Transcription Factor Binding Profiles Reveal Cyclic
Expression of Human Protein-coding Genes and Non-
Chao Cheng1,2*, Matthew Ung1, Gavin D. Grant1, Michael L. Whitfield1
1Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America, 2Institute for Quantitative Biomedical Sciences,
Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, United States of America
Cell cycle is a complex and highly supervised process that must proceed with regulatory precision to achieve successful
cellular division. Despite the wide application, microarray time course experiments have several limitations in identifying cell
cycle genes. We thus propose a computational model to predict human cell cycle genes based on transcription factor (TF)
binding and regulatory motif information in their promoters. We utilize ENCODE ChIP-seq data and motif information as
predictors to discriminate cell cycle against non-cell cycle genes. Our results show that both the trans- TF features and the
cis- motif features are predictive of cell cycle genes, and a combination of the two types of features can further improve
prediction accuracy. We apply our model to a complete list of GENCODE promoters to predict novel cell cycle driving
promoters for both protein-coding genes and non-coding RNAs such as lincRNAs. We find that a similar percentage of
lincRNAs are cell cycle regulated as protein-coding genes, suggesting the importance of non-coding RNAs in cell cycle
division. The model we propose here provides not only a practical tool for identifying novel cell cycle genes with high
accuracy, but also new insights on cell cycle regulation by TFs and cis-regulatory elements.
Citation: Cheng C, Ung M, Grant GD, Whitfield ML (2013) Transcription Factor Binding Profiles Reveal Cyclic Expression of Human Protein-coding Genes and Non-
coding RNAs. PLoS Comput Biol 9(7): e1003132. doi:10.1371/journal.pcbi.1003132
Editor: Sarah A. Teichmann, EMBL-European Bioinformatics Institute & Wellcome Trust Sanger Institute, United Kingdom
Received February 26, 2013; Accepted May 24, 2013; Published July 11, 2013
Copyright: ? 2013 Cheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the American Cancer Society Research Grant, #IRG-82-003-27 and by the start-up funding package provided to CC by the
Geisel School of Medicine at Dartmouth College. MLW and GG were supported by the V Foundation for Cancer Research, ACS-IRG 82-003-17, and NIH grants R01
CA130795 and R01 HG004499. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
As one of the most important cellular processes, the cell division
cycle is under precise regulation in all organisms. Mis-regulation of
the cell cycle can lead to catastrophic cellular events, e.g.
premature apoptosis or abnormal proliferation of cells, which
are the causes of some human diseases such as cancer [1,2]. Cell
cycle regulation has been studied intensively, with focuses mainly
on two aspects. First, cell cycle regulated genes have been
identified systematically using microarrays to detect periodic
expression of genes in cell cycle time course data [3,4]. Second,
the genes, particularly, the transcription factors (TFs) that
modulate cell cycle have been investigated, e.g. identifying their
genomic occupation using chromatin immunoprecipitation fol-
lowed by microarray hybridization (ChIP-chip) or massively
parallel sequencing (ChIP-seq) [5,6]. These studies have provided
many insights into cell cycle regulation during normal biological
processes and in cancers.
Genome-wide gene expression during the cell cycle has been
investigated using DNA microarrays in a wide range of organisms,
including bacteria , yeast [3,8–10], mouse , human
[4,12,13] and Arabidopsis . Microarray cell cycle time course
data has been very successful at identifying a wide range of cell
cycle-regulated genes. Despite its success, the microarray-based
method has a few limitations. First, it is not effective for
determining if a gene expressed at low levels is periodic due to
low signal/noise ratios. Second, the synchronization procedure
itself may change the expression pattern of some genes during the
cell cycle, leading to false positive or false negative results. Third,
limited by probe design, it is often difficult to distinguish
expression patterns of different transcripts from the same gene.
For example, for a gene with alternative promoter usage, it is
possible that one isoform is cell cycle regulated while others are
not. Consequently, the two isoforms may not be distinguished by a
microarray based method if they share most of the exons.
Moreover, previous microarray-based studies have focused on
identification of cell cycle regulated protein-coding genes, while
the non-coding RNAs have been largely overlooked. These issues
can be overcome by measuring cell cycle gene expression using
RNA-seq experiments, which unfortunately has not been per-
The cell cycle is under precise gene regulation at different levels
of expression [15,16]. Particularly, at the transcriptional level it
has been shown that a series of TFs act at different phases of the
cell cycle and coordinate the sequential transcription of cell cycle
genes [17–19]. The periodic expression pattern of cell cycle genes
is encoded in cis in their promoters and can be manifested in trans
by the TFs that bind to them. Namely, we would expect cell cycle
genes to be bound by cell cycle regulating TFs. In this work, we
raise and verify the hypothesis that cell cycle genes can be
predicted by their genomic features (the motif occurrence in their
promoters) and TF binding features (binding affinity of TFs).
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Recently, the ChIP-seq genomic binding data for a large number
of human TFs have been published. In particular, the ENCODE
(the Encyclopedia of DNA Elements) project has published
binding profiles for more than 120 human TFs in different cell
lines and more ChIP-seq data are being produced . Motivated
by these data, we aim to construct a model that integrates
microarray cell cycle expression data with ChIP-seq TF binding
data to predict new cell cycle genes and to understand the function
of TFs in cell cycle regulation.
In this article, we present a computational method that predicts
human cell cycle genes based on genomic and TF-binding features
of genes. The model uses a supervised machine learning approach
to integrate microarray cell cycle data, ChIP-seq TF binding data
and motif information from sequence analysis (Figure 1). We first
apply the model to all human RefSeq genes, which are well
annotated with high accuracy. We validate the effectiveness of the
model for cell cycle gene prediction by cross-validation, and we
explore the relative importance of different predictors in the
model. We then apply the model to the GENCODE TSS
annotations, which provide a more comprehensive list of human
promoters for both protein-coding genes and non-coding RNAs
. This systematic analysis enables us to explore the human
genome to predict a full list of cell cycle-driving promoters. Our
approach is effective in identifying cell cycle genes with low
expression levels and is not sensitive to synchronization treatment.
