Mini-Review

Deep cap analysis gene expression (CAGE):

genome-wide identification of promoters, quantification of their expression, and network inference
Michiel de Hoon and Yoshihide Hayashizaki
BioTechniques 44:627-632 (25th Anniversary Issue, April 2008)
doi 10.2144/000112802

In cap analysis gene expression (CAGE), short (20 nucleotide) sequence tags originating from the 5 end of full-length mRNAs are
sequenced to identify transcription events on a genome-wide scale. The rapid increase in the throughput of present-day sequencers
provides much deeper CAGE tag sequencing, where CAGE tags can be found multiple times for each mRNA in a given experiment.
CAGE tag counts can then be used to reliably estimate the cellular concentration of the corresponding mRNA. In contrast to microarray and SAGE expression profiling, CAGE identifies the location of each transcription start site in addition to its expression level.
This makes it possible for us to infer a genome-wide network of transcriptional regulation by searching the promoter region surrounding each CAGE-defined transcription start site for potential transcription factor binding sites. Hence, deep CAGE is a unique
tool for the construction of a promoter-based network of transcriptional regulation. CAGE-based expression profiling also allows us
to identify dynamic promoter usage in time-course experiments and the specific promoter regulated by a given transcription factor
in disruption experiments. The sheer size of the short-tag datasets produced by modern sequencers spurs a need for new software
development to handle the amount of data generated by next-generation sequencers. In addition, new visualization methods will be
needed to represent a promoter-based transcriptional network.

INTRODUCTION
Cap analysis gene expression (CAGE)
was introduced in 2003 as a method to
determine transcription start sites on
a genome-wide scale by isolating and
sequencing short sequence tags originating
from the 5 end of RNA transcripts (1).
Mapping these tags back to the reference
genome identifies the transcription start
sites from which the transcripts originated.
CAGE relies on a cap-trapper system to
capture full-length RNAs while avoiding
rRNA and tRNA transcripts. First, an oligodT primer is used to reverse-transcribe
poly-A terminated RNAs. Alternatively,
a random primer can be used for RNAs
without a poly-A tail, which may constitute
almost half of the transcriptome (2). RNA/
DNA double-stranded hybrids that contain
a mature mRNA are selected by biotinylating their 5 cap structure, allowing
capture by streptavidin-coated magnetic
beads. Ligation of a linker sequence
containing an MmeI recognition site to
the 5 end of the full-length cDNA creates
a restriction site about 20 nucleotides

downstream, producing a short CAGE tag

starting at the 5 end of eukaryotic mRNAs
(3). CAGE tags that map just upstream
of known genes may be derived from
the corresponding full-length mRNAs,
whereas others may reflect the existence
of currently unknown transcription start
sites or genes. Due to their short size,
sequencing CAGE tags is more efficient
at detecting transcription start sites than
sequencing full-length cDNAs.
In early CAGE experiments, the
throughput of sequencers limited the
achievable sequencing depth such that
many CAGE tags were found only once
in a given experiment. More recently, a
new generation of sequencers excelling at
high-throughput sequencing of short tags
has enabled deep CAGE tag sequencing,
generating upward of a million tags from
a single experimental condition. The tag
counts found in such experiments are
typically much larger than one, allowing
an accurate estimate of the cellular
concentration of the RNA molecule corresponding to each CAGE tag. Deep CAGE
thus detects both the transcription start site

as well as its expression level, making it

a unique tool in the analysis of transcriptional regulatory networks.
Characteristic Features of CAGE
High-throughput gene expression
experiments based on microarrays (4),
massively parallel signature sequencing
(MPSS) (5), serial analysis of gene
expression (SAGE) (6), and CAGE give
us a snapshot of the RNA concentrations
in the cell at a particular time in a specific
experimental condition. Quantitative realtime PCR (qRT-PCR) (710), while not
a high-throughput method, can provide a
valuable standard for validation because
of its accuracy and wide dynamic range.
Whereas these methods are complementary to each other, the characteristic
features of CAGE expression profiling
make it particularly suitable for investigating the transcriptional regulatory
network that drives the expression of genes
and noncoding RNAs.
First, CAGE tag counts allow us to
calculate the cellular amount of the corre-

