Biology Reference
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experimental procedure is quite complex and is aimed at capturing DNA-
bound proteins engaging physical contacts with other protein-bound DNA
complexes. The procedure can be summarized as follows (
Fig. 10.7
): DNA-
bound protein complexes are captured by immunoprecipitation with a spe-
cific antibody, following chromatin fragmentation (this step is similar to that
of a regular ChIP). However, as opposed to conventional ChIP, cross-
linking is not reversed after this step. Instead, a double-stranded DNA linker
containing the binding site of the restriction enzyme
Mme
1, which cuts
DNA 27 bp downstream of its binding site is ligated to each end of the
DNA fragment. The DNA fragments are then ligated. Two kinds of ligation
events can take place:
cis
-ligation (self-ligation of a DNA fragment, provid-
ing ChIP-Seq like data but no information on long range interactions) and
trans
-ligation (ligation between two fragments far from each other on the
genome but brought close to each other within the large protein-DNA
complex). After ligation, the complexes are de-cross-linked, and the ligated
DNA fragments are restricted with
Mme
1 to produce fragments containing
pairs of ends ligated to the linker. After amplification and deep sequencing,
they are mapped to the reference genome. One of the strengths of ChIA-
PET relative to 3C and 4C is that it can probe “many to many” interactions
of DNA regions associated with the protein, be it via interactions of the pro-
tein with other transcription factors (
Fullwood, Liu, et al., 2009
) or core
components of the chromatin such as histones or other factors such as
insulator-binding proteins (CCCTC-binding protein) (
Chepelev, Wei,
Wangsa, Tang, & Zhao, 2012; Handoko et al., 2011
). As a consequence,
in addition to profiling DNA-bound transcription factors, ChIA-PET also
provides experimental evidence for physical interactions between DNA re-
gions far away from each other, and thus can unambiguously identify direct
target genes.
Using ChIA-PET on TR would help document with unprecedented
details the topology and dynamics of regulatory networks of TH regulated
genes. Importantly, this would not only provide a genome-wide map of
TR binding but also identify direct TR target genes. Perhaps most of
all, ChIA-PET will provide us with the opportunity to go beyond the local
chromatin organization and probe higher orders of chromatin architecture.
Our preliminary data show that indeed, TR can act over large genomic
distances and that the local chromatin context might be marginally relevant
to understand the mechanisms of TH-induced transcription cascades
(unpublished data).
