A minimal model for the rhythmic protein per expression in Drosophila

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A minimal model for the rhythmic protein per expression in Drosophila

DEGLI AGOSTI, Robert

DEGLI AGOSTI, Robert. A minimal model for the rhythmic protein per expression in Drosophila.

In: Greppin H., Penel C., Broughton W, Strasser R.J. Integrated plant systems . Genève : University of Geneva, 2000. p. 229-236

Available at:

http://archive-ouverte.unige.ch/unige:42875

Disclaimer: layout of this document may differ from the published version.

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A minimal model for the rhythmic protein per expression in Drosophila

R. DEGLI AGOSTI

Laboratory of Plant Biochemistry and Physiology, Plant Physiomatics, University of Geneva, 3 Place de l'Université, CH-1211 Genève 4, Switzerland E-mail:

Robert.DegliAgosti@bota. uni ge. ch

Introduction

Metabolic models with negative feedback regulation have been since a long time proposed to explain sorne rhythmic phenomena's [ 4, 12]. They were temporarily discarded from consideration in circadian rhythms on the basis of theoretical grounds [see 2, for a discussion of this aspect]. However, it clearly appears now from experimental results obtained in the insect Drosophila, the fungus Neurospora and man that such kind of feedback's are involved in circadian rhythms or even constituting the core of biological clocks [ 1,2,5,8]. Among the current most studied systems is the Drosophi/a one. An important gene has been identified early and found to be closely involved in the timing mechanisms: the per gene. Its mRNA and the corresponding per protein oscillate in a circadian ( -24 h) manner. Since then, other genes have also been identified [7,9,11]. We use here the concept of minimal modeling approach to model the per rhythmic circadian expression with the minimum number of variables (elements or state variables). W e will use the method of metabolic network modeling with a negative feedback regulation [2].

The purpose of this paper is to illustrate how we can construct such models, starting from experimental data, to fmally obtain coherent simulations on computers. This is not so trivial since from the experimental model (Fig.1) it is not intuitively clear how this can effectively lead to a correct simulation of the observed (measured) variables.

In other words, how many people when observing Figure 1 could positively answer to the question: is this model oscillating with a circadian (-24 h) rhythmicity? To answer this question one must construct a mathematical model, obtain the evaluation of parameters and then simulate it. To be precise, we should mention here that methods do exist that extract values of parameters from raw data with just the mathematical model only ( e.g. by non linear multiple least squares regression). This method will allow to estimate parameters where often not enough experimental data is available and is therefore very useful. However, the drawback of this is that the simulation will be a little biased to necessarily work with artificially estimated parameters. ln this

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paper we will follow the frrst method, namely obtain successively each parameters estimation from experimental data available.

1. The model

From an early "experimental" biological model presented in the literature for the per gene regulation in Drosophi/a the following network is presented in Figure 1.

Figure 1. Early model of the per protein regulation involved in circadian rhythms in Drosophila. Very simplified metabolic network of the of protein per regulation according to [5]. RNA and per represent the relative concentrations of mRNA for the per protein and the protein per itself, respectively. RNA is synthesized with a basic flux (v o> controlled by the inhibition with the per protein (negative feedback f(pery).

The RNA is degraded with a first order kinetic law (k1). The synthesis of the protein per depends on the. RNA concentration according to a first order kinetics reaction law (k2), a delay has been experimentally observed in this reaction (-8 h). Finally the per protein is destroyed with a first order kinetics reaction law (k3).

The corresponding differentiai equations that allow the modeling and the simulation of the dynamics of the concentrations of the 2 substances may be written as follows:

d.RNA(t) v0f(per(t))- k1RNA(t) dt

dper(t) = k

2RNA(t- delay)- k

3per(t) dt .

2. Evaluation of the model parameters

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The data which will allow us to evaluate the parameters of the models are available in the literature. W e will describe this important aspect in details here for illustrating purposes. Data for RNA is found in [5] and for per protein in [13]. We have normalized the original values to "normalized units of concentrations (n.u.c)", by eliminating baselines and dividing each protein or mRNA per value by their respective maximal values. They are presented in Figure 2.

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Subj. Light

-

~

=! 0.8

.s

tA 1: 0.6

ii

0 Loo

-

1: cu u 0.4

1: 0 0.2

0

0.0

0 4

a

12 16 20 24

Time (hours)

Figure 2. Experimental data expressed in normalized units of concentrations (n.u.c) of the per protein and per mRNA according to [13] and [6]. One might observe that the synthesis and degradation of per rn RNA are triggered around a threshold of 0.2 n.u.c. of perprotein.