Since it is applied at the TSS level, it can distinguish the different
isoforms of a gene regulated by alternative promoters. Further-
more, it can also be used to predict cell cycle regulated non-coding
RNAs, which we believe will substantially promote our under-
standing of cell cycle regulation.
Transcription factor regulatory scores can discriminate
cell cycle from non-cell cycle genes
With the rationale that periodic expression of cell cycle genes is
driven by a subset of transcription factors (TFs), we first examined
whether cell cycle genes and non-cell cycle genes show different
binding strength by TFs. We collected the ChIP-seq data from the
ENCODE (The Encyclopedia of DNA Elements) project ,
which provided high-resolution binding events of more than 120
human TFs in multiple cell lines. The binding strength of a TF to
the promoters of genes was calculated by a probabilistic model
called TIP (Target Identification from Profiles) we proposed
previously . This model provides a significantly more accurate
measure of TF binding affinity to particular genes than the peak-
based method used in many studies .
We prepared a dataset of cell cycle genes and non-cell cycle
genes in HeLa cells that have been experimentally verified with
high confidence based on the meta-analysis described in Cyclebase
. In the 424 ENCODE ChIP-seq TF binding profiles, 46 were
performed in HeLa cells, for which we calculated the regulatory
scores using TIP and the average binding signals in a 2 kb DNA
region centering at the TSS of all genes (see Methods for details)
(Figure 1). Corresponding values of each measurement method
were compared between cell cycle genes and non-cell cycle genes
using Student’s t-test. The regulatory scores for cell cycle genes are
significantly contrastive (generally higher than) from those of non-
cell cycle genes. As shown in Figure 2A, comparative analysis of
the regulatory score distributions of both CMYC and E2F1 show
that cell cycle genes tend to have substantially higher regulatory
scores than non-cell cycle genes (P=2e-55 and P=1e-50,
respectively). This is indicative of the significant regulatory roles
CMYC and E2F1 have on the expression of cell cycle genes, thus
suggesting that they are important features to be used in a cell
cycle prediction model [25,26].
In comparison, the average TF binding signals can also
discriminate cell cycle versus non-cell cycle genes, but with much
lower significance levels. For example, when average signals of
CMYC and E2F1 binding were calculated, we observed less
significant difference in values between cell cycle and non-cell
cycle genes. The P-values of average signal comparisons are 2e-8
for CMYC and 9e-7 for E2F1 (Figure 2B), indicating that average
signals are less effective classifiers for predicting cell cycle genes
than regulatory scores.
Other than CMYC and E2F1, many other TFs also reflect
significant differences in binding strengths between cell cycle and
non-cell cycle genes, especially when TIP is utilized (Figure 2C
and Suppl. Table S1). This suggests that the discriminatory
efficacy of regulatory scoring is maintained throughout a high
percentage of TFs and is not confined to a particular subset of cell
cycle regulatory TFs. Thus, we will use regulatory scoring of TF to
genes to predict cell cycle genes.
It should be noted that cell cycle genes tend to have higher
expression levels than non-cell cycle genes; some of the TF binding
difference may reflect the expression level difference rather than
their involvement in cell cycle (see Discussion for details). We also
note that the cell cycle regulatory function of a TF may not be
reflected at the transcriptional level. Among the 46 TFs we
investigated, only 6 showed significant periodical expression
pattern in cell cycle: E2F1, BRG1, CJUN, RAD21, GABPB and
CTCF. The known cell cycle regulators, E2F4 and E2F6, are not
significant at the transcriptional level (P.0.01). The model that
relates TF binding with cell cycle expression pattern, however, can
be used to elucidate the function of TFs in cell cycle regulation by
calculating their relative importance.
Genomic features are predictive of human cell cycle
Under the presumption that certain genomic features can
discriminate cell cycle genes from non-cell cycle genes, we
constructed Random Forest classification models to predict cell
Cell cycle is a complex and highly supervised process that
must proceed with regulatory precision to achieve
successful cellular division. Microarray time course exper-
iments have been successfully used to identify cell cycle
regulated genes but with several limitations, e.g. less
effective in identifying genes with low expression. We
propose a computational approach to predict cell cycle
genes based on TF binding data and motif information in
their promoters. Specifically, we take advantage of ChIP-
seq TF binding data generated by the ENCODE project and
the TF binding motif information available from public
databases. These data were processed and utilized as
predictor for predicting cell cycle genes using the Random
Forest method. Our results show that both the trans- TF
features and the cis- motif features are predictive to cell
cycle genes, and a combination of the two types features
can further improve prediction accuracy. We apply our
model to a complete list of GENCODE promoters to predict
novel cell cycle driving promoters for both protein-coding
genes and non-coding RNAs such as lincRNAs. We find that
a similar percentage of lincRNAs are cell cycle regulated as
protein-coding genes, suggesting the importance of non-
coding RNAs in cell cycle division.
Model for Cell Cycle Gene Prediction
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cycle genes using ENCODE ChIP-seq-derived TF-binding data
and TRANSFAC-derived motif matching data as predictors.
More specifically, we calculated the regulatory scores for all
human RefSeq genes as described above, resulting in 424 TF
binding profiles, each corresponding to a ChIP-seq dataset from
the ENCODE project. These binding profiles represent binding
strength of TFs to RefSeq genes in a number of different cell lines
such as K562, HESC, HeLa, etc. In addition, we also examine the
existence of all TRANSFAC TF binding motifs in the promoters
of RefSeq genes (from TSS to upstream 1 kb), resulting in a total
of 546 motif matching score profiles (see Methods for details). To
train the model, we used the cell cycle and non-cell cycle genes
identified by microarray experiments in HeLa cells . Consis-
tently, from the 424 TF binding profiles we only included the 46
profiles from HeLa cells in our model.
