The Coherent Feedforward Loop Serves as a Sign-sensitive Delay Element

1. The Coherent Feedforward Loop Serves as a Sign-sensitive Delay Element In Transcription Networks S. Mangan, A. Zaslaver and U. Alon Weizmann Institute of Science, Israel Presented by Anushri Prabhu

2. background • DNA makes up the human genome. DNA is subdivided into information bytes called genes. Genes encode unique proteins. Each protein performs a specialized function in the cell. • HOW DOES A GENE PRODUCE A PROTEIN? • Cells use the two-step process of TRANSCRIPTION and TRANSLATION to read each gene and produce the string of amino acids that make up the unique protein. • TRANSCRIPTION– The cell’s machinery in the nucleus copies the gene sequence into the messenger RNA or mRNA. • TRANSLATION– The protein making machinery reads the mRNA sequence and translates it into the amino acid sequence of the protein.

3. These patterns are called Network Motifs. Cells respond to external stimuli. HOW? By using Regulatory Networks. WHAT? A network of DNA segments in a cell that interact with each other and with substances in the cell. WHY? Helps govern the expression levels of mRNA and proteins. WHAT DO SCIENTISTS WANT TO DO? Understand the structure of these networks. They know these networks are complex. HOW CAN THEY BE SIMPLIFIED, THEN? By studying the network in terms of recurring circuit patterns.

4. introduction • TRANSCRIPTION REGULATORY NETWORK • A transcription regulatory network works to control the rate of transcription. • While analyzing the structure of this network, the authors identified several network motifs. • Recurring regulatory patterns • Resemble circuits • Have specific functions • Occur more frequently than in randomizedcontrol networks Production of Gene Products

5. They studied Escherichia coli and Saccharomyces cerevisiaeand found several highly significant network motifs. • The most significant network motif they found present in both E.coli and yeast is the FEEDFORWARD LOOP (FFL). • Before we can learn about the FFL, we need to learn about another concept – transcription factor. • TRANSCRIPTION FACTOR • It is a substance such as a protein that binds to specific DNA sequences. • This helps control the flow of genetic information from DNA to mRNA. • In other words, it helps control transcription.

6. Feedforward loop • The FFL is composed of a transcription factor X, which regulates a second transcription factor Y, such that X and Y jointly regulate the gene Z. The transcription factors X and Y usually have inducers Sx and Sy. These inducers are small molecules, protein partners or covalent modifications that activate or inhibit their transcriptional activity. The action of X and Y is integrated at Z with a cis-regulatory input function. The FFL has eight possible structural types, because each of the three interactions in the FFL can be activating (positive) or repressing (negative).

7. CIS-regulatory logic • Cis-regulatory logic also comes into play when trying to understand the regulation of Z. • WHAT IS A CIS-REGULATORY MODULE? • A stretch of DNA where a number of transcription factors can bind and regulate expression of nearby genes. • Labelled cisbecause the transcription factors are typically located on the same DNA as the genes they control. cis-regulatory module Transcription factors Transcription machinery Rate of gene transcription

8. X Z Y X Z Y Cis-regulatory logic combines the two inputs X and Y such that: X and Y are both needed to activate Z : AND-gatelogic Either X or Y is sufficient to activate Z : OR-gate logic

9. COHERENT FFL • Four of the 8 FFL are termed coherent FFL because: • The sign of the direct regulation path (from X to Z) is the same as the sign of the indirect regulation path (from X through Y to Z) • They act as sign-sensitive delays • This means they slow down the response time of the target gene expression • But this happens by following stimulus steps only in one directione.g. OFF to ON, but not in the other direction OFF to ON

10. INCOHERENT FFL • The other four FFL are termed incoherent FFL because: • The sign of the direct regulation path (from X to Z) is opposite to the sign of the indirect regulation path (from X through Y to Z) • They act as sign-sensitive accelerators • This means they speed up the response time of the target gene expression • But this happens by following stimulus steps only in one directione.g. OFF to ON, but not in the other direction OFF to ON

11. COHERENT & INCOHERENT FFL X through Y to Z X to Z Coherent Incoherent +  activating -  repressing

12. SIGN-sensitive delay • The authors suggest 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. • The response time to stimulus is not symmetric. • Thus, the FFL is a sign-sensitive delay element: it responds rapidly to stimulus of Sx in one direction (ON to OFF), and at a delay in the opposite direction (OFF to ON).

