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How multiple NFAT5 sites can impact to the NFAT5-inducible gene expression: an approximate solution of an ill-posed inverse problem Petr M. Ponomarenko 1,*, Mikhail P. Ponomarenko 1, Niko lay A. Ko lchanov1
1

,2,3

,

Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia; 2Novosibirsk State University, Novosibirsk, Russia; 3National Research Centre "Kurchatov Institute", Moscow, Russia; *pon@bionet.nsc.ru

Birgit Eisenhaber4 and Frank Eisenhaber
4

4,5,6

Bioinformatics Institute; A*STA R; Singapore; 5Department of Biological Sciences; National University of Singapore; 6School of Computer Engineering; Nanyang Technological University; Singapore; franke@bii.a-star.edu.sg

The transcript ion factor NFAT5 (Nuclear Factor of Activated T-cells 5; syno nyms: TonEBP and OREBP as Tonicit y E lement-Binding Protein and Osmot ic Response Element-Binding Protein, respectively) is a master-gene for the osmotic stress response. It is, therefore, crit ica l for many developmental and regeneration processes such as wound healing and it is known that certain drugs interfere with this pathway [ 1]. Mult iple response elements (RE) within the regulatory gene regio ns are characteristic of the glucocorticoid, steroid, thyro id, auxin (AuxRE), and many other hormones as well as of the heat shock, hypertonic, osmotic, and many other stresses. There is few data on the mo lecular mechanisms underlying how mult iple REs contribute to gene expressio n whereas a bulk of the data on the only single RE is available. In our previous work [2], we have introduced an approximate solution of an ill-posed inverse problem, of dissect ing the expressio n, , of a given gene originat ing from different variants of mult iple REs and to decode the sequence-activit y relat ionships of each of them: ; here: 0, a basal expressio n level; Pk, a probabilit y of k fro m amount N REs, 1
... k NPk

(1) 1; N>1;

is an impact of a given set of -th, -th, ... and -th REs into the induced expressio n level. {2, 3,

As for N REs Eq. (1) contains 2N variables, the optimal size of independent experimental data for its estimat ion by the mult iple linear regression is 2 2N, i.e. 16, 64, 256, ... at N 4, ...} that is unavailable. But two extreme cases, addit ive {P1=1; P
k>1

=0} and mult iplicat ive


{P

k
=0; PN=1}, are solvable by the standard tools STATISTICA, the last case by Sandwich

theorem. By this way we found the insignificant addit ive and significant mult iplicat ive impacts of the mult iple AuxREs into the expressio n level magnitudes o f three auxin-responsive genes in plants, and a repressio n of these genes wit hout auxin [ 2] that is impossible to do otherwise. In this work we analyzed six independent datasets on the mult iple NFAT5 binding sites both wild-typed and subjected to genetic manipulatio ns wit hin promoters of six genes, namely: HSP70-2 (heat shock protein 70-2) in human and in mouse, AR (aldose reductase) in huma n and in rat, SMIT (sodium/myo-inosito l cotransporter) in human, AQP2 (aquaporin) in mouse. As an example, upon 19 probes of four TonREs - A, B, C, D, - of murine HSP70-2 promoter, x, upstream Luc (luciferase) gene of pGL2 plasmid by which the murine IMCD cells were transfected [3], we found that both addit ive (a) and mult iplicat ive (m) impacts of these TonREs into luciferase activit y, , are significant: {
D (a) 0 (a)

=1.0,
D (m)

(a) A

=5.2,

(a) B

=3.6,

(a) C

=-6.3,
(a)

=2.7} and
(m)

0

(m)

=0.5,
(a)

(m) A

=7.3,
(m)

(m) B

=3.8,

(m) C

=0.2,

=2.3}. There are the significant (x))

correlat ions between and (

and

values (r=0.98, <0.025), between reconstructions (

(x)) based on each of them (r=0.96, <0.05), and, also (r=0.98, <0.025), between and their jo int reconstruction (x) using Eq.(2), (true)=1 and (false)=0, as: (2)

the magnitudes

Both negat ive

(a) C

=-6.3 addit ive and fract ional

(m) C

=0.2 mult iplicat ive impacts mean the

repressive affect of TonRE-C to murine HSP70-2 that was consciously lost earlier [3] because it is immeasurable experimentally against three rest TonREs activat ing this gene. Finally, we confirmed the repressio n of HSP70-2 using an independently derived human dataset [4]. We thank grants RFBR 11-04-01888, 13-04-00359, SciSh-5278.2012.4, Russian Ministry of Education & Science Project 8740, and RAS Programs "Mo lecular and cellular bio logy". 1. L.K.Schenk et al. (2010) Cell Physiol Biochem, 126:887-900. 2. V.V.Mironova et al. (2013) J Bioinform Comput Biol., 11:1340011. 3. S.K.Woo et al. (2002) Mol Cell Biol., 22: 5753-5760. 4. J.I.Heo et al. (2006) Exp Mol Med., 38:295-301.