SHORT REPORT Open Access
Functional and topological characterization of
transcriptional cooperativity in yeast
Daniel Aguilar*and Baldo Oliva
Background: Many cellular programs are regulated through the integration of specific transcriptional signals
originated from external stimuli, being cooperation between transcription factors a key feature in this process. In
this work, we studied how transcriptional cooperativity in yeast is aimed at integrating different regulatory inputs
rather than controlling particular cellular functions from a organizational, evolutionary and functional point of view.
Findings: Our results showed that cooperative transcription factor pairs co-evolve and are essential for the life of
the cell. When organized into a layered regulatory network, we observed that cooperative transcription factors were
preferentially placed in the middle layers, which highlights a role in regulatory signal integration. We also observed
significant co-activity and co-evolution between members of the same cooperative pairs, but a lack of common
Conclusions: Our results suggest that transcriptional cooperativity has a specific role within the regulatory control
scheme of the cell, focused in the amplification and integration of cellular signals rather than control of particular
cellular functions. This information can be used for better characterization of regulatory interactions between
transcription factors, aimed at determining the spatial and temporal control of gene expression.
Keywords: Regulatory network, Transcription factor, Gene regulation, Gene expression, Transcriptional cooperativity
Many cellular programs are regulated through the inte-
gration of specific transcriptional signals originated from
external stimuli. In order to understand these programs,
it is necessary to explore modes of interaction between
transcription factors (TFs) such as transcriptional coop-
erativity. Particularly in eukaryotes, the process of coop-
erativity enables a small number of TFs to combine their
regulatory influences to execute a large number of regu-
latory decisions [1,2]. This can be achieved through dif-
ferent mechanisms, such as interaction between adjacent
TFs on the promoter, interaction between distantly
mechanisms devoid of protein contacts [3-6].
Previous studies have devised methods for computa-
tionally detecting and measuring transcriptional coop-
erativity based on different mechanisms of cooperation
such as co-expression, co-binding to the promoter or
TF-TF interaction. These works produced different lists
of cooperative transcription factor pairs (CTFPs) [3-9].
In a previous work we found that a core of the predicted
CTFPs actually shared some particular characteristics
when analyzed in terms of their placement in the protein
interaction network and in the regulatory network .
In this work, we characterize transcriptional coopera-
tivity from a functional and evolutionary point of view.
Also, we explore how transcriptional cooperativity in
yeast is aimed at integrating different regulatory inputs
and the transmission of the regulatory signal to other
TFs which control particular cellular functions [10,11].
To do so, we examined the role of known CTFPs in the
hierarchical layout of the regulatory network.
Are cooperative TFs essential for the life of the cell?
We observed that the probability that both members of
a CTFP are synthetic lethals is 144-fold larger than for
random expectation, which is statistically significant
(Fisher's test; p-value=1.63*10-6). For comparison, TF
pairs regulating similar functions (i.e. co-functional, see
* Correspondence: firstname.lastname@example.org
Structural Bioinformatics Group (GRIB/IMIM), Departament de Ciencies
Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona Biomedical
Research Park (PRBB), c/Dr. Aiguader 88, 08003 Barcelona, Spain
© 2012 Aguilar and Oliva; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Aguilar and Oliva BMC Research Notes 2012, 5:227
Methods) were synthetic lethals 4.74 times more often
than random expectation. This suggests that the dele-
tion of a member of a cooperative TF might disturb the
transcriptional profiles of some genes but renders the
cell viable. The deletion of both members of a CTFP,
however, is critical. This highlights the importance of
cooperativity as a transcriptional coordinative process.
Do cooperative TFs co-evolve?
It is known that essential TFs tend to evolve slower that
non-essential ones . Furthermore, it is known that
interacting proteins pairs are likely to co-evolve [13,14].
