Dissecting the logical types of network control in gene expression profiles
© Marr et al; licensee BioMed Central Ltd. 2008
Received: 25 September 2007
Accepted: 19 February 2008
Published: 19 February 2008
In the bacterium Escherichia coli the transcriptional regulation of gene expression involves both dedicated regulators binding specific DNA sites with high affinity and also global regulators – abundant DNA architectural proteins of the bacterial nucleoid binding multiple sites with a wide range of affinities and thus modulating the superhelical density of DNA. The first form of transcriptional regulation is predominantly pairwise and specific, representing digitial control, while the second form is (in strength and distribution) continuous, representing analog control.
Here we look at the properties of effective networks derived from significant gene expression changes under variation of the two forms of control and find that upon limitations of one type of control (caused e.g. by mutation of a global DNA architectural factor) the other type can compensate for compromised regulation. Mutations of global regulators significantly enhance the digital control, whereas in the presence of global DNA architectural proteins regulation is mostly of the analog type, coupling spatially neighboring genomic loci. Taken together our data suggest that two logically distinct – digital and analog – types of control are balancing each other.
By revealing two distinct logical types of control, our approach provides basic insights into both the organizational principles of transcriptional regulation and the mechanisms buffering genetic flexibility. We anticipate that the general concept of distinguishing logical types of control will apply to many complex biological networks.
One important objection to Lamarckian evolution by inheritance of acquired characteristics emphasized by Bateson over forty years ago is the reduction of adaptational flexibility upon progressive specialization, necessitating the occurrence of genotypic changes compensating for this limitation . In unicellular organisms such as bacteria, in keeping with Batesons' prediction the same acquired mutations beneficial in one environment can be restrictive in another . At the same time, evolving Escherichia coli populations can demonstrate remarkable flexibility in genetic adaptation . The mechanisms sustaining this flexibility remain unclear. In order to understand the genetic flexibility it is essential to decipher the organizational logic of transcriptional control. For the classical model organism E. coli the largest electronically accessible network integrating the data on the transcriptional regulation of genes is available . The interlinked elements form a complex structure, which is essentially of digital nature (digital refers here to the fact that the network provides static information on the connections between unique, discontinuous components , e.g. a particular pair of regulating and regulated gene). Notably, such pair-wise connections are not necessarily reflected in genomic expression profiles [6, 7] indicating that not all the interactions given in the network occur at all times. Furthermore, this type of network does not account for the analog mode of gene regulation via alterations of DNA topology – a long known control mechanism revived by recent DNA microarray analyses [8–10] (analog refers here to the fact that the expression of specific genes is under the control of continuous information provided by spatial distributions of supercoiling energy in the genome ). Indeed, transcriptional responses to alterations of DNA superhelicity reveal non-trivial spatial patterns, raising new questions on the coordination of genomic transcription [9, 11] and the interplay between chromosomal organization and patterns in gene expression is now becoming the focus of computational analyses [12, 13]. From these considerations it is obvious that a holistic theory of transcriptional regulation has to include the relationships between these two logically distinct (digital-binary and analog-continuous) types of information and therefore has to distinguish them in the first place. Although other mechanisms of gene regulation between the binary and continuous extremes can be considered, for understanding the organizational principles of transcriptional regulation we assume a working model here in which the impacts of the two distinct logical types of control – one of digital and another of analog type – are to be clearly distinguished and related to each other.
In the following, we will translate the patterns in gene expression changes observed under systematic variation of the two types of control into effective networks and study their connectivity. The effective networks are derived as subnetworks of two larger (static) networks: (1) the transcriptional regulatory network based upon the action of dedicated transcription factors; (2) spatial proximity of two genes on the circular chromosome.
We will statistically compare the properties of these effective networks with those obtained by random sampling of the static networks with a certain number of expression changes. The core quantity derived from these comparisons is the ratio of connected to isolated nodes (control ratio) and, furthermore, its z-score with respect to the random networks. This z-score we denote the confidence level of the particular control type (control type confidence, CTC).
A unifying approach enabling to combine the data derived by different methodologies is essential for understanding the basic organizational principles of transcriptional regulation, especially since recently transcriptional sub-networks with organizationally distinct architectures have been described . In this study we dissect the logical types of information derived by two established methodologies studying transcriptional regulation based either on TRN analyses, or on the analyses of transcriptional supercoiling response of genomic expression patterns. We denote the information retrieved by assessing directional interactions between the genes in TRN as digital, whereas we denote the information retrieved by assessing the influence of superhelical density on expression patterns as analog. This dissection enables us to present a generic approach allowing both, to distinguish and to assess the relationships between two logically distinct types of transcriptional control.
While this network is intimately involved in spatial organization of transcription in E.coli, spatial organisation of transcription is observed in both, prokaryotes and eukaryotes [21, 22]. In E. coli this phenomenon can be readily rationalized on the basis of topological domains of variable size underlying the organization of bacterial chromosome [23–25]. Indeed, both FIS and H-NS have been directly implicated in formation of topological barriers to supercoil diffusion . Thus the preponderance of analog-type control in the wild type cells compared to mutants lacking FIS and H-NS (see Figure 5) is in keeping with the property of these proteins to stabilize supercoils and modulate the distributions of effective superhelicity in the genome [17, 18]. Furthermore, observed alterations of spatial connectivity by mutations of fis and hns genes are also consistent with long-range effects of these proteins on the three-dimensional structure of DNA [16, 18, 25]. Finally, our GPNs analyses reveal that the control by FIS is more pronounced at low superhelical densities, whereas in the case H-NS the spatial control is more pronounced under conditions of high superhelical density (Figure 4c). These observations are fully consistent with the property of FIS to buffer upon DNA relaxation the activity of strong supercoiling-dependent promoters, such as those involved in ribosome production , whereas H-NS has been shown to predominantly repress the genes requiring high negative superhelicity during the exponential growth phase . We note however, that these differences might also include some variations of mRNA stability. These effects are beyond the concepts (particularly the distinction between the two logical types of control) outlined here and may very well account for some of the expression changes not explained by the TRN or the GPN.
