Biology Reference
In-Depth Information
The canonical T
3
RE sequence is well defined and it could be tempting
to perform an exhaustive search of this motif genome wide with tools such as
NHRscan (
Sandelin & Wasserman, 2005
). Unfortunately, given its small
size and degeneracy, search for this motifs will be flooded by strong statistical
noise (
Fig. 10.6
A). For example, more than 200,000 DR4 could be found
across the genome. However, it is improbable that there are this many bind-
ing sites across the
X. tropicalis
genome, given that so far, the number of nu-
clear receptor binding site in a genome has been estimated between 2000
and 10,000 (
Hamza et al., 2009; Nielsen et al., 2008; Welboren et al.,
2009
). In addition, identifying canonical DR4 motifs does not guaranty
finding functional TR-binding sites. Indeed, TR was shown to bind other
motifs (e.g., ER6 and DR1, see
Desvergne, 1994
). Nuclear receptors can
also bind to motifs that depart significantly from the canonical motif
(
Zirngibl, Chan, & Aubin, 2008
). Thus, it is imperative to use methods
such as chromatin immunoprecipitation (ChIP) to provide experimental
evidence of TR binding at specific genomic locations. To this end,
protein-protein and protein-DNA interactions are stabilized by cross-
linking, and chromatin is fragmented by sonication to a size range of
200-400 bp. The DNA fragments bound to the protein of interest are cap-
tured by immunoprecipitation with a specific antibody against the protein.
After de-cross-linking and purification, the resulting DNA sample is
enriched with the fragments originally associated with the protein. These
samples can be subject to different detection techniques. With conventional
ChIP-qPCR, the abundance of a known DNA fragment is measured by
real-time quantitative PCR (qPCR). This method, which requires to know
a priori
the genomic region being monitored, has proved to be remarkably
helpful in the context of amphibian metamorphosis to measure the differen-
tial binding patterns of TR in the vicinity of the TR
b
gene, as well as the
dynamics of histone modifications (
Bilesimo et al., 2011
). Although it is a
very efficient technique, its throughput is very low and labor intense, since
every location has to be tested individually by qPCR, and it requires to
know
a priori
where to look for binding. With the ChIP-Seq technology,
the detection method involves sequencing the purified DNA fragments.
In this case, the number of sequence reads per genomic location provides
a quantitative measure of DNA binding. This approach is naive and does
not rely on prior knowledge of the putative binding sites, and rather pro-
vides genome-wide binding profiles (
Fig. 10.6
B).
The profiling of a few chromatin modification marks as well as RNA-
Polymerase II occupancy has been carried out on
X.
tropicalis
early
