Biology Reference
In-Depth Information
The functionality of enhancers can be studied in two
complementary ways: either by using reporter constructs
to determine enhancer activity, or by perturbing (deleting
or mutating) enhancers in the genome. Perturbing CREs is
relatively straightforward in S. cerevisiae, where highly
efficient homologous recombination permits investigators
to create deletions or mutant versions of CREs (or genes
encoding the TFs that bind them) in the genome, and
examining the effect of such deletions on gene expression
or more global phenotypes. Such studies are much more
challenging in metazoans, where homologous recombi-
nation is less efficient. Instead, heterologous reporter
assays that measure enhancer activity are typically per-
formed at non-native chromosomal loci or using plasmid-
based assays
[82,83]
. Drosophila has been a major model
organism for the study of enhancers in animals. In
particular, significant efforts aimed at identifying tran-
scriptional enhancers in D. melanogaster have focused on
the blastoderm stage of development
[84
e
86]
. In addi-
tion, a GFP reporter assay has been employed to examine
44 DNA fragments, each ~3-kb long, for enhancer
activity in the adult fly brain
[87]
. Other groups have
taken a two-tiered approach whereby TF-occupied regions
identified by ChIP were subsequently tested in reporter
assays
[88,89]
. Altogether, it has been estimated that there
are ~50 000 transcriptional enhancers in the Drosophila
genome
[87]
. Despite all these efforts, at the time of
writing there are still fewer than 900 known transcrip-
tional enhancers in D. melanogaster
[90]
. Except for
a handful of examples
[82]
, CREs in C. elegans have
been poorly characterized, and even basic trends about
CRE complexity in C. elegans, such as the extent of
combinatorial TF input and whether tissue-specific gene
expression is regulated by complex transcriptional
enhancers as in in Drosophila and mammals, remain
unknown.
Testing of mammalian enhancers by in vivo knockout at
a native chromosomal locus is a slow, laborious and costly
approach that limits testing to just one or a few enhancers
within a study
[91]
. Therefore, candidate mammalian
enhancers are instead typically tested by reporter assays.
Currently the largest-scale testing of mammalian
enhancers can examine of the order of ~100 predicted
enhancers in a single study. Such studies generally employ
one of two methods: whole-mount LacZ reporter staining
in mouse
[70,92
e
94]
, or luciferase reporter assays in
mammalian cell culture
[95,96]
. Mammalian enhancers
can also be tested in zebrafish, which offers the advantage
that it can be done at higher throughput than testing in mice
[97]
. However, a negative result can be more difficult to
interpret, as the transcriptional regulatory networks in
mammals might be substantially diverged from those in
zebrafish, and because fish are not useful for assessing
enhancer activity that may be specific to cell
found only in mammals or primates, such as certain
neurons
[98]
.
GRN Edges: Physical Interactions Between
TFs and DNA
High-throughput experimental methods that can be used for
the identification of physical TF
e
target gene interactions
(
Box 4.1
) can be classified into two conceptually comple-
mentary categories
[99]
. The first are 'TF-centered'
(protein-to-DNA) methods that identify DNA sequences or
loci bound by individual TFs. Target genes are then usually
attributed to a TF by their proximity to a TF-binding event.
ChIP, combined with either microarrays (ChIP-chip) or
high-throughput sequencing (ChIP-seq), is the most widely
used method for the TF-centered identification of in vivo
TF-DNA interactions, and has been used in a variety of
model systems. For example, ChIP has been comprehen-
sively used in S. cerevisiae to map physical interactions
between most of the TFs and gene promoters both in rich
media and under a set of different conditions
[100,101]
.
An in vitro TF-centered approach is protein-binding
microarray (PBM) technology, in which a TF is applied
directly to double-stranded DNA microarrays covering
a wide range of possible DNA-binding site sequences.
PBMs have been used to determine the in vitro DNA-
binding specificities of a variety of different classes of TFs
from a wide range of organisms, including yeast
[102]
,
worms
[18]
and mice
[103,104]
. Based on the PBM results,
position weight matrices (PWMs) can be constructed that
capture the binding specificity of each TF. Such PWMs can
then be used to identify potential binding sites for the TF in
the relevant genome. Two additional TF-centered methods
for TF
e
DNA interaction identification, SELEX and
DamID, are described in
Box 4.1
.
The second category is comprised of 'gene-centered'
(DNA-to-protein) methods that identify the repertoire of TFs
that can interact with a regulatory genomic region of interest.
Yeast one-hybrid (Y1H) assays provide such a gene-centered
method, and have been mostly applied to the delineation of
GRNs in the nematode C. elegans
[28,52,105
e
107]
,though
more recentlyY1H resources have become available for other
organisms as well
[24,26,52]
.
TF- and gene-centered approaches are complementary
because they enable a researcher to address different types
of questions related to the systems biology of gene
expression
[44,99,108]
. For instance, ChIP is highly useful
when one is interested in identifying the in vivo DNA
targets for one or a few TFs, e.g., involved in development
or in a particular disease. Y1H assays, on the other hand,
are particularly useful when one is interested in a single
gene or a set of genes involved in a system or process of
interest. For instance, when one would like to know the TFs
that bind to a promoter of interest, it would not be practical
types
