ChIA-PET

手法

ChIA-PET法は、クロマチン免疫沈降法(Kuo & Allis, 1999)および3C法を組み合わせた手法である(The ChIA-PET method combines ChIP-based methods (Kuo & Allis, 1999), and Chromosome_conformation_capture (3C), to extend the capabilities of both approaches.)。
ChIP-Seq法が転写因子結合領域(TFBS)を決定するのに広く用いられる手法である一方、染色体間の大域的相互作用の測定には3C法が用いられていた(Dekker et al., 2002)(ChIP-Sequencing (ChIP-Seq) is a popular method used to identify TFBS while 3C has been used to identify long-range chromatin interactions (Dekker et al., 2002).)。 However, both suffer from limitations when used independently to identify de-novo long-range interactions genome wide. While ChIP-Seq is typically used for genome-wide identification of TFBS (Barski et al., 2007; Wei et al., 2006), it provides only linear information of protein binding sites along the chromosomes (but not interactions between them), and suffers from high genomic background noise (false positives). Additionally, only a small amount of sequences generated by ChIP-Seq uniquely map to the genome, and an even smaller amount are functional TFBS (Johnson et al., 2007).
While 3C is capable of analyzing long-range chromatin interactions, it cannot be used genome 
wide and, like ChIP-Seq, also suffers from high levels of background noise. Since the noise increases in relation to the distance between interacting regions (max 100kb), laborious and tedious controls are required for accurate characterization of chromatin interactions (Dekker et al., 2006).
The ChIA-PET method successfully resolves the issues of non-specific interaction noise found in ChIP-Seq by sonicating the chip fragments in order to separate random attachments from specific interaction complexes. 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). 
The ability of 3C approaches to identify long-range interactions is based on the theory of proximity ligation. In regards to DNA inter-ligation, fragments that are tethered by common protein complexes have greater kinetic advantages under dilute conditions, than those freely diffusing in solution or anchored in different complexes. ChIA-PET takes advantage of this concept by incorporating linker sequences onto the free ends of the DNA fragments tethered to the protein complexes. In order to build connectivity of the fragments tethered by regulatory complexes, the linker sequences are ligated during nuclear proximity ligation. 
Therefore, the products of linker-connected ligation can be analyzed by ultra-high-throughput PET sequencing and mapped to the reference genome. Since ChIA-PET is not dependent on specific sites for detection as 3C and 4C are, it allows unbiased, genome-wide de-novo detection of chromatin interactions (Fullwood et al., 2009).

歴史

Fullwood et al. (2009), used ChIA-PET to detect and map the chromatin interaction network mediated by oestrogen receptor alpha (ER-alpha) in human cancer cells. The resulting global chromatin interactome map revealed that remote ER-alpha-binding sites were also anchored to gene promoters through long-range chromatin interactions suggesting that ER-alpha functions by extensive chromatin looping in order to bring genes together for coordinated transcriptional regulation.