Open Journal of Genetics, 2012, 2, 1-4 OJGen
Published Online December 2012 (h ttp://
Published Online March 2012 in SciRes.
Bioinformatic screening of the binding transcription sites
in the regulatory regions of genes up-regulated in response
to oxidative stress
Shkurat TP, Ponomareva NS, Aleksandrova AA, Shkurat MA, Butenko AI, Panich AE
Southern Federal University, Rostov-on-Don, Russia
Received 2012
This study focuses on bioinformatics search for new
regulatory structures in the non-coding DNA, located
around the patterns of gene expression levels
changed significantly in response to oxidative stress.
Hypothesized that all of the genes increase the ex-
pression in response to oxidative stress may have the
same motifs in non-coding DNA. To search for motifs
created an integrated collection database of tran-
scription binding sites - JASPAR, TRANSFAC, Ho-
comoco TF Homo sapiens, Uniprobe TF Mus mus-
culus. Two types of regulatory regions: the promoter
region and the sequence with the capture of potential
cis-regulatory modules. In the regulatory regions of
genes increase the expression in response to oxidative
stress, in contrast to the gene expression level did not
change, families of transcription factors identified
SOX (1-30) and HX (A, B, C, D).
Keywords: Gene Expression; DNA Microarrays;
Noncoding DNA; Oxidative Stress; Transcription Factor;
Sites of Transcription Factor Binding; DNA Motif
Non-coding DNA sequences are considered to be a
new challenge for molecular genetics, genomics,
transcriptomics and proteomics. Studying them is
exceptionally important for full understanding of
biological processes under normal and pathological
conditions. According to the report published by the
international project ENCODE in September, 2012,
more than 80% of the non-coding DNA is represented
by different types RNA, regions responsible for histone
modifications, open chromatin regions and transcription
factor binding sites [1]. However, it is not yet fully
understood how the regulatory information is encoded in
DNA in order to define the positions of enhancers,
silencers and other distantly operating regulatory
elements (Noonan J.P., McCallion A.S., 2010;
Lindblad-Toh K, e.a., 2011). Systematic analysis of the
transcription profile provides us with the data on
regulatory mechanisms and screening for the linked
scenario of coding and non-coding DNA interactions
that are basically important for understanding of system
biology and molecular pathogenesis of a variety of
human diseases.
We chose oxidative stress as a model of non-specific
pathogenesis. It was previously shown that a single-time
treatment of newborn rats with high oxygen pressure
modifies intracellular metabolism and results in
formation of basically new ratio of pro- and antioxidant
activities in the organism. Moreover, this newly formed
ratio seems to be stable and may be observed in the first
generation progeny [2, 3]. Cells of newborn animals
appear to be better used to variable physiological
conditions compared to mature cells. It is suggested that
treatment of newborn animals with low dose hyperbaric
oxygenation will facilitate the formation of a wide
spectrum oxygen sensitivity of a neural network that, in
turn, will lead to increase in resistance of animals to
oxidative stress in further ontogenesis [4]. Full genomic
analysis of the transcribed sequences in the brain tissue
of these animals revealed that genes, which form at least
one expression genetic pattern and regulate six key
processes including oxidative metabolism, synaptic
transmission, intracellular transport, apoptosis and
proliferation, membrane permeability membrane
potentials and intercellular contact formation, are
involved into the early genetic mechanisms of
preadaptation to oxidative stress [7].
Objectives: The present study was aimed at
bioinfo rmatics screening of new regulatory structures in
the promoter and cis-regulatory regions of the
non-coding DNA, which participate in gene expression
in response to oxidative stress.
Shkurat TP et al. / Open Journal of Genetics 2 (2012) 1-4
Copyright © 2012 SciRes. OJGen
The object of the study were white outbreed rats
Rattus norvegicus treated with high oxygen pressure (0.2
MPa, 1 h). Animals were sacrificed in 3 h after the
treatment and gene expression was studied in the frontal
lobe. The results of full genomic transcriptome study
were previously described in [5].
To perform bioinformatic analysis two groups of
genes were formed. The first group involved genes
up-regulated in response to oxidative stress. The second
group consisted of genes with unchanged expression.