One prediction from the observed interdependence between digital and analog types of transcriptional control is that adaptive mutations in E. coli will affect the determinants of global DNA architecture. Indeed, a recent study of long-term experimental evolution in E. coli unmasking DNA topology as a key target for selection identified fitness-enhancing mutations in topoisomerase and fis genes . Furthermore, such "evolved" populations possess high adaptational flexibility . We propose that the buffering of transcriptional regulation by balancing effects of analog and digital types of control can counteract the reduction of adaptational flexibility caused by accumulation of mutations in bacteria . In this respect it is revealing, that fis is a relatively late acquisition in bacterial evolution , whereas H-NS is implicated in regulating "adaptive" gene rearrangements and minimizing the cost of competitive fitness during horizontal gene transfer [19, 29].
We believe that the general concept of distinguishing logical types of control developed in this study will apply to many complex biological networks. We shall also emphasize that based on our data, reinterpretation of the interactions contained in the E. coli TRN database RegulonDB with respect to both, their digital and analog control characteristics – for example, consideration of the supercoiling sensitivity of the genes – might be a worthwhile extension of this database.
Microarray and network data
Transcript profiling for wild type, fis and hns LZ strains was carried out using E. coli K12 V2 OciChip™ DNA microarray. The genetically engineered E. coli LZ41 and LZ54 strains contain drug-resistant topoisomerase gene alleles enabling to selectively inhibit either DNA gyrase or topoisomerase IV activity and respectively induce either relaxation or high negative supercoiling . The fis and hns mutants of the LZ41 and LZ54 strains were obtained by phage P1 transduction. Introduction of the fis and hns mutations in the LZ41 and LZ54 strains does not substantially alter the global supercoiling response to drug (norfloxacin) addition . Each experiment was performed as two biological replicates with two technical replicates each, resulting in 28 cDNA microarray hybridisations. Scanned array images were quantified and normalized by applying a LOWESS (locally weighted scatterplot smoothing) algorithm to the data within print-tip groups using the TM4 software package . A one-class t-test was applied to replicated experiments to obtain genes with significant changed expression. For all results presented in our article, we used a significance level α = 0.05. However, we find that the results remain unaffected over a wide range of significance levels (0.05 > α > 0.02). DNA microarray data sets have been deposited in the Array Express data bank with the accession number E-TABM-86. For detailed DNA microarray data description and analyses see .
The latest version of the RegulonDB 5.6 data sets  "gene product"  and "regulatory network interactions"  were used for gene proximity network (GPN) and transcriptional regulatory network (TRN) generation, respectively.
Preceding the construction of effective TRNs, dimeric regulatory gene identifiers in the microarray data (flhC, flhD; gatR_1, gatR_2; hupA, hupB; ihfA, ihfB; rcsA, rcsB) were replaced by unique Regulon DB identifiers (flhCflhD; gatR_1gatR_2; hupAhupB; ihfAihfB; rcsArcsB). The effective TRN subnet of a DNA microarray transcript profile is the set of affected genes in the TRN and their regulatory interactions contained in RegulonDB (see Additional file 1 for edge lists of the resulting effective TRNs). Connected components of an effective TRN emerge, if both regulating and regulated genes are affected in the transcript profile (see subnet analysis and Figure 2). Connected and unconnected subnet components were further analysed [see Additional file 1].
Preceding GPN subnet construction, the inter-strain transcript profile data was split up into genes with positive and negative log ratios, respectively. Genes with positive log ratios refer to high transcript levels in wild type background, genes with negative log ratios refer to high transcript levels in fis or hns mutant background. GPN subnets of the split DNA microarray transcript profiles were generated based on genomic position of affected genes together with the proximity threshold t, given in in nucleotide bases (b). All affected genes with spatial distance (here distance is relating to ORF start and stop position) below the selected proximity threshold t were considered as connected. GPN subnets were generated for a meaningful range of 1b <t < 10 kb, resulting in connected genes within an operon scale at t ≈10b, up to completely conntected GPNs for t > 10 kb. Connected and unconnected subnet components were further analysed [see Additional file 2].
For each subnet, the control ratio R was calculated as the number of connected nodes Nconnected (i.e. the size of the connected subnet component) over the number of isolated nodes Nisolated (i.e. the size of the unconnected subnet component), R = Nconnected/Nisolated. The control type confidence, CTC, is the z-score of R, calculated from the mean R and its standard deviation obtained from 10000 runs of the corresponding null model. In the case of the digital null model, the same number of affected nodes was mapped randomly on the TRN (see Figure 2). For the analog null model, the same number of affected genes was mapped randomly on the positions in circular genome.
The robustness of calculated ratios and CTCs was verified by 10% random data replacement with data of all affected genes from the remaining DNA microarray sets (see Figure 3).
control type confidence, GPN, gene proximity network, TRN, transcriptional regulatory network.
CM was supported by a grant of the Darmstadt University of Technology. MG is supported by the DFG grant DFG-MU-2FIS.
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