We examined three models for classifying cell cycle versus non-
cell cycle genes using Random Forest method. In a TF model, the
trans TF-binding features were used as predictors; in a Motif
model, the cis motif features are used as predictors; and a
TF+motif model uses a combination of all the features. The
performance of these models was evaluated by 10-fold cross-
validation (see Methods for details). Our results suggest that both
TF binding features and motif features are informative for cell
cycle gene prediction, with a prediction accuracy AUC=0.768
achieved by the TF model and AUC=0.642 achieved by the
motif model (Figure 3A). This also suggests that the ChIP-seq
derived TF binding features are considerably more predictive than
motif features from in silico sequence analysis. Strikingly, a
combination of both sets of features results in a prediction
accuracy that surpasses that of both TF and Motif models, leading
to an AUC=0.861 by the TF+motif model. This indicates that the
trans- information captured by ChIP-seq data and the cis-
information provided by the motif analysis complement each
other during cell cycle prediction.
In a Random Forest model, the contribution of an individual
feature to the overall predictive power of the model can be
estimated by its relative importance, measured as the Mean
Decrease in its Gini Coefficient (MDG) (see Methods for details).
Hence, we calculated the relative importance for all TF binding
(Figure 3B) and motif features (Figure 3C) in the TF+motif model.
Overall, TF features exhibit higher relative importance than motif
features, with the best TF feature achieved by SYDH_E2F4
(SYDH is the Lab ID) (Figure 3B) and the best motif feature
achieved by V$GEN_INI2_B (Figure 3C). These data confirm
that TF-binding regulatory scores are much better predictors than
motif matching scores. The high relative importance of E2F4 is
consistent with the critical roles it plays in cell cycle regulation
To investigate whether the predictive accuracy of the model is
predominantly determined by a few features or by many, we
removed features one by one from the model and examine the
change in prediction accuracy. In each step we removed the most
predictive feature based on their relative importance, then
recalculated the accuracy of the new model and re-estimated the
relative importance of all remaining features. Our results show
that many TF binding features are predictive of cell cycle genes. As
shown in Figure 3D, removing the most predictive feature one by
one only slowly reduce the AUC score of the model. Such a
situation changes until most of the TF binding features have been
removed, which leads to a sudden drop in prediction accuracy. At
Figure 1. Schematic diagram of our analysis for predicting human cell cycle genes. The predictive model integrates three types of data
from microarray, ChIP-seq experiments and computational TF binding motif analysis.
Model for Cell Cycle Gene Prediction
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this point, most of the predictors remained in the model are motif
features. In fact, we can achieve fairly accurate predictions by
selecting a small set of predictors. For instance, when the top 10
TF binding features and the top 10 motif features with highest
relative importance in the full TF+Motif model are selected as
predictors, we achieve a AUC=0.850, only slightly lower than the
full model (AUC=0.862).
Apart from the Random forest model, we also implemented
other machine learning methods, including support vector
machine (SVM) and penalized logistic regression (PLS). Results
from all these methods confirm the conclusions from the Random
Forest model, e.g. higher predictive accuracy of TF binding
features than motif features. Overall, Random Forest gives rise to
the best predictive accuracy and thus in this paper we focus on this
method in our analysis.
Cell cycle genes are tissue specific as suggested by
Since experimentally verified cell cycle and non-cell cycle genes
(required to train the model) were determined based on
microarray experiments with HeLa cells, we restricted our analysis
to HeLa cells in that we only include HeLa TF binding profiles as
features in our models. In fact, in the 424 profiles from ENCODE
ChIP-seq data, there are 68 from GM12878, 94 from K562, 37
from HESC and 55 from HEPG2 cell lines, respectively (Suppl.
Table S2). We thus examined the cell line specificity of our cell
cycle gene prediction model. If cell cycle regulation is cell line
specific, we would expect to achieve the best prediction accuracy
using HeLa TF binding profiles; and otherwise a similar accuracy
throughout different cell lines. Our results exhibit highest
prediction accuracy when the TF binding features from the
HeLa cell line are used for predicting HeLa cell cycle genes,
which is the case in both the TF+motif model (Figure 4A) and the
TF only model (Figure 4B). The TF sets with ChIP-seq profiles in
distinct cell lines contains different TFs. We thereby compared
the prediction accuracy of models using the 32 common TFs in
HeLa and K562 as predictors. ChIP-seq data from HeLa cells
achieve AUC=0.756 in the TF only model and AUC=0.860 in
TF+Motif model, whereas ChIP-seq data from K562 cells
achieves AUC=0.722 and AUC=0.831, respectively. These
results suggest that at least a subset of cell cycle genes is cell line
Furthermore, we investigate the binding strength of TFs to their
target gene promoters in different cell lines. As shown in Figure 4C,
the regulatory scores from E2F4 binding to the known cell cycle
genes (those used in our model as positive set) are used as a metric
to compare differential cell cycle regulation in K562 and HeLa cell
lines. Although the scores calculated in HeLa and K562 cells are
highly correlated, there is a small set of genes that show differential
binding by E2F4, most of which show higher regulatory scores in
the HeLa cell line. In addition, when the target genes of E2F4
identified by TIP method in K562 and HeLa are compared, we
find that many targets are unique to a single cell line (Figure 4D).
This indicates cell line specific binding of TFs to genes and as such,
it is not surprising to observe cell line specificity of our cell cycle
gene prediction model.
Figure 2. Regulator scores of TFs on genes can discriminate cell cycle (CC) versus non-cell cycle (non-CC) genes. (A) Distributions of
regulatory scores for CMYC and E2F1 are significantly different between CC and non-CC genes (P=2e-55 and P=1e-50, respectively). (B) The average
signals of CMYC and E2F1 show similar distributions between CC and non-CC genes (P=0.03 and P=0.05, respectively) (C) The t-scores for CC versus
non-CC genes calculated by comparing regulatory scores and average signals of TFs. SYDH, UTA and HAIB are the Lab IDs of a dataset.
Model for Cell Cycle Gene Prediction
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Prediction of phase specific cell cycle genes
Due to the periodicity of cell cycle genes, genomic features may
vary in predictive power across cell cycle phases. Therefore, we
examined whether genomic features are phase-specific by applying
the Random Forest classifier model to categorize genes expressed
in each phase. To generate training data, known cell cycle genes
were partitioned into G1/S, G2, G2/M, M, and S categories
based on the annotation in Cyclebase . Model accuracy was
assessed via 10-fold cross-validation to yield an ROC curve for
each cell cycle phase (Figure 5A). Phase-specific cell cycle gene
classification via Random Forest proved to be robust as shown by
relatively high AUC scores for each phase (Figure 5A). AUC
scores of 0.858, 0.793, 0.864, 0.859, and 0.858 were obtained for
G1/S, G2, G2/M, M/G1, and S cell cycle phases, respectively.
