gene DNase I footprints can be used to directly identify transcription factor binding sites, and thereby infer their motifs. We found previously unknown binding sites in the genome for well-characterized factors, and observed other annotated binding sites that appear not to be protected from nuclease digestion under our conditions. This approach has the potential to characterize the transcriptional regulatory network of a poorly characterized organism given only its genome sequence.
•Jay Hesselberth, Zhihong Zhang
Functional Chromosomal Interactions
Recent advances have led to complete genome sequences for an increasing number of organisms. In contrast, for any single organism we understand significantly less about how these sequences and the chromosomes they compose are arranged within the nucleus. We also do not understand how the positions of individual genetic loci respond to or establish changes in gene expression.
It is increasingly clear that nuclear location does, indeed, matter (Cell 132:929). However, studying single loci in isolation is not sufficient to gain a complete understanding of the contribution of nuclear organization to gene expression. Therefore it is necessary to determine spatial relationships between genetic elements as they occur in vivo. Spatial relationships between genetic elements are most commonly determined using a technique called chromosome conformation capture (3C) (Science 295:1306).
The compact and well-annotated genome of the unicellular model eukaryote Saccharomyces cerevisiae makes it well suited for 3C analysis. Additionally, years of careful study have precisely measured changes in transcriptional output due to treatment of S. cerevisiae with exogenous factors. We hope to couple 3C and high throughput DNA sequencing technologies to determine how the relative positions of a subset of loci change in response to transcriptional activation.
•Kevin Schultz