Genome Exploration Research Group, RIKEN Genomic Sciences Center, Yokohama Institute, Yokohama, Kanagawa, Japan
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Mini-Review
sponding RNA molecule in a digital form.
As one mRNA is not preferentially detected
over another, expression profiling based
on tag sequencing is unbiased, allowing
a direct comparison of the expression
values of different genes measured in
a single deep CAGE experiment. In
contrast, microarray fluorescence levels
are affected both by the mRNA concentration and by the probe-dependent mRNA
affinity, precluding a direct comparison
between genes. In addition, tag counts as
a measure of mRNA concentrations have a
dynamic range that is orders of magnitude
larger than microarray expression levels.
The accuracy and the dynamic range of
CAGE- and SAGE-derived expression
levels as well as the sensitivity of detecting
lowly expressed transcripts can be
improved further by deeper sequencing.
Importantly, microarray and qRT-PCR
expression profiling are restricted to those
transcripts for which a probe or primer
pair is available, whereas methodologies
based on tag sequencing can also measure
the expression of currently unknown
transcripts.
Deep CAGE expression profiling is
unique among high-throughput expression
profiling methods because the 5 end of the
CAGE tag identifies the corresponding
transcription start site. Hence, deep CAGE
allows us to determine the promoter driving
the transcription of each transcript in
addition to its expression level. In contrast,
tags generated by SAGE or MPSS are
located at the 3 end of the transcript and
do not identify the promoter, which may
lie tens of kilobases or more upstream in
the genome sequence. A variant of SAGE

has been developed that uses oligo-capping

to create tags at the 5 end of the transcript
(5 SAGE) (11), but it is currently not in
common use. Whereas expressed sequence
tags and full-length mRNA sequencing do
identify the promoter, the throughput of
these techniques is insufficient to allow
genome-wide expression profiling and
may not be able to detect lowly expressed
transcripts.
Next-generation High-throughput
Sequencers Revolutionize
Transcriptomics
In recent years, advances in sequencing
chemistry have led to innovations such
as pyrosequencing, Solexa sequencing
by synthesis (Illumina, Inc., San Diego,
CA, USA), SOLiD sequencing by oligonucleotide ligation and detection with
2-base encoding (Applied Biosystems,
Foster City, CA, USA), and true single
molecule sequencing by Helicos (Helicos
BioSciences Corp., Cambridge, MA,
USA). The number of bp that can be read
in 1 day by high-throughput sequencers
based on these techniques is orders of
magnitude larger than conventional
sequencers based on Sanger sequencing
(see Table 1). These sequencers are
particularly suitable for short sequence
reads of the kind generated by CAGE, in
contrast to genome sequencing, for which
longer sequence reads are preferred. This
makes transcriptome sequencing one of
the primary targets of high-throughput
sequencers.
The increased throughput of sequencers
enables CAGE sequencing at a much

deeper scale than previously possible. On

the one hand, this implies that it is increasingly possible to detect RNA molecules
present at very low-copy numbers in the
cell. This is critical in time-course experiments, for example, where transcripts
coding for regulatory molecules may be
present only transiently and at low concentrations. Approximately, the probability to
detect an RNA molecule that is present in
k copies per cell is 1 exp(-nk/
nk/m
/m), where n
is the number of CAGE tags extracted and
m is the total number of RNA molecules
in the cell. A 454 sequencer (454 Life
Sciences, Branford, CT, USA) generating 400,000 reads per run of a length of
about 250 bases can produce 1,000,000
mappable CAGE tags in one run. Assuming
a total mRNA concentration of 200,000
molecules per cell, the probability to detect
an RNA molecule present in one copy per
cell is 99.33%. The Solexa sequencer can
generate about 5
more CAGE tags per
run, making it possible for us to detect an
RNA molecule present in one copy per
five cells at the same probability. Deeper
CAGE also increases the CAGE tag count
for each particular mRNA, enabling more
precise expression profiling also for lowly
expressed transcripts, with the sampling
error decreasing as the square root of the
number of mappable CAGE tags.
Transcriptome and Regulation
Probed by Deep CAGE Experiments
Due to the inherent variability in
transcription start sites, CAGE tags are
typically found scattered over short
genomic regions. In previous shallow

