ChIA-PET

By creating ChIA-PET interactome maps for DNA-binding regulatory proteins and promoter regions, we can better identify unique targets for therapeutic intervention (Fullwood & Yijun, 2009).

While ChIP-Seq is able to identify TFBS genome-wide,[4][5] it provides only linear information of protein binding sites along the chromosomes (but not interactions between them), and can suffer from high genomic background noise (false positives).

While 3C is capable of analyzing non-linear, long-range chromatin interactions, it cannot be used genome wide and, like ChIP-Seq, also suffers from high levels of background noise.

The next step, which is referred to as enrichment, reduces complexity for genome-wide analysis and adds specificity to chromatin interactions bound by pre-determined TFs (transcription factors).

ChIA-PET takes advantage of this concept by incorporating linker sequences onto the free ends of the DNA fragments tethered to the protein complexes.

[8] Compared to Hi-C, the use of an antibody pulldown limits the number of sequenced fragments to chromatin interactions bound by the protein of interest which also can ease the data analysis.

Proposed mechanism showing how distal regulatory elements can initiate long-range chromatin interactions involving promoter regions of target genes.

Small loops might package genes near the anchoring center in a tight sub-compartment, which could increase the local concentration of regulatory proteins for enhanced transcriptional activation.

This mechanism might also enhance transcription efficiency, allowing RNA pol II to cycle the tight circular gene templates.

The original ChIP method is an antibody-based technology that identify and bind proteins selectively in order to offer information regarding chromatin states and gene transcription.

Figure 1. Biotinylated universal linkers with Mme1 restriction endonuclease sites are introduced.
Figure 2. Biotinylated universal linkers are ligated to the free DNA ends.
Figure 3. Ligation of linkers during proximity ligation.
Figure 4. Pull down of biotinylated linkers by streptavidin-beads, and amplification of DNA tags.
Figure 5. Conformations of universal linkers.
Figure 6. Intra and inter-ligated PETs are clustered around TFBS when mapped to the reference human genome.
Figure 7. Proposed DNA looping mechanism between distal regulatory proteins and the promoter region
Chromosome conformation techniques