Journal of Molecular Biology
Volume 334, Issue 2, 21 November 2003, Pages 197-204
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The Coherent Feedforward Loop Serves as a Sign-sensitive Delay Element in Transcription Networks

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Abstract

Recent analysis of the structure of transcription regulation networks revealed several “network motifs”: regulatory circuit patterns that occur much more frequently than in randomized networks. It is important to understand whether these network motifs have specific functions. One of the most significant network motifs is the coherent feedforward loop, in which transcription factor X regulates transcription factor Y, and both jointly regulate gene Z. On the basis of mathematical modeling and simulations, it was suggested that the coherent feedforward loop could serve as a sign-sensitive delay element: a circuit that responds rapidly to step-like stimuli in one direction (e.g. ON to OFF), and at a delay to steps in the opposite direction (OFF to ON). Is this function actually carried out by feedforward loops in living cells? Here, we address this experimentally, using a system with feedforward loop connectivity, the l-arabinose utilization system of Escherichia coli. We measured responses to step-like cAMP stimuli at high temporal resolution and accuracy by means of green fluorescent protein reporters. We show that the arabinose system displays sign-sensitive delay kinetics. This type of kinetics is important for making decisions based on noisy inputs by filtering out fluctuations in input stimuli, yet allowing rapid response. This information-processing function may be performed by the feedforward loop regulation modules that are found in diverse systems from bacteria to humans.

Introduction

Cells compute responses to external stimuli using networks of regulatory interactions. A major goal of biology is to understand the dynamics of these complex networks.1., 2., 3. Great simplification would occur if the network could be studied in terms of recurring circuit elements,4., 5. each with a defined signal-processing function.

Recently, an approach for discovering significantly recurring patterns in networks was introduced.2., 3. This was based on detecting network motifs: connectivity patterns that occur much more frequently than in randomized “control” networks built of the same components as the real network. The transcription network of Escherichia coli was found to contain several highly significant motifs.2 It was then found that Saccharomyces cerevisiae shares the same network motifs.3., 6. One of the most significant motifs in both E. coli and yeast is the feedforward loop (FFL).2 The FFL appears in hundreds of diverse, non-homologous gene systems.2., 3., 6.

The FFL is composed of a transcription factor X, which regulates a second transcription factor Y, such that X and Y jointly regulate gene Z (Figure 1(a)). The transcription factors X and Y usually have inducers, Sx and Sy, respectively, which are small molecules, protein partners or covalent modifications that activate or inhibit their transcriptional activity (Figure 1(a)). Each of the three transcription interactions in the FFL can be either positive (activation) or negative (repression). There are therefore eight possible structural configurations of connection signs. Four of these configurations are termed “coherent”: the sign of the direct regulation path (from X to Z) is the same as the overall sign of the indirect regulation path (from X through Y to Z).2 The other four structures are termed “incoherent”: the signs of the direct and indirect regulation paths are opposite. The FFL with three positive interactions, termed type-1 coherent FFL, is by far the most common configuration in E. coli.7 To understand the regulation of Z, one needs also to specify the cis-regulatory logic8., 9., 10. that combines the two inputs X and Y, such as AND-gate logic, in which both X and Y are needed, and OR-gate logic in which either X or Y is sufficient to activate Z.

In a previous study,2 based on numerical simulations, we suggested that the coherent FFL with AND logic is a processing element that functions as a persistence detector. Only a persistent stimulus of Sx can activate both X and Y, and lead to expression of Z. On the other hand, even a temporary removal of the Sx stimulus leads to a rapid turn-off of Z expression. An equivalent and more intuitive description is that the FFL is a sign-sensitive delay element7: it responds rapidly to step-like stimuli of Sx in one direction (ON to OFF), and at a delay to steps in the opposite direction (OFF to ON). By sign-sensitive delay, we mean that the response time to step-like stimuli is not symmetric and depends on the sign of the step.

Here, we present experimental results that support this premise. We select a representative gene system with an FFL connectivity, we show that this system is a coherent AND-gate FFL (in the sense that Y is regulated significantly by X, and that both X and Y are needed for Z expression). Then, we show that the system displays sign-sensitive delay kinetics. We discuss the biological function of sign-sensitive delay as a filter that can protect the target gene from fluctuations in the input stimuli.

Section snippets

The experimental system

To experimentally study the FFL, we selected one of the best-characterized systems in E. coli, the l-arabinose (ara) utilization system (Figure 1(b)).11., 12., 13., 14., 15. The ara system includes the catabolism operon araBAD, and transporters such as araFGH. Both araBAD and araFGH are regulated transcriptionally by two transcription factors, AraC and CRP. AraC acts as a transcriptional activator when it binds the sugar l-arabinose, and as a repressor in its absence. CRP acts as an activator

Discussion

We chose the most common coherent FFL configuration,7 the type-1 coherent FFL, for experimental study. We employed the ara feedforward loop system, and experimentally studied its dynamic response to cAMP input variations. We found that the ara system responded as a sign-sensitive delay element, with delayed responses to ON steps of cAMP and rapid responses to OFF steps.

Summary

The present study demonstrated experimentally that the FFL network motif carries out a signal-processing function. It would be important to experimentally study other systems with FFL connectivity. The present approach could be used to study the signal-processing roles of other network motifs.

Plasmids and strains

Promoter regions were PCR amplified from MG1655 genomic DNA with the following start and end genomic coordinates:36lacZYA (365438–365669), araC (69973–70452), araBAD (70452–69973), araFGH (1984067–1984952). This included the entire region between open reading frames (ORFs) with an additional 50–150 bp into each of the flanking ORFs. The promoter regions were sub-cloned into XhoI and BamHI sites upstream of a promoterless gfpmut237 gene in a low-copy pSC101-origin plasmid as described.19 The

Acknowledgements

We thank C. Bargmann, D. Ginsberg, O. Hobert, S. Leibler, M. Magnasco, S. Quake, R. Schleif, E. Sontag, and all members of our laboratory for discussions. This work was supported by the Israel Science Foundation, a Minerva Junior Research Group, and the Human Frontiers Science project.

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