DNase-chip is a robust method to identify DNase I hypersensitive sites by hybridizing DNase I–treated, end-captured material to tiled microarrays. This method has many of the same strengths of traditional Southern blot strategies for identifying regulatory regions. By using three DNase I concentrations, the ability to distinguish DNase I hypersensitive sites that display different levels of sensitivity is enhanced. The protocol for DNase-chip is straightforward, sensitive and specific at identifying valid DNase I hypersensitive sites.
Although we have validated the approach in the ENCODE regions, this method can make use of custom tiled microarrays to readily focus on any portion of the genome from any organism. The method is fully scalable and should be amenable to whole-genome scans, as was recently done with ChIP-chip13
One of our original concerns regarding mapping DNase I hypersensitive sites in replicating cells was that replicating DNA forks would introduce background5,14
. This was an early justification for using CD4+
T cells, which are nonreplicating when derived by aphoresis. Even though there were slightly elevated background levels of DNase I digestion in the cycling GM06990 lymphoblastoid cell line, this method appears to be quite effective regardless of the status of cell division. But to reduce the background even further, it may be helpful to synchronize or block cell division.
As with any array-based method, DNase-chip has limitations. First, the resolution size for DNase-chip is limited by the extent of sonication-based shearing (about 200–500 bp). But because DNase I hypersensitive sites are typically around 250 bp in size, we believe this level of resolution is acceptable. Second, tiled microarrays exclude repetitive DNA. Therefore, combining DNase-chip with other methods, such as MPSS with long sequence reads, might be needed to discover DNase I hypersensitive sites within repetitive elements. Third, presently DNase-chip currently requires a large number of cells (~5 × 107). Finally, the cost of performing DNase-chip is considerable, if performed on the whole genome, but will become more affordable as arrays become less expensive.
The genomic coordinates of all DNase I hypersensitive sites described in this manuscript are publicly available (http://research.nhgri.nih.gov/DNaseHS/chip_2006
). The locations of these DNase I hypersensitive sites correlate well with other annotated regions of the genome known to mark gene regulatory elements, such as 5′ ends of genes, CpG islands and highly conserved sequences. Notably, the lowest density of DNase I hypersensitive sites mapped to gene-poor regions of the genome that were highly conserved between human and mouse. Future studies aimed at this latter set of conserved elements will determine what type of functional elements these regions may represent. Genes with a DNase I hypersensitive site nearby were more likely to have elevated gene expression, but the presence of a DNase I hypersensitive site was not sufficient for higher expression levels. The small number of outliers (genes that have elevated expression levels, but do not have a nearby DNase I hypersensitive site) could be due to the false negative rate of DNase-chip, the presence of repetitive elements nearby the transcription start (thereby not included on the arrays), or an incorrectly mapped transcription start site. We were also surprised that we were unable to detect significant changes in gene expression between CD4+
and GM06990 cells for genes that had a nearby DNase-chip signal present in only one of the two cell types. One explanation for this could be the similar gene expression patterns of these two lymphocyte cell types. Future expression studies using more diverse cell types may help clarify how chromatin structure and gene expression are related.
In the future, whole-genome DNase-chip from several cell types can be used to identify ubiquitous versus cell type–specific DNase I hypersensitive sites. It should also be possible to apply this approach genome-wide to different states of the same tissue, including normal versus diseased, resting versus stimulated, undifferentiated versus differentiated, and untreated versus drug-treated, to identify global changes in regulation. But even though methods such as DNase-chip will help identify the functional regions of the genome, determining the type of regulatory function for each DNase I hypersensitive site remains a daunting challenge. Clues can be gleaned from correlating DNase I hypersensitive sites with sequence conservation, transcription factor binding sites and histone modifications (ChIP-chip), motif discovery, promoter or enhancer activity, DNA methylation and more detailed gene expression analysis. Groups such as the ENCODE consortium are beginning to compare and contrast these global data sets in an effort to better understand how the genome is regulated1
. DNase I hypersensitive site identification can be an important component of such ground breaking efforts to understand the complete function of the genome.