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    Open AcceResearch articleFunctional genomic delineation of TLR-induced transcriptional networksRan Elkon1, Chaim Linhart2, Yonit Halperin2, Yosef Shiloh1 and Ron Shamir*2

    Address: 1The David and Inez Myers Laboratory for Genetic Research, Department of Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel and 2School of Computer Science, Tel Aviv University, Tel Aviv 69978, Israel

    Email: Ran Elkon - ranel@post.tau.ac.il; Chaim Linhart - chaiml@post.tau.ac.il; Yonit Halperin - yonithal@post.tau.ac.il; Yosef Shiloh - yossih@post.tau.ac.il; Ron Shamir* - rshamir@post.tau.ac.il

    * Corresponding author Equal contributors

    AbstractBackground: The innate immune system is the first line of defense mechanisms protecting thehost from invading pathogens such as bacteria and viruses. The innate immunity responses aretriggered by recognition of prototypical pathogen components by cellular receptors. Prominentamong these pathogen sensors are Toll-like receptors (TLRs). We sought global delineation oftranscriptional networks induced by TLRs, analyzing four genome-wide expression datasets inmouse and human macrophages stimulated with pathogen-mimetic agents that engage variousTLRs.

    Results: Combining computational analysis of expression profiles and cis-regulatory promotersequences, we dissected the TLR-induced transcriptional program into two major components: thefirst is universally activated by all examined TLRs, and the second is specific to activated TLR3 andTLR4. Our results point to NF-?B and ISRE-binding transcription factors as the key regulators ofthe universal and the TLR3/4-specific responses, respectively, and identify novel putative positiveand negative feedback loops in these transcriptional programs. Analysis of the kinetics of theinduced network showed that while NF-?B regulates mainly an early-induced and sustainedresponse, the ISRE element functions primarily in the induction of a delayed wave. We furtherdemonstrate that co-occurrence of the NF-?B and ISRE elements in the same promoter endowsits targets with enhanced responsiveness.

    Conclusion: Our results enhance system-level understanding of the networks induced by TLRsand demonstrate the power of genomics approaches to delineate intricate transcriptional webs inmammalian systems. Such systems-level knowledge of the TLR network can be useful for designingways to pharmacologically manipulate the activity of the innate immunity in pathological conditionsin which either enhancement or repression of this branch of the immune system is desired.

    BackgroundImmune systems in vertebrates have two basic arms:

    innate and adaptive immunity. The innate immune sys-tem is the first line of defense protecting the host from

    Published: 29 October 2007

    BMC Genomics 2007, 8:394 doi:10.1186/1471-2164-8-394

    Received: 4 July 2007Accepted: 29 October 2007

    This article is available from: http://www.biomedcentral.com/1471-2164/8/394

    2007 Elkon et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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    invading pathogens such as bacteria and viruses. It con-sists of various types of leukocytes (e.g., blood monocytes,neutrophils, tissue macrophages, dendritic cells) that spe-cialize in phagocytosis (ingesting and digesting patho-gens) and in evoking a complex response at the site ofinfection, collectively known as inflammation. The adap-tive immunity arm is capable of specifically recognizingand selectively eliminating foreign microorganisms andmolecules. It relies on T and B lymphocytes that expressantigen-specific receptors. Upon encountering their spe-cific antigens, these lymphocytes undergo extensive pro-liferation (clone expansion), maturation and activation.There are multiple cross-talks between the innate andadaptive immunity arms. For example, the phagocyticcells are intimately involved in the activation of the adap-tive arm by functioning as antigen presenting cells (APCs)required for the activation of T lymphocytes, and TH lym-phocytes secrete stimulatory cytokines that enhancephagocytosis by the specialized phagocytic cells.