The first grouped was formed by genes
NM_017138:Lamr1, NM_053440:Stmn2,
NM_057207:Sv2b, NM_053339:Acox3, Scn7a, Trpc3,
Nid2:NM_213627, Herc1, Ssc1:BC085795,
Golph2:NM_023977, Actr1a, Crebzf, Pdk4:NM_053551,
Mrpl3, Api5, Zfhx1b, Snrpb:BC083694, Snrpb. The
second group included all genes of the chromosome 20
of R. norvegicus.
Regulatory regions of 264 genes available in
KnownGene with exception of the first and the last gene
were studied.
Two types of regulatory elements were studied in both
groups. These included the promoter regions (1000, 800
b. p. upstream the start point and 200 b. p. downstream
the termination point) and the sequences, overlapping
the potential cys-regulatory elements (5000, 4000 b. p.
upstream the start point and 1000 b. p. downstream the
termination point)
The bioinformatic screening was seeking DNA motifs
located near the first group genes. Here, the “recognition
motif” implies the way to describe a set of similar
oligonucleotides, which can be specifically recognized
and bound by a certain regulatory protein.
To search the motifs we created an integrative
collection of the binding transcription sites on the basis
TF Homo sapiens and Uniprobe TF Mus musculus
databases. The integrative collection Uniprobe included
272 motifs for transcription factors of M. musculus. The
collection Нocomoco TF H. sapiens, which was
obtained by means of integration of the data from
different sources, included 332 motives for 321
transcription factor. To perform bioinformatic analysis
we chose the release of R. norvegicus genome rn4
(Baylor 3.4/rn4)
[http :// genome. uc sc.e du/ c gi-bi n/h gGat e wa y].
The conception of homotypic transcription factor
binding site clusters, which were represented by a group
of transcription factor binding site, was used as a model
of a binding region. To assess the statistical confidence
of a binding site cluster the “r-scan” model with fixed
motive acceptance threshold was used (Papatsenko D.,
2007). The statistical confidence of the “r-scan” was
assessed as a probability to find at least the expected
number of transcription factor binding site clusters in a
sequence of certain length on account that at least 1
transcription factor binding site was already found, i.e.
one cluster contains at least one binding site.
In order to perform reliable assessment of the
presence of homotypic clusters they were counted in the
first and second group. We assessed the chance (p) to
choose such a gene, the regulatory region of which
would contain at least one cluster (for promoters) and a
cluster with a confidence of at least 0.0005 for
cis-regulatory elements.
The p value can be assessed using the binomial
distribution as a confidence of the fact that in the studied
sample the transcription factor binding site clusters
contain at least k number of regulatory regions, where k
corresponds to experimentally found number of
sequences which contain the transcription factor binding
site clusters (for promoters) / at least 0.0005 confident
transcription factor binding site clusters for
cis-regulatory elements.
It was shown that transcription factor binding sites of
the SOX subfamily more often preceded the first group
of genes (Table 1). The SOX subfamily involves 30
transcription factors, which contain heptamerous
sequence (A/T) (A/T) CAA(A/T)G (Fig. 1) [5]. It is
considered to be one of the most important subfamilies,
which regulate development of both vertebrate and
invertebrate animals. Biological functions of these
proteins were investigated in a variety of mammalian
tissues and cells during embryogenesis and
postembryonic development. It is suggested that
differentiation of the proteins for groups is typically due
to specificity of their functioning in different tissues.
They initiate differentiation program and activate tissue
specific expression. The SOX subfamily is represented
by multifunctional proteins. Among multiple roles that
SOX proteins play in cells the transcription factors can
either induce or suppress a variety of cellular processes.
Moreover, it was shown that SOX proteins are involved
in DNA reparation processes.
HXA3, hepoxilin A3; in the absence of cytosol glu-
tathione peroxidase is transformed into 12S-hydroper-
oxyeicosatetraenoic acid on biologically active epoxides.
The process is catalyzed by lipoxygenase.