These observations made us wonder whether CTFPs
shared similar selection processes. We found a posi-
tive correlation between the protein evolution rate of
members ofthe same
r=0.464, p-value<2.2*10-16). This suggests that muta-
tions in only one of the members of a CTFP are enough
to have a deleterious effect and suffer from a negative se-
lection, which highlights the functional dependence be-
tween both members of a CTFP. This result is interesting
because only 5 of the 32 CTFPs in our set (16%) are
known to physically interact. We have to note that co-
evolution does not imply a particular substitution rate
along evolution, but that both members of the same CTFP
evolve at a similar rate (in fact, we did not observe a sig-
nificant trend for CTFPs towards a preferential evolution-
ary rate). If we assume that the known yeast interactome
comprehensively covers the protein-protein interactions
between transcription factors, the value of this observation
lies in the fact that the rate of evolution of a TF is influ-
enced by its cooperative interactions, regardless of a phys-
ical interaction between them.
Are cooperative TFs co-expressed or co-active?
We did not observe any significant correlation between
the mRNA expression profiles of the members of the
same CTFP in the different cellular conditions under
study (see Methods). We did not did find any significant
correlation either between expression levels (number of
copies/cell), half-lives of the transcripts or transcrip-
tional frequency for members of the same CTFP. This
lack of correlation may be explained by the presence of
post-transcriptional mechanisms regulating the func-
tional activation of TFs and by the average window of
activity of TFs inside the cell. We then investigated
whether the activity profiles of the members of the same
CTFP were correlated (since the actual activity of a TF
can be uncoupled from its expression profile ). We
did find a significant correlation between the activities of
TFs belonging in the same CTFP for 15 of the 17 experi-
ments (Table 1). This suggests that post-transcriptional
modifications have a stronger influence than expression
regulation in transcriptional cooperativity. We also
examined co-activity in a layer-by-layer basis (Additional
file 1). Finally, we measured the correlation between
evolutionary rate and expression levels (copies/cell) for
members of the same CTFP. We observed a strong anti-
correlation of ρ=−0.72 (Spearman's test; p-value=
0.021). These results agree with previous observations
Analysis of the regulatory hierarchy
It is known that the transcriptional regulatory network
has a multi-layered hierarchical structure acting as a
decision-making system (Figure 1), where the topmost
layers is where external stimuli reach the regulatory net-
work (e.g. through a signalling cascade) . The regula-
tory signal is then integrated with other signals and
amplified as it travels down the hierarchy through the
middle layers (which act as bottlenecks in the informa-
tion flow). In response, TFs in the lower layer execute
the transcriptional response by turning on/off the activ-
ity of different groups of genes. This is known as the
Is cooperativity associated with the cogitation process?
Because members of the same CTFP do not share simi-
lar regulatory inputs, we suggested in a previous work
that cooperativity might have a role in integrating mul-
tiple regulatory signals . If cooperativity provides in-
tegration and amplification of regulatory signals, then
CTFPs should be preferentially placed in the middle
layers of the hierarchy, since this is where incoming sig-
nals from the global modulators are integrated before
the activation of the different sets of genes which carry
out particular functions.
We found that CTFPs were slightly (but significantly)
under-represented in the layer-1 of the hierarchy (odds
ratio=0.75; p-value=1.17*10-4), and were clearly over-
represented in the layer-2 (odds ratio=5.1; p-value=
1.73*10-7). The presence of cooperative TFs in layer-1
may be surprising because TFs in layer-1 only control
non-TF genes. However, we have to take into account
dependent on regulation of expression, but may be based
on co-binding to the promoter  or in protein-protein
interactions . This explains the presence of coopera-
tive TFs in this layer.
These findings confirm that cooperativity is mainly
related not to the process of execution of transcriptional
responses (if so, CTFPs would be preferentially placed in
layer-1), nor to the reception of external stimuli (their
presence is not relevant in the upper layer). Instead,
cooperativity seems to be associated mainly with the in-
tegration of regulatory signals and their transmission to
the lower layers.