    Innate immune responses to pathogens are triggered byrecognition of prototypical pathogen components, calledpathogen-associated molecular patterns (PAMPs),through cellular pattern recognition receptors (PRRs).Prominent among these pathogen sensors is the family ofToll-like receptors (TLRs). To date, ten and thirteen TLRgenes have been cloned in human and mouse, respec-tively; each of the TLRs appears to recognize a unique setof PAMPs [1,2]. TLR1, 2, 4, 5 and 6 are expressed on thecell surface membrane and recognize bacterial and fungalproducts, while TLR3, 7, 8 and 9 reside in intracellularendosomes and specialize in detection of pathogens'nucleic acids [3]. For example, lipopolysaccharide (LPS),which is a common structure of the cell wall of Gram-neg-ative bacteria, is recognized by the extracellular TLR4,whereas double-stranded RNA (dsRNA), which is a viralPAMP, triggers the intracellular TLR3 signaling. The func-tion of the other TLRs is less characterized.

    After recognition of their ligands, TLRs trigger intricate cel-lular signaling pathways that endow the cells with antivi-ral and antibacterial states, which are acquired by theinduction of protein effectors that impede viral replica-tion and bacteria growth, and of inflammatory cytokines,chemokines and co-stimulatory molecules that enhancethe activation of the adaptive immune response [2,4]. Theactivation of this broad response is mediated by a signal-ing cascade that leads to stimulation of several transcrip-tion factors (TFs), primarily NF-?B, IRF3/7, and AP-1.Important among the induced cytokines are the interfer-ons (IFNs), whose secretion results in the induction of aset of IFN-stimulated genes (ISGs), which are vital compo-nents in the development of antiviral and antimicrobialcellular states [5]. The transactivation of the ISGs is con-trolled via the JAK/STAT signaling pathway either by an

    IFN?/?-activated TF complex termed ISGF3 (composed ofSTAT1, STAT2 and IRF9), which binds to a regulatory ele-ment denoted as ISRE (IFN-stimulated response element)[5,6], or by an IFN?-activated STAT1 homodimer com-plex, which binds primarily to the GAS regulatory element[7].

    The transcriptional program spanned by activated TLRsencompasses hundreds of genes. The advent of geneexpression microarrays and the availability of completesequences of the mouse and human genomes enablestudy of these networks on the system level. Here, we ana-lyzed four publicly available genome-wide datasets thatrecorded expression profiles in mouse and human macro-phages stimulated with various pathogen-mimetic agents,with the goal of obtaining global delineation of the tran-scriptional network activated by TLRs. Combining com-putational analyses of gene expression profiles and cis-regulatory promoter sequences, we dissected the TLR-induced transcriptional program into two major compo-nents: the first is universally activated by all examinedTLRs, and the second is specific to TLR3 and TLR4. Ourresults identify NF-?B as the key regulator of the universalTLR response and the ISRE element as the key control siteof the TLR3/4 specific component, and reveal, on agenomic scale, known and novel target genes regulated bythese elements. We also identify novel putative positiveand negative feedback loops in these transcriptional pro-grams, further increasing the complexity of the knowntightly regulated network induced in response to patho-gen invasion. Analysis of the kinetics of the induced net-work showed that while NF-?B regulates mainly an early-induced and sustained response, the ISRE element func-tions primarily in the induction of a delayed wave. Inaddition, we demonstrate that the pair of NF-?B and ISREelements constitutes a cis-regulatory module that endowsits targets with enhanced responsiveness to TLR3/4 activa-tion. By combining expression and promoter analyses, wesubstantially reduced the high level of noise inherent ingenome-wide analysis of such data, and obtained highlyreliable results supported by independent datasets fromboth human and mouse.

    ResultsWe sought to obtain a global view of the transcriptionalprograms that are induced by activated TLRs, and to iden-tify components common to all TLRs and those specific tosome of them. To this end, we used four large-scale geneexpression datasets that examined global response inmouse and human macrophages stimulated with variousTLR stimulators [8-10] (Table 1). Our analysis flow isschematically sketched in Figure 1 and is described indetail in the sections below. In brief, starting with themouse datasets, we first partitioned the induced genesinto disjoint groups according to the subset of stimulators

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    to which the genes were responsive. Applying computa-tional analysis of cis-regulatory promoter elements wesought to discover the major TFs that control each of theidentified response groups. Next, we analyzed the kineticsof the transcriptional network indu