13. Revisiting the title • The Coherent Feedforward Loop Serves as a Sign-sensitive Delay Element in Transcription Networks • Coherent FFL – sign of the path from X to Z is the same as the sign of the path from X through Y to Z. • Sign-sensitive Delay – They slow down the response time of the target gene expression i.e. Z in only one direction. • Transcription Networks – A transcription regulatory network works to control the rate of transcription.

14. Where’s the proof? • The authors set up an experiment to prove their premise. • They found that the FFL with three positive interactions, termed the type-1 coherent FFL, is the most common configuration in E.coli. They chose the L-arabinose (ara) utilization systemin E.coli. araBAD and araFGH are regulated by two transcription factors araC and CRP. AraC acts as an activator when it binds L-arabinose, and as a repressor in its absence. CRP acts as an activator when it binds the inducer cyclic AMP (cAMP). CRP binds araC and enhances transcription.

16. CONTROL SYSTEM • The authors needed a control for the experiment. • It needed to be a non-FFL system with the same input Sx. They chose the lactose (lac) utilization system. lacZYAis regulated by two transcription factors CRP and lacl. There is no transcription regulation of Lacl by CRP.

17. Output measure • They generated low-copy reporter plasmids in which the araBAD, araFGH and lacZYA control the green fluorescent protein (gfp) gene. • The gfp variant was chosen because of its ability to become fluorescent within a few minutes of transcription. This allowed for rapid responses to be measured. Green fluorescent protein (GFP) fluorescence and optical absorbance (A600 nm) were measured about once per minute. These measurements were used to determine the dynamics of GFP concentration produced by araBAD, araFGHand lacZYA.

18. RESULTS • The arasystem is a type-1 coherent FFL with AND logic • To function as a type-1 coherent FFL, araC(Y) needs to activated significantly by CRP (X) in the presence of cAMP(Sx). • They measured araC promoter activity with and without cAMP, in the presence of L-arabinose (Sy). • cAMP is a molecule that is produced within the cell upon glucose starvation (e.g. when glycerol is the only carbon source the cell can grow on). • Levels of cAMP were controlled in two ways: • Cells growing on glucose (low cAMP) were compared to cells growing on glycerol (high cAMP) • Cells growing on glucose (low cAMP) were compared to cells growing on glucose with saturating exogenouscAMP(high cAMP)

19. The ara system is a type-1-AND-gate FFL. Similar results were observed in both cases. araC exhibited a measurable basal level of expression in the presence of L-arabinose and in the absence of cAMP. araC promoter activity increased at least four- to five-fold above this basal level in the presence of cAMP. The activity of araBAD, araFGH and lacZYA under 0 to saturating levels of the inducers was measured. The expression in the presence of both inducers is greater than when either or both inducers are missing. It behaves as an AND-gate with respect to inputs Sx = cAMP and Sy = L-arabinose (or Sy = IPTG in case of lac system).

20. + cAMP Low cAMP High cAMP OFF ON X Y Z X Y Z + glucose Low cAMP High cAMP OFF ON X Y Z X Y Z The ara system acts as a sign-sensitive delay with respect to cAMP steps The authors generated the ON step by adding saturating cAMP to cells growing exponentially on glucose minimal medium (low cAMP). The authors generated the OFF step by adding saturating glucose to cells growing exponentially in glycerol minimal medium (high cAMP).

21. They found the following: The ON response of araBAD and araFGH was significantly slowerthan the ON response of lacZYA.

22. Asymmetric behavior with delayed responses to cAMP ON steps but not OFF steps is hallmark of sign-sensitive delay. The OFF response of araBAD and araFGH was more rapid, and identical with that of lacZYA.

23. At a large basal level of Y: The response tends to become more symmetric. The delay in onset of Z expression reduces. The sign-sensitive delay effect is lost. Simulations on the effect of araC basal level The delay following an ON step decreases as the basal level of Y in the expression increases.

24. DISCUSSION • Biological function of sign-sensitive delay • Why has it not been lost by evolutionary forces? • Coherent FFL in transcription networks of microorganisms and animals • Coherent FFL in E.coli appear in systems that respond to stimuli like glucose, nitrogen and drugs. • Systems responsive to similar stimuli in S. cerevisiae also display FFL. • Possibility of convergent evolution to the same regulatory circuit? • FFL in other biological networks • FFL is found in the network of synaptic connections of C.elegans neurons • Found in protein-protein interaction networks

25. THE END • And they lived happily ever after! THANK YOU