2.1 The type of regula tory fonction

The regulatory function (negative feedback) of per mRNA synthesis may be identified directly by a closer look to these experimental results. Indeed, when a concentration higher of 0.2 n.u.c. of per is present the synth~sis is switched off.

Whereas, when a concentration lower than 0.2 n.u.c is present the synthesis is switched on (degradation is always active). This corresponds typically to one· of the most simple threshold regulatory function as follows:

f(per(t)) = {1 if per(t) ~ 0.2 0 if per(t) > 0.2 2.2 Estimating k1

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When the synthesis ofmRNA is inhibited (whenper> 0.2 n.u.c), then using (3), the frrst term in equation (1) becomes 0, and we have:

dARN(t) -k ARN(t)

dt 1 (4)

Which after integration gives the weil known solution:

(5) This is a frrst order decreasing kinetics. From the original data one plots the natural logarithm as function of the time and estimates k1 from the slope (Fig. 3) as 0.402 ± 0.049 h-1 (correlation= 0.95).

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R. Degli Agosti

0.5 . . . . - - - -- - - - -- - - , -0.5

~ -1.5

~

.5

-2.5

-3.5

corr. = 0.95 0

0

-4.5 +---.----r---r----r---~---1

0 5 10 15 20 25 30

Time (hours)

2.3 Estimating vo

Figure 3. Evaluation of the per mRNA degra- dation kinetics constant k1 by the slope of the RNA degradation. Data is the same as in Figure 2, but plotted on a natural logarithmic scale. The correlation coef-ficient is 0.95.

When the synthesis ofper mRNA is switched on (i.e.,f(per(t)= 1), the equation (1) becomes:

dRNA(t) = U

0 - 0.402RNA (t) dt

Which after integration gives:

(1-eo.402t) RNA(t) = V0 ~----'-

0.402

This could be linearized in:

RNA(t)

=

u0B(t)

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(8) Thus, the plot of experimentally measured RNA as a fonction of calculated B(t) should be aline and its slope will be v0• It equals to 0.358 ± 0.015 n.u.c. h-1 (Fig. 4).

1 ~ 0~

Figure 4. Plot of the

,

Cl)

...

::1 en 0.8 ~ corr.

=

0.93 from Figure 2) as a function measured per mRNA (data

0.6 0 of theoretical B(t) (see text).

"'

Cl)

E 0.4 The slope of the line is v0.

<( The correlation coefficient is

z 0:::

0.2- 0.93.

0 0

0 0.5 1.5 2 2.5 3

B(t) function

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2.4 Estimating the delay

The delay value is an experimentally observed feature. It represents a time latency between the observed maxima of RNA and per protein (see [5]), delay= -8 h.

2.5 Evaluation of k2 and k3•

These evaluations are more difficult to obtain, since datais far less precise (see Fig.

2). It is possible to obtain an estimation of k3 (per protein degradation) from Figure 2 by estimating that the half-life (t112) of the degradation kinetics is -2 h. This represents a k3 of 0.35 h-1 (k3 = ln(2)/t112). A rough estimation of k2 is obtained if we consider that a steady state is observed at 1.0 n.u.c. Indeed, at per steady state (per(teq)), equation (2) becomes:

(9) per(teq) = 1.0 n.u.c, k3 =0.35 h-1, the value of RNA(teq-8) can be estimated directly from Figure 2, since per(teq) is observed at- 22 to 24 h, then RNA(teq-8) at 14 to 16 his 1.0 n.u.c. Substituting these numbers in (9), it cornes that k2 = 0.35 h-1.

3. Model simulation

Once each parameter has been estimated, the dynamics of the system is described by the following equations:

~:(t)

0.358/(per(t))- 0.402RNA(t)

dper(t) = 0.35RNA(t-8)- 0.35 per(t) dt

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The regulation function f(per(t)) is defmed by (3). It is clear that, from these equations, it is rather difficult to intuitively see if they oscillate and, more difficult even, is at which period. One way is to simulate their resolution by numerical methods (numerical integration by a runge-kutta 4th order method, e.g.) in order to obtain the RNA and per dynamics.

The simulation is presented in Figure 5: the system oscillates with a periodicity of -26 h, which is very near the one experimentally observed (-24 h). A rough sensitivity study has been realized by varying sorne parameters to check the stability of the model. With a delay of 6 h instead of 8 h, the period is still circadian (21.6 h).

Changing k2 = k3 (actually at 0.35 h-1) to 0.175 h-1 orto 0.70 h-1 gives a periodicity of 29.8 h and 23.2 h respectively. The model is thus robust with respect to changes in cri ti cal parameters (particularly the ones that were the most difficult to evalua te).

