DNA microarray technologies for measuring protein-DNA interactions

Abstract

DNA-binding proteins have key roles in many cellular processes, including transcriptional regulation and replication. Microarray-based technologies permit the high-throughput identification of binding sites and enable the functional roles of these binding proteins to be elucidated. In particular, microarray readout either of chromatin immunoprecipitated DNA-bound proteins (ChIP-chip) or of DNA adenine methyltransferase fusion proteins (DamID) enables the identification of in vivo genomic target sites of proteins. A complementary approach to analyse the in vitro binding of proteins directly to double-stranded DNA microarrays (protein binding microarrays; PBMs), permits rapid characterization of their DNA binding site sequence specificities. Recent advances in DNA microarray synthesis technologies have facilitated the definition of DNA-binding sites at much higher resolution and coverage, and advances in these and emerging technologies will further increase the efficiencies of these exciting new approaches.

Figure 1. ChIP-chip

(a) Types of DNAs used in ChIP-chip. Oligonucleotide tiling genomic regions (left); PCR amplification of genomic regions (right). (b) ChIP-chip experimental design. A large variety of protein-DNA and protein-protein crosslinks are created nonspecifically, due to the nonspecific nature of formaldehyde crosslinking. An antibody (orange) either specific for the protein of interest (blue) or specific for an epitope tag fused to the protein of interest is used in immunoprecipitation (IP) in the experimental sample. This IP will enrich for the target protein, including protein directly bound to genomic DNA binding sites, and also protein indirectly associated with DNA via protein-protein interactions. A control (“mock”) IP is performed using either no antibody, an irrelevant antibody, or pre-immune IgG antibodies. This mock IP is not expected to enrich for the target protein of interest.

Figure 2. DamID

(a) Types of DNAs used in DamID microarray hybridizations. Oligonucleotide tiling genomic regions (left); cDNAs or PCR amplification of genomic regions (right). (b) DamID experimental design. The protein of interest (blue) is overexpressed in vivo from a plasmid as a fusion to Dam (purple). Wherever the protein binds DNA, Dam will methylate adenines within GATC sites in the vicinity of the binding sites. The methylated sites are digested with the methyl-specific restriction enzyme DpnI, which cuts only at methylated GATC sites. The smaller DpnI digestion fragments, corresponding to the methylated regions, are either purified by sucrose gradient centrifugation or specifically amplified using a methylation-specific PCR protocol. (Panel (b) was adapted from [7] and [51] with permission from Nature Publishing Group.)

Figure 3. Protein binding microarrays (PBMs)

(a) Types of DNAs used in PBMs. Short double-stranded oligonucleotides created by primer extension using a primer (red), complementary to a sequence present on spots either on an oligonucleotide array [8] or in solution [9, ··10] (left); regions of short double-stranded DNA created by self-hairpinning oligonucleotides on an oligonucleotide array [··50] (center); longer double-stranded DNAs resulting from PCR amplification of genomic regions [··10] (right). (b) PBM experimental design. Double-stranded DNA microarrays can be either bound by an epitope-tagged TF and labeled by a fluorophore-conjugated (green) antibody specific for the tag [··10] (left), or bound by a directly fluorophore-labeled (green) TF [··50] (right). (Panel (b) was adapted from [··10] with permission from Nature Publishing Group.)