The normalized relative importance of each genomic feature
was calculated to deduce its predictive differentiability in each cell
cycle phase (see Methods for details). In all phases, TF features
show significantly higher relative importance than motif features.
Out of all TF features measured through all cell cycle phases,
E2F4 is predominantly the most important predictor in G2/M,
G2, S, G1/S phases. However, in the M/G1 phase the prediction
accuracy is driven by multiple TF features; interestingly E2F4 still
has high relative importance but is not the most predictive feature
any more (Figure 5B). In line with these results, we observed that
E2F4 targets were enriched in cell cycles genes with peak
expression around G2/M and G1/S (Suppl. Figure S2). We note
that the ChIP-seq data were performed in unsynchronized cells
and reflect TF binding status in a mixed population of cells. We
would expect an improvement of phase specific cell cycle gene
prediction if phase specific TF binding features were available and
utilized as predictors.
Identification of novel human cell cycle genes
Having shown the effectiveness of our model in predicting cell
cycle genes using cross-validation, we applied it to identify new
RefSeq genes that are potentially cell cycle regulated. The model
was trained and then utilized to predict the cell cycle regulation of
a total of 17,023 unclassified RefSeq genes (gene dataset used in
model training were excluded). Each gene was assigned a
probability indicating the likelihood of a gene to be cell cycle
regulated. By setting the threshold to 0.7, we predicted 726 new
cell cycle genes with a precision of 92% (positive predictive value,
PPV=0.92). Many of them are subunits of a protein complex that
is known to be cell cycle regulated. For instance, Whitfield et al.
measured the expression patterns of 12 centromere-associated
proteins  in HeLa cells, among which 6 were identified as
Figure 3. Statistical models for predicting cell cycle genes using Random Forest method. (A) The ROC curves for 3 classification models
that use TF-only, motif-only features or a combination of them as predictors. (B) The relative importance (measured as MDG, Mean Decrease in Gini
coefficient) of TF features in the combined model (TF+Motif). (C) The relative importance of motif features in the combined model. (D) The change of
prediction accuracy (measured as AUC scores) when remove the most important predictor from the full model one by one. Note that cell cycle genes
in the training data are from data in Hela cells, and thus we use only TF binding data from the same cell line in our model.
Model for Cell Cycle Gene Prediction
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periodically expressed in the cell cycle (CENPA, CENPF,
CENPM, CENPL, CENPO, CENPQ and CENPT) . Our
analysis predicts 2 additional subunits, CENPK and CENPN, to
be cell cycle regulated, suggesting that the model is complemen-
tary to microarray based analysis.
To further evaluate the reliability of these predicted cell cycle
genes, we carried out Gene Ontology (GO) enrichment analysis on
them. The results strongly support cell cycle related functions of
these new predicted genes (Suppl. Table S3). As shown, the top
enriched GO categories are all cell cycle related, such as
chromosome (GO:0005694), cell cycle (GO:0007049), cell cycle
process (GO:0022402), cell cycle phase (GO:0022403), M phase
Another method of evaluating prediction reliability is to
compare them with RNAi knockdown experimental datasets.
We downloaded two genome-wide RNAi knockdown datasets
published by Mukherji et al.  and Kittler et al. , in which
cell cycle regulators are identified by knocking down individual
genes and examining cell division defects that may result. We find
that the novel cell cycle genes we predict tend to exhibit increased
likelihood of cell division defect upon RNAi-induced loss-of-
function perturbation. In fact, the new cell cycle genes are highly
enriched in the cell cycle regulators identified by the two knock-
down experiments. A total of 686 and 901 cell cycle regulating
genes were identified by Mukherji et al. and Kittler et al.,
respectively, among which 47 were identified by both experiments
(P=4e-4). Out of the 726 novel cell cycle genes we predicted, 50
and 55 were reported to be cell cycle regulating genes by Mukherji
et al. (P=2e-4, Fisher’s exact test) and by Kittler et al. (P=5e-3,
Fisher’s exact test) (Figure S1).
Moreover, we examined the interaction partners of known cell
cycle genes, the predicted cell cycle genes, and the predicted non-
cell cycle genes. We expect that cell cycle genes are more likely to
interact with one another and will therefore have more cell cycle
partners in the protein-protein interaction (PPI) network. As
shown, the known cell cycle genes interact with more partners
than other genes (Figure 6A), presumably due to the fact that they
are more intensively studied in their interactions, e.g. by yeast two
hybrid experiments. Moreover, the known cell cycle genes tend to
have more cell cycle partners in terms of both number (Figure 6B)
and percentages (Figure 6C). We note that after excluding these
known cell cycle genes, the remaining genes used for prediction
have substantially fewer partners, cell cycle partners and lower
percentage of cell cycle partners. However, compared with
Figure 4. Tissue specificity of cell cycle predictive models. (A) The ROC curves when TF binding data from different cell lines are used as
predictors in the combined model. (B) Similar to (A), but results are from TF-only model. (C) The regulatory scores of E2F4 on Hela cell cycle genes in
HelaS3 versus K562 cells. Note that a small subset of genes shows strong E2F4 binding only in Hela cells. (D) E2F4 regulates overlapping but different
target genes in HelaS3 versus K562. (C) and (D) are based on ENCODE ChIP-seq data.
Model for Cell Cycle Gene Prediction
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predicted non-cell cycle genes (Probability ,0.3 in our model), the
predicted cell cycle genes (Probability .0.7) interact with
significantly more and higher percentage of cell cycle partners
(Figure 6B and 6C), implying their functions in cell cycle control.