Aguilar and Oliva BMC Research Notes 2012, 5:227
Page 2 of 6
We also found that the protein functions regulated by
cooperative TFs in the layer-1 were significantly enriched
in metabolism-related functions such as Metabolism,
Regulation of metabolism and protein function or Cellular
transport (Table 2). However, other housekeeping func-
tions (e.g. cell-cycle-related and communication-related)
were under-represented. This suggests that metabolism is
the cellular function to be cooperatively regulated at the
bottom level of the hierarchy. Conversely, cooperative TFs
in layer-2 were responsible of cell-cycle related functions,
which is consistent with a role in coordination of broad
cellular processes. Also, the function Interaction with the
Table 1 Co-activity between CTFPs
Condition Average co-activity Co-activity increase (in n-fold)
Anearobic N-C-P-S chemostats
C-S-P-N chemostat limitation
Compounds and stress
Regulation by PDR1
TCA cycle mutants
Titratable promoter alleles
Average co-activity (calculated using a squared Spearman's correlation coefficient) between members of the same CTFP. Increase in co-activity is calculated as the
ratio of average correlation in CTFPs vs average correlation in 1000 non-cooperative TF pairs.
Figure 1 Regulatory hierarchy. TFs are represented as nodes and regulatory interactions as directed edges. The two topmost layers were
merged for the analysis. Blue nodes: non-cooperative TFs. Red nodes: cooperative TFs.
Aguilar and Oliva BMC Research Notes 2012, 5:227
Page 3 of 6
environment is significantly over-represented, thus imply-
ing that cooperative TFs in this level are responsible for
passing external signals down to the lower layers of the
hierarchy. Finally, although no communication-related
functions were significantly over-represented in layer-3,
we observed a clear under-representation of housekeeping
functions (such as metabolism), preferentially regulated in
the bottom layer. The same analysis using Gene Ontology
terms instead of FunCat categories yielded very similar
results (Additional file 2).
In this work, we studied the role of transcriptional
cooperativity in the control of regulatory programs in
yeast. Our results suggest that transcriptional coopera-
tivity has a specific role focused in the amplification
and integration of cellular signals rather than control
of particular sets of genes or detection of external
stimuli. We also show a functional dependence be-
tween members of a cooperative TF pair (both are
synthetic lethals, co-evolve and have similar activity
profiles). This information can be used for better
transcription factors, aimed at determining the spatial
and temporal control of gene expression.
extracted from Beyer et al. . We used the subset of
between TFs andtargetgenes were
TF-regulated gene associations labeled as highly confident
by the authors. We built a set of CTFPs based on the
compilation of computationally-predicted CTFPs by four
different methods [6-10]. We selected those TF pairs pre-
dicted as cooperative at least by two methods. The result-
ing amount of CTFPs was 32, composed by the pairing of
26 distinct TFs (Additional file 3).
Following the Breadth-First Search algorithm described
by Yu & Gerstein , we built a directed network of
TFs as a multi-layered hierarchical structure (Figure 1).
We merged the two upper layers of the network (with 8
and 2 TFs, respectively) in order to avoid a low number of
TFs that would hinder statistical calculations. The final
network had four layers (the bottom layer termed layer-
1, the topmost layer termed layer-4) composed by 148
TFs and 96 regulatory interactions (Additional file 4).
This network will be referred to as regulatory hier-
archy. A slightly different implementation of the al-
gorithm places all targets of a TF in the same level,
thus forcing all interactions in the hierarchy to
point downward or horizontally, but never upwards
. We also built and analyzed this hierarchy
(Additional file 5, Additional file 6). The enrichment
in CTFPs for level n was calculated as the ratio of
the probability of finding a CTFP in that level vs
random expectation. The statistical significance was
calculated using 10  random hierarchies where
the target genes were randomly exchanged between
Table 2 Functional enrichment in the regulatory hierarchy
Biogenesis of cellular components0.02
Cell cycle and DNA processing 0.01
Cell rescue, defense and virulence1.58*10-5
Cell type differentiation
Cellular communication/signal transduction mechanism 0.01
Cellular transport, transport facilities and transport routes 6.56
Interaction with the environment
−2.8Protein with binding function or cofactor requirement
Regulation of metabolism and protein function6.1
Cell type differentiation1.95
Cellular transport, transport facilities and transport routes 0.01
Interaction with the environment 2.28
Regulation of metabolism and protein function0.03
Cell type differentiation0.01
Interaction with the environment4.43*10-3
Regulation of metabolism and protein function 0.03
This table shows all significantly over-represented or under-represented functional categories for cooperative TFs in regulatory hierarchy. Negative z-scores mean
under-representation, positive z-scores mean over-representation.