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1.0 0.8 0.6 0.4 0.2

0.0

0 10 20 30 40 50

Time (hours)

Figure 5. Simulation of the per protein and its mRNA dynamics in the Drosophila model. The period of oscillation is circadian (26 h). The conœptual and mathematical (before and after evaluation of the parameters from the experimental data) are presented in Figure 1, equations (1 & 2) and equations (3,10 & 11) respectively.

4. Comparison between real and simulated data

Figure 6 shows the comparison between the original and simulated data for per mRNA (see also Figure 2). From visual inspection the fitting seems to be rather correct.

• 0 1.0

~

"2

0.8

5-ro

~ :J 0.6

cv

E E lii 0.4

<(<(

z z

0.2

~~

0.0

0 10

Time (hours)

Figure 6. Comparison between the original experimental data of per mRNA and the results of the simulated per mRNA according to equations (3, 10, 11). Units of mRNA are in n.u.c.

Conclusion

More recent works have shown that the oscillation of the per protein is also dependent of another essential element: the protein tim [7, 9, 11 ], that possess a specifie gene distinct from per. As it is possible to observe in this work, the model we have elaborated doesn't contain these informations. However, it seems to rather correctly

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describe the endogenous oscillation. The model is systemic, it possess a great power of simplification (aggregation) of a complex phenomenon. This choice is deliberate.

More precisely, the existence of a delay factor is actually corresponding to a lot of molecular 1 physiological 1 biochemical reactions that were introduced in this kind of model only by their fmal simplified result, but with a correct dynamical equivalent.

IndeecL as sorne authors have suggested, this delay might be explained by the necessity for per to be stabilized by tim for the transport of this complex from the cytoplasm to the nucleus [9].

In other words, in order to elaborate a functional (imperfect, but useful) systemic model, it is not necessary to know ali the detailed molecular mechanisms of the phenomenon, but only sorne essential dynamical characteristics, only sorne aggregating parameters are needed. It is clear that the identification of such key parameters is of the utmost importance for modeling complex phenomena's with minimal systemic modeling. Other important domains were complexity is the rule like ecological modeling are using this kind of approach with success since a long time now [10].

There are little doubts that once any system has been somehow reduced in sorne of its components, one needs again someway to integrate them in order to test the modeled "behavior" with the experimental observations. One method, among others, to achieve integrated approaches is by modeling and simulating techniques.

References

1. Aronson BD, Johnson KA, Loros JJ, Dunlap JC (1994) Negative feedback defming a circadian clock: autoregulation of the clock gene frequency. Science 263: 1578-1584

2. Degli Agosti R, Greppin H (1996) Simulation ofrhythmic processes: rhythms as a result of network properties. In: Greppin H, Degli Agosti R & Bonzon M, eds, Vistas on Biorhythmicity, University ofGeneva, Geneva, pp 157-172

3. Goldbeter A (1990) Rythmes et chaos dans les systèmes biochimiques cellulaires, Masson, Paris

4. Goodwin B (1963) Temporal Organization in Cells, Acad Press, London

S. Hardin PE, Hall JC, Rosbash M (1990) Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature 343: 536-540 6. Hardin PE, Hall JC, Rosbash M (1992) Circadian oscillations in period gene

mRNA levels are transcriptionally regulated. Proc Nat Acad Sei USA 89: 11711- 11715

7. Lee C, Parikh V, Itsukaichi T, Bae K, Edery 1 (1996) Resetting the Drosophila clock by photic regulation of PER and a PER-TIM complex. Science 217: 1740-

1744

8. Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P (1993) lnducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75: 875-886

9. Myers MP, Wager-Smith K, Rothenfluh-Hilfiker A, Young MW (1996) Light- induced degradation of TIMELESS and entertainment of the Drosophila circadian clock. Science 217: 1736-1740

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10. Odum HT (1994) Ecological and General Systems. Revised edition, Univ Press Colorado, Colorado

11.Seghal A, Priee JL, Man B, Young MW (1994) Loss of circadian behavioural rhythms and per RNA oscillations in the Drosophila mutant timeless. Science 263:

1603-1606

12. Viniegra-Gonzales G (1973) Stability properties of metabolic pathways with feedback inhibition. In: Chance B, Pye EK, Ghosh AK & Hess B, eds, Biological and Biochemical Oscillators, Academie Press, New York, pp 41-59

13.Zerr DM, Hall JC, Rosbash M, Siwicki KK (1990) Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila. J Neurosci 10: 2749-2762

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