Prediction of GENCODE promoters that drive periodical
Having shown the effectiveness of our model for predicting cell
cycle genes, we then applied it to the GENCODE annotation
data, which provides a complete list of human transcripts
including protein-coding genes, several categories of non-coding
RNAs and so on. For all these transcripts, the precise positions of
their TSSs were determined and the expression level associated
with each TSS was quantified by CAGE (Cap Analysis of Gene
Expression) experiments [30,31]. We calculated the regulatory
scores of these TSSs based on the ENCODE ChIP-seq data and
their motif-matching scores for all motifs as we have done for
RefSeq promoters (see Methods for details). Finally, the TF+motif
Random Forest model trained using the above-mentioned RefSeq
cell cycle and non-cell cycle genes was applied to the GENCODE
dataset. Thus, by using the regulatory scores and motif-matching
scores as features, the model predicts whether a TSS is cell cycle
regulated and assigns a probability score to each TSS.
We predicted the probability of cell cycle regulation for all
GENCODE annotated human TSSs using our model. These
TSSs are associated with different genomic feature categories
Figure 5. Prediction of phase specific cell cycle genes. (A) ROC curves of models that classify cell cycle genes at specific phase against non-cell
cycle genes. (B) The relative importance of different TF features in the 5 phase specific models.
Figure 6. Predicted cell cycle genes are more likely to interact with cell cycle partner in protein-protein interaction network. (A) the
average number partners; (B) the average number of cell cycle partners; (C) the average percentage of cell cycle partners. Note all known cell cycle
genes are excluded from the predicted cell cycle gene set. The P-values for difference in numbers of partners or cell cycle partners between two gene
classes are calculated by Chi-squared test.
Model for Cell Cycle Gene Prediction
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including protein-coding genes, microRNAs, lincRNAs, snRNAs,
snoRNAs and pseudogenes. As negative controls, we also included
10,013 randomly selected genomic locations (i.e. artificial TSSs)
from the genome and predict their probability to be cell cycle
regulated using our model. Certainly, the number of positive
predictions is determined by the threshold setting and the
precision (also called PPV, positive predictive value, the percent-
age of true positives in all predicted positive predictions) at each
threshold can be estimated by cross-validation in our training data
(Figure 7A). To have a confident set of predictions, we set up a
stringent threshold (Probability score .0.7) in the following
analysis, corresponding to a PPV=0.92. At this threshold, we
identify 3,322 protein-coding, 83 lincRNA, 6 miRNA, 8 snoRNA,
4 snRNA, 16 pseudogene, and 9 artificial TSSs that are predicted
to be cell cycle regulated (Suppl. Table S4). The percentage of cell
cycle regulated TSSs for each genomic feature category is shown
in Figure 7B. As shown, the percentage of positive artificial TSSs is
very low (,0.1%), indicating a high precision of our predictions.
Similarly, the percentage of positive pseudogene TSSs is also very
low (1%), since most of them are untranscribed ‘‘junk DNA’’. But
compared to the randomly selected artificial TSSs, it is possible
that some pseudogene TSSs are actually active and expressed in
cell cycle a dependent manner. Strikingly, lincRNA and protein-
coding genes show similar percentage of cell cycle regulated TSSs
(,3%) (Figure 7B), indicating that lincRNAs might also be
important in cell cycle regulation. Cell cycle regulated miRNA,
snoRNA, and snRNA are identified in relatively low percentages,
possibly due to low quality of annotation in their TSSs. For
instance, annotation of miRNAs usually begin at the +1 start site of
the corresponding pre-miRNA (,110 bp) as opposed to the
genuine TSS of pri-miRNA.
GO enrichment analysis was performed on the predicted cell cycle
regulated TSSs associated with GO terms, most of which are for
protein-coding genes. The results suggest that these positive
predictions are highly enriched in gene categories involved in or
related to cell cycle functions (Suppl. Table S5). Almost all of the top
enriched GO terms are cell cycle related, e.g. cell cycle
(GO:0007049), chromosome (GO:0005694), mitosis (GO:0007067),
Many genes possess multiple transcript isoforms with alternative
TSSs and our model can predict the probability of each TSS to be
cell cycle regulated. In fact, our results indicate that different
isoforms of the same gene may be either cell cycle regulated or not
cell cycle regulated, namely have distinct functions with respect to
cell cycle regulation. For example, the gene DBF4 (with Ensembl
ID ENSG00000006634) is annotated to have 8 different TSSs by
GENCODE, which forms two TSS clusters. The first cluster
contain 6 TSSs, which are all predicted to be cell cycle regulated
with a probability score .0.7; whereas the second cluster (11 kb
away from the first cluster) contains 2 TSSs with probability score
of 0.296 and 0.190 respectively. The DBF4 protein is known to be
essential for initiation of DNA replication  and the transcrip-
tion of its promoter is activated through cell-cycle box (MCB)
transcription elements . Assuming the TSS annotation is
correct, our analysis imply that only the first cluster of transcript
isoforms are regulated in a cell cycle dependent manner; and that
the two isoforms in the second cluster may not be periodically
expressed during the cell cycle, either not being involved in cell
cycle regulation or impacting cell cycle in a different way from the
first cluster of isoforms.
Compared to the average binding signals of TFs in promoters,
the regulatory scores we define are more informative for predicting
cell cycle genes (Figure 1). Regulatory scores can be regarded as
weighted average binding signals of TFs around the TSS of genes.
For each TF, a specific weight is assigned to each nucleotide
position in the 10 kb DNA region centering at TSS based on the
characteristic binding profile of the TF. Thus regulatory scores can
more accurately capture the regulatory potential of a TF to genes
than average signals. When utilized as predictors for classifying cell
cycle versus non-cell cycle genes, they generally reveal greater
differentiability between the two gene classes, suggesting they are
more powerful classifiers. In fact, the Random Forest model that
utilizes average signals for the same set of TFs as predictors
achieves a classification accuracy AUC=0.683, which is similar to
the accuracy of the motif only model (AUC=0.642), and is
significantly lower than the TF only model that is based on
regulatory scores (AUC=0.768). Thus, it seems that by combining
with machine-learning methods, the regulatory scores calculated
from ChIP-seq data might also be promising in other applications,
for example, predicting tissue specificity or conditionally expressed
Figure 7. Prediction of cell cycle related promoters. Model is applied to ,138,000 GENCODE annotated promoters to identify novel cell cycle
genes of different types. (A) The number of cell cycle related genes identified the model when different threshold is used. The precision (1-FDR) is
shown as the increasing grey line. (B) The percentage of different types of genes that are predicted to be cell cycle related at threshold of 0.7
(Prob.0.7). FDR: false discovery rate.
