Biology Reference
In-Depth Information
development (
Akkers et al., 2009
), but not on amphibian metamorphosis.
It would be of great interest to derive genome-wide TR-binding profiles
to help understand the underlying mechanistic events of amphibian
metamorphosis.
However, as seducing as it may sound, ChIP-Seq suffers from a major
limitation as it provides no experimental evidence of the target gene(s)
whose transcription is regulated by the DNA-bound transcription factor,
if any. At best, one assumes that the target gene is located nearby the
DNA-bound transcription factor, and infers such functional connection.
Although this might be true for a few genes, this postulate is clearly not true
genome wide (see below). It is clear that DNA-bound transcriptional
regulators can act over large genomic regions and physically interact with
the basal transcription machinery through DNA looping (
Kim, Bresnick,
& Bultman, 2009
). Also, transcriptional regulators can interact with one
another, thus adding more complexity (
Fullwood, Liu, et al., 2009
).
4.2. A need for 3D analysis
Recent technological breakthrough bypass the ChIP-Seq limitations and
proceed a step further in the analysis of functional interactions between
DNA-bound proteins. Ten years ago, the development of the chromosome
conformation capture (3C) technology allowed to assess physical interac-
tions between chromosome loci (
Dekker, Rippe, Dekker, & Kleckner,
2002; de Wit & de Laat, 2012
).The throughput was quite low in that 3C
was used to detect the physical proximity of two DNA fragments in the nu-
cleus in a “one to one” manner. It also required a prior knowledge of the
genomic locations to probe. The first improvements of the 3C technology
resulted in the circularized chromosome conformation capture (4C) tech-
nique, which allows to probe physical interactions in a “one to many” man-
ner (
Simonis et al., 2006; Zhao et al., 2006
). In 2009, the Genome Institute
of Singapore developed the chromatin interaction analysis by paired-end
tags sequencing (ChIA-PET) (
Fullwood, Liu, et al., 2009; Fullwood &
Ruan, 2009
), which combines the principles of ChIP-Seq and 4C, and pro-
duces two lines of evidence from a single experiment: a genome-wide bind-
ing profile together with a map of the physical interactions between protein-
bound DNA regions. It is important to note that this method only reveals if
two regions of DNA are close to each other but does not tell which or if any
proteins is involved in the interaction. It does not either reveal any physical
interactions among proteins, especially if no other proteins are analyzed. The