Table 1. Sequencing Capacities of Current and Near-future Next-generation Sequencers, and the Estimated Corresponding Deep CAGE Throughput and Sensitivity

Throughput

CAGE

Sequencer
Base/Run

Base/Read

Read/Run

Base/Day

Mapped Tag/Run

Sensitivity at 99%

454

100,000,000

250

400,000

100,000,000

1,100,000

1 per 1 cell

Solexa

500,000,000

25

20,000,000

120,000,000

5,000,000

1 per 5 cells

SOLiD

1,400,000,000

35

40,000,000

180,000,000

13,000,000

1 per 14 cells

Helicos

10,000,000,000

30

300,000,000

1,000,000,000

150,000,000

1 per 160 cells

The sensitivity of deep CAGE is assessed as the concentration per cell of an mRNA molecule that can be detected at 99% probability, assuming a total mRNA
concentration of 200,000 molecules per cell.

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CAGE experiments, promoters were
constructed by clustering the 5 ends of
the individual CAGE tags based on the
distance between them on the genome (12).
In deep CAGE experiments, transcription
start site clustering may also take into
account the similarity in expression
profiles. Deep CAGE thus allows us to
define the individual promoters from
which genes are transcribed based on the
promoter activities.
Previous CAGE experiments indicate
that transcription units in mammalian
genomes are characterized by alternative
transcription start sites and multiple splice
forms that are active in different cellular
contexts (1216). In any given CAGE
experiment, some of the CAGE tags correspond to previously identified promoters,
whereas others are due to novel promoters
and transcripts that may give rise to novel
protein variants or contain alternative
localization or degradation signals. CAGE
has also led to the discovery of novel
noncoding RNAs (12,17).
CAGE expression profiling has
produced a wide variety of new insights
into the mammalian transcriptome and
its regulation. An analysis of CAGEdefined transcription start sites in human
and mouse illustrated that promoters
characterized by a TATA-box tend to
have a clear, single transcription start
site, whereas promoters associated with
CpG islands tend to have transcription
start sites distributed over a broad area
(14). CAGE expression profiling, with its
ability to localize the promoter as well as
determine its expression level, is ideally
suited to study the regulation of bidirectional promoters (18,19), which may be
coregulated by shared transcription factor
binding sites. Antisense transcripts, which
are transcribed in the opposite direction
to the coding strand, may play a role in
regulation via RNA interference as well as
gene silencing at the chromatin level (20
24). A genome-wide analysis of CAGE
transcriptome data revealed frequent
concordant regulation of sense/antisense
pairs (25).
While genes can have multiple
promoters in principle, their usage will
likely vary depending on cellular conditions. Time-course CAGE experiments
can be used to study the dynamic usage of
promoters by comparing the distribution
of expression levels over transcription
start sites in the upstream region of a gene
to discover which promoters are switched

on and off between time points during the

experiment. Similarly, we may find tissuespecific promoter usage, or promoters that
are only used in specific cell lines.
A major goal of the analysis of deep
CAGE sequence data is to infer the
regulatory network that orchestrates
transcription in a cell (26). With the target
of each regulatory interaction being a
promoter instead of a gene, such a network
is qualitatively different from current genebased networks inferred from microarray
or SAGE expression profiling. Each of the
promoters may be associated with one or
more coding or noncoding transcripts.
A promoter-based network can be
constructed by first obtaining matrix
models to describe the binding affinities
of transcription factors, and then using
these matrices to search for potential
transcription factor binding sites in
promoter regions. Both of these steps
are facilitated by the availability of the
promoter locations and expression levels
measured by deep CAGE.
Matrix models of transcription factors
can be derived either from the literature
or by aligning the upstream regions of
coregulated genes. Such coregulated genes
can be found by clustering the genomewide expression profiles measured in
microarray experiments. However, the
expression profile measured by microarrays is an aggregate of the expression
profiles of the individual promoters from
which transcripts originate, hampering a
clean clustering result. In addition, these
promoters are typically regulated by a
different set of transcription factors. Deep
CAGE expression profiling enables us to
separate the expression of a gene into the
contribution of the individual promoters,
which can then be clustered based on their
expression profiles to generate tighter
clusters of co-expression. Each of these
clusters of promoters is likely regulated by
a smaller set of transcription factors than
conventional clusters of genes. In addition,
the CAGE-defined promoter identifies
the genome regions to be aligned to find
overrepresented sequence motifs.
Similarly, we can restrict the genome
region to be searched for potential
transcription factor binding sites to the
promoter region identified by CAGE,
significantly reducing the possibility
of false positives. While comparative
genomics may also be used to identify
the promoter region, it does not pinpoint
the exact transcription start site. In addition,