Model for Cell Cycle Gene Prediction
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Our analysis indicates that cell cycle regulation in different cell
lines may not be exactly the same but shows certain cell line
specificity: cell cycle genes identified in Hela cells can be best
predicted by ChIP-seq TF binding profiles from the same cell line.
Although the binding strengths of E2F4 to HeLa cell cycle gene
promoters in both HeLa and K562 cell lines are comparable, there
exists an observable small subset of genes exhibiting highly
differential E2F4 binding affinities; with the majority of them
showing more vigorous binding in HeLa cells. (Figure 4C). In fact,
a large percentage of E2F4 target genes identified by ChIP-seq
experiment are HeLa or K562 specific (Figure 4C). Moreover, we
compared the cell cycle genes identified via cDNA microarray
experiment in HeLa cells by Whitfield et al. (588 genes)  and in
fibroblast cells by Iyer et al. (480 genes) , and discover that
only 155 are cell cycle genes in both cell lines. Contrastingly, TF
binding data from other cell lines also prove predictive to HeLa
cell cycle genes with reasonably high accuracy, indicating
somehow a similar language of cell cycle regulation between cell
lines. From these observations, it seems that to some extent, cell
cycle regulation is cell line specific yet there may exist a core set of
genes that are cell cycle regulated across all cell lines.
The relative importance of predictors in our model suggests that
E2F4 is essential in cell cycle gene regulation. In addition, the TF
binding profiles for E2F1 and E2F6 also show significantly
differential binding strengths between cell cycle and non-cell cycle
genes, and exhibit high relative importance in our model. These
results are in accordance with existing literature, which assert that
the E2F family of transcription factors plays an inextricable role in
driving and regulating cell cycle . E2Fs are regulated by the
pRB-family and pRB-related proteins (e.g. p130 and p107), that
are inactivated upon CDK-mediated phosphorylation .
Additionally, E2F exhibit dual properties in that E2F1–E2F3 act
as activators and E2F4–E2F8 as repressors . In particular,
E2F4 is shown by ChIP-chip analysis to have a plethora of gene
targets involved in every phase of the cell cycle [26,34,37].
Principal targets of E2F4 include genes involved in cell cycle
regulation, DNA replication, DNA repair, chromatin remodeling,
and cell cycle checkpoints . Evidently, these cellular processes
are all associated with cell cycle genes thereby forming an
integrated network of gene regulation . Because E2F4 is a
negative regulator, it must be constantly repressed by pRb and
only expressed intermittently to allow the cell cycle to progress.
This allows cellular processes controlled by E2F4 to occur in a
phase-specific fashion (i.e. DNA repair during S/G2, DNA
replication during S, and chromosome remodeling during G2/
M) . Additionally, a comparative genomics study carried out
by Linhart et al. proposes that there is substantial decrease in E2F4
binding during M/G1 phase of the cell cycle . This is in
accordance with our results which show a decrease in normalized
relative importance of E2F4 during the M/G1 phase (Figure 5B).
Overall, these results suggest that E2F4 is repressed upon
termination of mitosis and subsequently de-repressed upon
initiation of G1 in daughter cells. The fact that E2F4 binding is
an effective discriminatory cell cycle-associated TF binding feature
demonstrates that our prediction model is indeed capable of
utilizing key inherent cellular predictors to classify a wide variety of
Previous studies have shown that TF binding signals are
predictive to the expression level of genes, accounting for .60%
variation of gene expression [39–42]. Here we show that TF
binding data can be used to predict cell cycle genes. The predictive
power of regulatory scores which capture the trans- information of
genes, can be further improved by the cis- information of these
genes, or the motif matching scores in their promoters. Our model
which uses TF+motif predictors achieves a classification accuracy
of AUC=0.861, suggesting the regulatory code for cell cycle genes
is largely harbored in their promoter regions. The TF binding data
and the motif information complement each other, because (1)
none of the two data are complete (e.g. the ChIP-seq data of many
critical cell cycle regulatory TFs are not available) and (2) the
trans- TF binding data from ChIP-seq captures regulatory
information not only at the transcriptional level but also at the
epigenomic level, since TF binding is significantly affected by
epigenomic modifications (e.g. histone modifications and DNA
methylation). The periodical expression pattern of cell cycle genes
is also regulated at the post-transcriptional level, e.g. by miRNAs,
and we believe that predictive accuracy of our model can be
further improved when such information are included. One caveat
of the model is that ChIP-seq experiment captures TF binding in a
population of unsynchronized cells, which limits our model from
more precisely elucidating the cell line specific and phase-specific
regulation of TFs.
As we have described, microarray-based methods are less
effective in identifying cell cycle genes expressed at low levels. For
this reason, cell cycle genes detected from microarray experiment
tend to have higher expression levels compared to those of non-cell
cycle genes. In fact, when we statistically compare the expression
levels of cell cycle genes versus non-cell cycle genes in HeLa cells,
we observe significant expression disparity (P=3e-42, Wilcoxon
rank sum test). Furthermore, it has been demonstrated previously
that TF binding is predictive of gene expression levels [39–42],
which makes expression level a confounding factor to account for
when classifying cell cycle versus non-cell cycle genes: the model
we propose here may be restricted to prediction of high versus low
gene expression rather than cell cycle genes. This also explains
why most of the TFs show very differential regulatory scores
between cell cycle and non-cell cycle genes (Figure 2), but only 6 of
them show periodical expression pattern during cell cycle in HeLa
cells. To address this confounding issue, we prepare a set of non-
cell cycle genes that have similar expression levels with cell cycle
genes in HeLa. When the regulatory scores are compared between
these genes and cell cycle genes, fewer TF features show significant
difference but the key cell cycle regulators (e.g. E2F1 and E2F4)
still maintain significant difference. This suggests that these key
regulators do in fact, bind differentially with cell cycle versus non-
cell cycle genes even after decoupling them from the influence of
expression levels. If average signals of TF features are compared,
none of the TFs show differential binding at the 0.001 significance
level, again demonstrating the advantage of regulatory scores.