Aguilar and Oliva BMC Research Notes 2012, 5:227
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We obtained mRNA expression data for the following
cellular conditions: diauxic shift , cell cycle ,
sporulation , and six environmental stress conditions:
heat, acid, alkali, peroxide, NaCl and sorbitol . Expres-
sion levels (copies/cell), apparent half-life of the tran-
(mRNAs/hour) were obtained from Holstege et al .
Correlation between expression levels was calculated
using a Spearman's correlation test. We downloaded the
protein activity profiles of the TFs in our sets from 17
experiments of the database RegulonProfiler, where TF ac-
tivity profiles are inferred from genomewide changes in
mRNA expression patterns of groups of genes with similar
regulation (called ChIP-based regulons), which allowed
the authors to quantify the post-translational activity of
TFs . Only activity profiles with E-value<0.05 were
considered. Correlations were calculated using a squared
Spearman's correlation test [26,27]. In all cases, the distri-
bution of the correlation of the activity levels of CTFPs
was compared against the distribution of the activity levels
of 1000 non-CTFPs by means of a KS test.
Information on the essentiality of yeast proteins was
downloaded from the Yeast Deletion Project . Infor-
mation on synthetic lethals was obtained from the Bio-
Grid database . Association between essentiality and
transcriptional cooperativity was calculated by means of
a Fisher's test.
Protein evolutionary rate for TFs was obtained from
Xia et al . The correlation between evolutionary
rates and expression levels for CTFPs was calculated as
the dn/ds ratio for members of the same CTFP and the
ratio of their expression levels. A Spearman's test was
used to calculate the correlation.
Protein functions were extracted from the FunCat
catalogue . Being FunCat a hierarchical classification,
we used first-level functions with experimental evidence,
which amounted to 16 different functions. Functional
similarity between TFs was calculated as in Aguilar &
Oliva . We defined two TFs as co-functional if their
functional similarity was larger than the 90thpercentile
of the distribution of the functional similarity values for
all TF pairs.
We first measured the enrichment for each function
in each level of the regulatory hierarchy by means of a
z-score, using a random model consisting in 10 
regulatory hierarchies where the gene functions con-
trolled by the TFs were randomly exchanged. We
repeated this analysis using the Gene Ontology func-
tional annotation at depth level 2 (which is roughly
equivalent to the first level of FunCat) . TFs anno-
tated at lower levels were re-annotated with the corre-
sponding parent terms of level 2. Only experimental
annotations were used. The R software was used for all
statistical tests .
Availability of supporting data
The data sets supporting the results of this article are
included within the article and its additional files.
Additional file 1: Co-activity between CTFPs in the regulatory
hierarchy. Average co-activity (calculated using squared Spearman's
correlation coefficient) between members of the same CTFP in the
different layers of the regulatory hierarchy.
Additional file 2: Functional enrichment in the regulatory hierarchy
in Gene Ontology terms. This document contains a table with the
functional enrichment in the regulatory hierarchy in Gene Ontology
Additional file 3: List of cooperative TF pairs. This file contains the
list of cooperative TF pairs.
Additional file 4: Regulatory hierarchy. This file contains the
Additional file 5: Regulatory hierarchy without upwards regulatory
interactions. This file contains the regulatory hierarchy with no upwards
Additional file 6: Regulatory hierarchy without upwards regulatory
interactions. This document explains the methods used to build this
hierarchy and the results of its analysis.
TF: Transcription factor; CTFP: Cooperative transcription factor pair;
The authors declare that they have no competing interests.
DA conceived the study and carried out the analysis. BO participated in the
design of the study and helped to draft the manuscript. Both authors read
and approved the final manuscript.
This work was supported by grants from Spanish Ministry of Science and
Innovation (MICINN) BIO2011-22568 & BIO2008-205.
Received: 9 February 2012 Accepted: 27 April 2012
Published: 10 May 2012
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Cite this article as: Aguilar and Oliva: Functional and topological
characterization of transcriptional cooperativity in yeast. BMC Research
Notes 2012 5:227.
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