Table 1 Genome-scale experimental methods to probe protein–DNA interactions

Adapted from [85].

ChIP-chip and ChIP-seqThe DNA-binding protein is tagged with an epitope and is expressed in a cell. The bound protein is covalently linked to DNA using an in vivo cross-linking agent such as formaldehyde. After cross-linking, DNA is sheared and the protein–DNA complex is pulled down using a tag-specific antibody. Reversal of the cross-link releases the bound DNA, allowing the sequence of the fragments to be determined by hybridization to a microarray (ChIP-chip) or by sequencing (ChIP-seq).
In ChIP-chip experiments, intergenic regions are spotted on to a microarray chip. Following the ChIP step, the cross-links are reversed and the bound DNA is hybridized on to the chip. Complementary sequences will bind to specific spots on the chip, thereby providing an indication of the intergenic region to which the protein was bound [10,11,242].
In ChIP-seq experiments, the bound fragments are sequenced directly using 454/Solexa/Illumina sequencing technology. The sequences are then computationally mapped back to the genome sequence. Fragments that were bound by the protein will be sequenced several times providing a direct measure of enrichment of binding [243245].
DamID [(Dam (DNA adenine methyltransferase) identification]To overcome any potential non-specific cross-linking of protein to DNA as could happen with ChIP-chip experiments, the DamID technique was introduced. The protein of interest is fused to an E. coli protein, Dam. Dam methylates the N6 position of the adenine in the sequence GATC, which occurs at reasonably high frequency in any genome (~1 site in 256 bases). Upon binding DNA, the Dam protein preferentially methylates adenine in the vicinity of binding. Subsequently, the genomic DNA is digested by the DpnI and DpnII restriction enzymes that cleave within the non-methylated GATC sequence, and remove fragments that are not methylated. The remaining methylated fragments are amplified by selective PCR and quantified using a microarray [246].
PBMs (protein-binding universal DNA microarrays)In contrast with the methods described above, this is an in vitro method to probe protein–DNA interactions. A DNA-binding protein of interest is epitope-tagged, purified and bound directly to a double-stranded DNA microarray spotted with a large number of potential binding sites. Labelling with fluorophore-conjugated antibody for the tag allows detection of binding sites from the significantly bound spots [247].