More importantly, when these expression-matched non-cell cycle
genes are used as the negative training set, we can still accurately
predict cell cycle genes with AUC=0.706 using the TF only
model and AUC=0.814 using the TF+motif model. Thus, we can
conclude that the model is effective for cell cycle gene prediction
when the influence of expression level is eliminated and a very
conservative training set is used.
Most previous cell cycle research is focused on protein-coding
genes, while in-depth systematic investigation of cell cycle non-
coding RNAs have not been conducted. A recent paper examined
the promoters of 56 cell-cycle genes using tiling array and revealed
extensive non-coding transcription near these genes . This
explorative study highlights the potential importance of regulation
by non-coding RNA during cell cycle division. We applied our
model to more than 130,000 human TSSs annotated by
GENCODE project and systematically predicted the probability
of these TSSs to act as cell cycle driving promoters. The
GENCODE TSS list contains TSSs for not only protein-coding
genes but also for several classes of non-coding RNAs such as
Model for Cell Cycle Gene Prediction
PLOS Computational Biology | www.ploscompbiol.org9July 2013 | Volume 9 | Issue 7 | e1003132
miRNAs, lincRNAs, snoRNAs, and snRNAs. Our predictions
suggest that there is at least equal percentage of lincRNAs that are
cell cycle regulated as there are protein-coding genes. Further
experimental investigation of these non-coding RNAs should
provide further insight into the non-coding world of cell cycle
The enormous amount of genomic data from the ENCODE
project provide valuable resources for biological studies. However,
how to more efficiently make use of such data to facilitate
hypothesis driven studies is still an open question. Here we show
an example that combines large-scale ChIP-seq data from
ENCODE with motif data from genome sequence analysis and
cell cycle microarray data from small-scale laboratory studies. The
framework introduced in this paper may also be applied to address
other biological questions such as identifying tissue specific
expression of genes, gene classes, and environment-induced gene
expression and so on.
Microarray cell cycle time course data
In this work, we used a supervised model to predict human cell
cycle genes. To train the model, we obtained the known cell cycle
genes and non-cell cycle genes from the data produced by
Whitfield et al , which measured gene expression during the cell
division cycle in HeLa cells using microarray experiments. The
data contain four different cell cycle time course series, each
providing a list of cell cycle genes. To have a confident cell cycle
gene list, we referred to the meta-analysis performed by Cyclebase
, which combined the results of all these four time courses.
The cell cycle genes (positive training set) were selected as those
with a significant combined P-value for periodicity (P,0.011),
while the non-cell cycle genes (negative training set) were selected
as those that were not significant in any of the four time courses
(P.0.1). In total, we obtained 853 cell cycle Refseq genes and
1051 non-cell cycle Refseq genes. The phase-specificity of cell
cycle genes were determined based on their peak expression time
provide by Cyclebase. In Cyclebase, each cell cycle genes is
assigned a value of 0–100 indicating their peak expression time
with G1 (0–47), S (47–70), G2 (70–90) and M (90–100).
Accordingly, we selected 138 M/G1 (95–100 or 0–20), 257 G2/
M (80–95), 253 G2 (70–90), 175 S (47–70) and 185 G1/S (20–60)
specific Refseq genes for model training.
Calculation of transcription factor regulatory score
We calculated the binding affinity of transcription factors to the
promoter of a gene based on their corresponding ChIP-seq data.
The ChIP-seq data provides the binding signal of a TF at each
nucleotide of the genome. We utilized the method called TIP
(Target Identification from Profile) to quantify the regulatory
relationships between TFs and target genes . Given the ChIP-
seq data of a TF, TIP builds a characteristic, averaged profile of
binding around the TSS of all genes and then uses this to weight
the sites associated with a given gene, providing a ‘regulatory’
score of this for each gene. From the ENCODE project , we
downloaded a total of 424 ChIP-seq data, representing the binding
data for about 120 different TFs in more than 10 cell lines such as
HelaS3, HESC, K562, etc. For each of them, we calculated the
regulatory scores for all RefSeq genes, giving rise to a matrix of
34,299 (RefSeq genes) rows and 424 columns (ChIP-seq datasets).
The average binding signals of a TF with a gene is calculated by
averaging the ChIP-seq signal of all nucleotide position in the
promoter DNA region (a 2 kb DNA region centering at the TSS)
of the gene.
Calculation of motif matching scores in promoter of
We downloaded 565 vertebrate motifs from the TRANSFAC
database , which represent the potential binding sites of DNA
binding proteins, mostly transcription factors. We also download-
ed the promoter sequences (from TSS to upstream 1000 bp of a
gene) of 34,229 human RefSeq genes from the UCSC Genome
Browser . For each promoter sequence, we used the MATCH
program  to examine the presence of these TF binding motifs.
The pre-calculated cut-offs provided by MATCH were used to
minimize the false positive rate. The MATCH program provides
all the potential binding sites and their matching-scores of all of the
RefSeq gene promoters. Based on these outputs, we constructed a
binding score matrix [B_i,j] of size N6M, in which each row
representing a RefSeq gene (N=34,229) and each column
corresponding to a motif (M=565). Each element B_ij was
calculated by aggregating the matching-scores of all the binding
sites of the motif j in the promoter of the gene i. The score was set
to 0 if there is no binding site in the promoter of a gene.
Predicting cell cycle genes using Random Forest
The Random Forest ensemble classifier was used to as a
machine-learning model to predict genes as cell cycle or non-cell
cycle. A prepared dataset containing known cell cycle genes
(annotation derived from RefSeq and Cyclebase) and their
associated TF features derived from ENCODE and TRANSFAC
databases was used to train the model. This dataset contained 863
known cell cycle genes and 1051 known non-cell cycle genes. To
generate the final training dataset, 81 TFs were chosen as pre-
selected features resulting in a total of 69,903 cell cycle TF-gene
pairs and 85,131 non-cell cycle gene pairs. Each cell cycle gene
was assigned a positive binary value (1) and each non-cell cycle
gene was assigned a negative binary value (0). Model accuracy was
assessed using 10-fold cross-validation in the following procedure.