HLX is expressed in mesodermal tissues in embryo-
genesis. Especially high expression was observed in
Shkurat TP et al. / Open Journal of Genetic 2 (2012) 1-4
Copyright © 2012 SciRes. OJGen
visceral mesenchyme, skeletal myoblasts and limb mes-
Gata 3 regulates the expression of genes, which are
involved in cell growth control, immune functions and
adipogenesis inhibition.
Table 1. Confident differences of transcription factor localization prior to the first group genes.
P <0.05
<0.05,r-skan >9
P <0.01
o (s1000)
HXA10, HXA7, DLX3, HXA9, BARH1, SOX15,
SP100, SOX30, GATA3, PO6F1, HXA1
SOX15, SOX30, ZN187, HXD13, PROP1,
SOX18, RX, SRY, SOX7, SOX14,
PO3F2, PO2F1,
SOX13, SOX17,
Figure 1. Consensus sequences of the SOX transcription factors SOX [(A/T) (A/T) CAA(A/T)G[5].
SP100 is a multifunctional protein factor often found
in brain
Therefore, we have found out that primary treatment
with hyperbaric oxygenation (0.2 MPa, 1 h) results in
Shkurat TP et al. / Open Journal of Genetic 2 (2012) 1-4
Copyright © 2012 SciRes. OJGen
up-regulation of a set of transcription factors, which are
associated with the studied genes and their promoters.
The obtained data support the idea of specific and
possibly cooperative interaction of transcription factors
in both promoters and cis-regulatory elements. The me-
chanism of simultaneous gene expression induced by
oxidative stress is not yet clear. It is suggested that the
neighbor sequences of non-coding DNA may be in-
volved in these processes. Our data allow us to conclude
that non-coding DNA somehow participates in
gene -gene interactions. The detailed study of all func-
tions of non-coding DNA is considered to be a challeng-
ing task for biological science.
This work was supported by the Federal Targeted Program by The
Ministry of education and science of Russian Federation, state contract
no. 14.740.11.0006
[1] The ENCODE Project Consortium. An integrated encyc-
lopedia of DNA elements in the human genome.- Nature
(2012) , Vol. 489: р. 57-74.
[2] EP Guskov, TP Shkurat, EI Shimanskaja, SI Guskova
Genetic effects of hyperbaric oxygen therapy// Mutation
Research/Genetic ToxicologyVolume 241, Issue 4, Au-
gust 1990, Pages 341–347.
[3] EP. Guskov, EV Mashkina, NI Belichenko, TV Varduni,
G I Volosovtsova, IO Pokudina, GE Guskov and TP
Shkurat Mutation processes in oxidative stress preadapted
animals\\ Russian Journal of Genetics: Applied Research
Volume 1, Number 2 (2011), 112-118.
[4] EP. Guskov, EV Mashkina, NI Belichenko, TV Varduni,
G I Volosovtsova, IO Pokudina, GE Guskov and TP
Shkurat Mutation processes in oxidative stress preadapted
animals // Ecological Genetics. —2009. V.VII, № 1.
[5] Guimont P, Grondin F, Dubois C: Sox9-dependent tran-
scriptional regulation of the proprotein convertase furin.
American journal of physiology. Cell physiology 2007,
[6] Allen, J.D., T. Lints, N.A. Jenkins, N.G. Copeland, A.
Strasser,R.P. Harvey, and J.A. Adams. 1991. Novel mu-
rine homeo box gene on chromosome 1 expressed in spe-
cific hematopoietic lineages and during embryogenesis.
Genes & Dev.5: 509-520.
[7] T. P. Shkurat, A. V. Shestopalov, G. E. Guskov, V. N.
Prokofiev, A. I. Butenko, T. V. Belik, M. Yu. Bibov, O.
V. Lyangasova, M. A. Shkurat, E. V. Mashkina, S. S.
Mandzhieva, E. V. Butenko, I. N. Sevastianova, P.
Ryzhkov, E. M. Vechkanov Study of novel mechanisms
of brain preadaptation to oxidative stress using DNA mi-
croarray // Problems of contemporary science and educa-
tion. 2011. № 5; URL: .