Biology Reference
In-Depth Information
accuracy
[127]
. This allows the expression patterns of
fluorescent protein reporter constructs such as GFP to be
mapped at cellular resolution in the L1 larva. To date, data
have been released for the expression patterns of GFP
reporters driven by the promoters of 93 different genes
[128]
. Mapping these expression patterns onto the lineage
fates predicts when their expression first starts during
development, and at which point cells diverge in fate
[128]
.
Third, an approach has been developed that allows for
the semi-automated tracking of all nuclei up to the ~350
cell stage of embryonic development, using time-lapse
confocal microscopy and histone
have been mapped genome-wide in multiple organisms. In
C. elegans two principal approaches have been used. The
first is a 'gene-centered' approach where the yeast one-
hybrid system is used to test for interactions between
a promoter of interest and each transcription factor from
a library expressed in the yeast nucleus
[135]
(see
Chapter 4). The second approach involves cross-linking
proteins to DNA, shearing chromatin into small fragments,
precipitating DNA bound to a protein of interest using
antibodies, and then identifying this DNA using micro-
arrays or deep sequencing (referred to as ChIP-CHIP or
ChIP-seq, respectively; see Chapter 4).
C. elegans protein
GFP reporter constructs
to visualize all nuclei
[129]
. The method can be used to
map gene expression patterns using a strain in which
histone
e
DNA interactions have been map-
ped quite extensively using the yeast one-hybrid system
and, when combined with perturbation experiments, have
provided insights into the regulation of intestine-expressed
genes
[135]
, the regulation of fat metabolism
[136]
,
neuronal development
[137]
, feedback interactions
between transcription factors and miRNAs
[138]
, and the
functional divergence of transcription factor gene families
during evolution
[139]
.
The mapping of C. elegans protein
e
e
GFP reporters are expressed in all nuclei and
a histone
mCherry reporter is expressed from the promoter
of a gene of interest. Although relatively low throughput in
capacity (for each gene a transgenic histone
e
mCherry
reporter strain must be constructed, and each dataset
requires long time-lapse recordings and a substantial effort
to correct the tracked lineages), such an approach has the
potential to map the expression patterns of genes of interest
at cellular resolution,
e
DNA interactions
in vivo by chromatin immunoprecipitation has been most
extensively undertaken as part of the modENCODE
project. Here the strategy has been to express GFP-tagged
transcription factors from large genomic constructs (to
recapitulate as closely as possible the endogenous regula-
tion of a gene) and to use ChIP-seq to determine sites of
enriched binding along the genome
[140]
. Analyzing the
enrichment sites of 22 transcription factors revealed
a surprising lack of specificity in their binding, with many
inferred binding sites detected in the upstream regions of
highly expressed genes
[140]
.
As for most of the gene expression datasets discussed
above, the current maps of transcription factor-binding sites
in C. elegans suffer from the major disadvantage that they
were performed from whole embryo or whole worm
extracts, and thus represent the average binding signals
from complex mixtures of cell types. Thus transcription
factor binding that is specific to only a subset of cells may
not be detected, and signals from different cell types may
be mixed. Moreover, information on how transcription
factor binding changes in different cell types is lacking. In
the future, approaches such as those employed in
Drosophila to perform ChIP from individual tissues
[141]
may prove useful. Moreover, at least in C. elegans, there is
still a big gap between the genome-wide maps of tran-
scription factor binding and an understanding of how genes
are differentially regulated in space and time: to date there
has been little success in using genome-wide transcription
factor-binding datasets to predict gene expression.
A second aim of the modENCODE project has been to
produce genome-wide maps of chromatin organization
[142]
. Here immunoprecipitation is performed with
e
including following perturbation
experiments
[130]
.
Fourth, spatiotemporal expression profiles have been
generated for ~900 different promoter
GFP constructs
using an adapted flow-cytometer (a 'worm sorter')
[131]
.
The advantages of this technique are the high-throughput
generation of profiles, and the quantitative expression data.
The main disadvantage of the technique is that the spatial
expression information is compressed from three dimen-
sions to one, with expression levels only quantified along
the long axis of the worm.
Fifth, an approach has been developed that uses multiple
(~50) fluorescently labelled probes to visualize individual
mRNA molecules in developing embryos (single molecule
fluorescence in situ hybridiazation, smFISH,
[132]
). To date
this method has only been used to quantify expression levels
in whole embryos, and in particular how this expression
varies across isogenic individuals
[133,134]
. There is,
however, the potential that smFISH could be quite widely
used to provide cell-resolution information on gene
expression patterns for many genes, although at the moment
the cost of the probes would limit such a project. A disad-
vantage of smFISH is that it uses fixed samples, so dynamic
changes in gene expression must be indirectly inferred.
e
Global Maps of Transcription Factor-Binding
Sites and Chromatin Organization
One central aspect of gene regulation is the physical
binding of transcription factors to specific regions of the
genome. Recently, binding sites for transcription factors