First, each fold was carried out by randomly dividing the training
dataset into 10 partitions, irrespective of binary assignment.
Second, 9 partitions were used to train the Random Forest model
and the remaining partition was used as a test set to determine
model performance. This is repeated 9 more times to yield the
averaged sensitivity ([#True Positives]/[#True Positives+#False
Negatives]) and specificity ([#True Negatives]/[#True Negati-
ves+#False Positives]) of the model. This allows construction of a
Receiver Operating Characteristic (ROC) curve, which is a direct
representation of the relationship between sensitivity and specific-
ity. The area under the ROC curve (AUC) is calculated via
Riemann summation of 100 trapezoidal partitions. Calculation of
the AUC is the main evaluator of Random Forest model accuracy
in this study; AUC=1 corresponds to 100% model accuracy and
AUC=0.5 corresponds to random classification by the model,
thus completely non-discriminatory.
The relative importance (RI) of a predictor in a Random Forest
model can be measured by the metric ‘‘%IncMSE’’ (increase of
mean squared error) . Given a trained model, ‘‘%IncMSE’’
measures the increase of prediction error in the test data when the
values for each individual predictor are permuted. The permuta-
tion of an important predictor will be expected to lead to a
considerable prediction error increase and therefore a large
‘‘%IncMSE’’. The relative importance of each genomic feature is
model specific. The relative importance of features for predicting
phase-specific cell cycle genes is calculated respectively from the
corresponding phase-specific model.
The R package ‘‘randomForest’’ was utilized to implement
Model for Cell Cycle Gene Prediction
PLOS Computational Biology | www.ploscompbiol.org10 July 2013 | Volume 9 | Issue 7 | e1003132
Predicting periodical expression driving GENCODE
Transcription start sites (TSS) information was derived from the
GENCODE gene annotation project  and the high confidence
TSS sets from GENCODE version 7 was used. This set includes a
total of 137,874 TSSs for different gene categories, including
protein coding genes (100,417), miRNAs (1,755), lincRNAs
(2,751), pseudogenes (13,164), etc. In the dataset, many genes
are associated with multiple TSSs which corresponds to these
genes having alternative promoters. As we have done for the
RefSeq genes, we calculated the TF regulatory scores and the
motif matching scores for all these TSSs using the above-described
We trained Random Forest using the RefSeq gene training data
described in the preceding section, and then apply the model to
predict the probability of these TSSs function as cell cycle driving
promoters. Meanwhile, we generated ,10,000 random TSSs that
are evenly distributed in the genome and fed them into the model
as controls. Given a cut-off, the false discovery rate (FDR) of our
model can be estimated by calculating the ratio of F_rand to
F_real, where F_rand and F_real are the fractions of predicted cell
cycle driving random TSSs and real TSSs (i.e. TSSs with
probability above the cut-off).
Enrichment analysis of E2F4 targets in cell cycle
We investigated the distribution of transcription factor target
genes in the cell cycle. First, we sorted the cell cycle genes in HeLa
cells according to their peak expression times. Then we examined
the enrichment of the target genes of a given transcription factor in
each sliding window of the cell cycle. We used a window size of 30
degrees with 10 degrees overlapping between neighboring
windows. We used the Fisher’s exact test to determine the
significance of enrichment of target genes for a transcription factor
in each cell cycle window (Suppl. Figure S2).
Other datasets and bioinformatic analysis
Systematic gene knockdown data for cell division genes
screening are available from Mukherji et al.  and Kittler et
al. . In the two studies, the majority of human protein-coding
genes were knocked down in U2OS and HeLa cells, respectively,
to identify cell cycle regulating genes. We examined and calculated
the significance the enrichment of our predicted cell cycle genes in
gene sets identified by Mukherji et al. and Kittler et al. using
Fisher’s Exact test (Suppl. Figure S1).
To examine the enrichment of genes of different gene ontology
(GO) categories in our predicted cell cycle gene set, we performed
GO enrichment analysis by using the web-based tool from
DAVID database (the Database for Annotation, Visualization
and Integrated Discovery), which calculated significance of
enrichment based on Fisher’s exact test . In the analysis, we
removed the cell cycle and non-cell cycle genes in the training set
to avoid their impact and limit bias
The human protein-protein interaction data is downloaded
from the Human Protein Reference Database (HPRD, Release 8)
. Human RefSeq gene annotations are obtained from the
UCSC Genome Browser database .
large-scale gene knockdown experiments. (A) Comparison of
predicated cell cycle genes with knockdown results from Mukherji
et al. (B) Comparison of predicated cell cycle genes with
knockdown results from Kittler et al. (C) Comparison of
knockdown results between Mukherji et al. and Kittler et al.
Validation of novel predicted cell cycle genes from
cycle. Human cell cycle genes are ordered based on their peak
expression time in the cell cycle, and enrichment of E2F4 targets in
each time window is calculated by using Fisher’s Exact test.
Enrichment of E2F4 target genes during the cell
factors for cell cycle and non-cell cycle genes.
Regulatory scores and average signals of transcription
The list of 424 ENCODE ChIP-seq experiment for
cycle Refseq genes.
Gene Ontology analysis results of novel predicted cell
of GENCODE annotated genes at different thresholds. PPV
(Positive Prediction Value) indicates the prediction precision
estimated by cross-validation.
The number of positive prediction of different classes
annotated protein-coding genes that are predicated to be cell
Gene Ontology analysis results of GENCODE
Conceived and designed the experiments: CC. Performed the experiments:
CC MU. Analyzed the data: CC MU GDG MLW. Contributed reagents/
materials/analysis tools: CC. Wrote the paper: CC MU MLW.
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PLOS Computational Biology | www.ploscompbiol.org12 July 2013 | Volume 9 | Issue 7 | e1003132