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many biologically functional sequences do

not seem to be evolutionarily constrained
across all mammals (16) and therefore
cannot be identified by comparative
approaches. Finally, deep CAGE profiling
identifies which promoters are active in a
particular biological context and therefore
suggests which transcription factor binding
sites may be biologically relevant.
Software Needs for High-throughput
Transcriptome Sequencing
The sheer size of the datasets generated
by high-throughput transcriptome
sequencing places new requirements on
the software tools used to analyze these
data. During extraction of CAGE tags
from the raw reads, care must be taken to
correctly distinguish the tag from linker
sequences. Mapping CAGE tags to the
genome is complicated by the possibility
of sequence mismatches as well as tags
mapping to multiple locations on the
genome. Whereas BLAST (27) can be
used to place CAGE tags on the genome,
this tool is likely too slow given the size of
the high-throughput datasets produced by
next-generation sequencers. In addition,
BLAST is based on the assumption
that the sequences to be compared are
evolutionarily related, which is clearly
not appropriate for CAGE tag mapping.
For this purpose, software specializing
in transcriptome tag sequencing such
as SSAHA (Wellcome Trust Sanger
Institute, Hinxton, Cambridge, UK) (28)
and Nexalign (RIKEN, Yokohama, Japan)
(29) may be more appropriate. The latter
is exceedingly fast for exact matches in
particular, and guarantees to find any tag
present in the genome.
Special care must be taken for
ambiguous tags that map to multiple
genome locations with equal scores. In
such cases, it may be possible to decide
between the mapping locations by considering the number of singly-mapped CAGE
tags in each genome neighborhood as
a prior probability (30). Many of the
ambiguous CAGE tags map to a large
number of genome locations, suggesting
that they originate from repeat regions
of the genome. This is consistent with
a considerable fraction of the human
transcriptome originating from repetitive
sequences, which may play a role in
the transcriptional regulation of gene
expression (31).
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The size of the datasets produced by
next-generation sequencers also poses a
challenge to data management systems. As
terabytes of image data may be generated
in a single run, saving all raw data
produced in one experiment may no longer
be an option. With billions of bases being
generated in a single run, even saving only
the sequence data will be a considerable
enterprise.
In addition to new software to analyze
deep CAGE data, the paradigm shift of
gene-based networks to promoter-based
networks of transcriptional regulation
requires new ways to visualize such
networks. Visualization software packages
such as Cytoscape (32) have previously
been developed to represent biomolecular
interaction networks and can be used
to draw gene regulatory networks. In a
promoter-based network, the targets of
regulatory interactions are the individual
promoters of a gene, which may be too
numerous for graphical representation
except in the most detailed drawings. The
situation is further compounded by the
multitude of transcripts that have been
identified for each gene. A multiscale
visualization approach in which users
can choose the level of detail at which
each gene is represented may be suitable
for visualizing promoter-based regulatory
networks.
ACKNOWLEDGMENTS
This work was supported by a
research grant from the RIKEN Genome
Exploration Research Project from the
Ministry of Education, Culture, Sports,
Science and Technology of the Japanese
Government to Y.H., a grant from the
Genome Network Project, also from the
Ministry of Education, Culture, Sports,
Science and Technology, and a grant from
the RIKEN Frontier Research System,
Functional RNA Research Program.
COMPETING INTERESTS
STATEMENT
The authors declare no competing
interests.
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Address correspondence to Yoshihide

Hayashizaki, Genome Exploration Research
Group, RIKEN Genomic Sciences Center,
Yokohama Institute, 1-7-22 Suehiro-cho,
Tsurumi-ku, Yokohama, Kanagawa 2300045, Japan. e-mail: yosihide@gsc.riken.jp
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