Computational Identification of Potential Transcriptional Regulators
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Genomics 103 (2014) 357–370 Contents lists available at ScienceDirect Genomics journal homepage: www.elsevier.com/locate/ygeno Computational identification of potential transcriptional regulators of TGF-ß1 in human atherosclerotic arteries Nedra Dhaouadi a,b, Jacques-Yuan Li a, Patrick Feugier a,Marie-PauleGustina, Houcine Dab b, Kamel Kacem b, Giampiero Bricca a, Catherine Cerutti a,⁎ a EA 4173 Génomique Fonctionnelle de l'Hypertension Artérielle, Université de Lyon, Université Lyon 1, Hôpital Nord-Ouest Villefranche-sur-Saône, 8 avenue Rockefeller, F-69373 Lyon, France b Unité de Physiologie Intégrée, Laboratoire de Pathologies Vasculaires, Université de Carthage, Faculté des Sciences de Bizerte, Bizerte, Tunisia article info abstract Article history: TGF-ß is protective in atherosclerosis but deleterious in metastatic cancers. Our aim was to determine whether Received 3 January 2014 TGF-ß transcriptional regulation is tissue-specific in early atherosclerosis. The computational methods included Accepted 3 May 2014 5 steps: (i) from microarray data of human atherosclerotic carotid tissue, to identify the 10 best co-expressed Available online 10 May 2014 genes with TGFB1 (TGFB1 gene cluster), (ii) to choose the 11 proximal promoters, (iii) to predict the TFBS shared by the promoters, (iv) to identify the common TFs co-expressed with the TGFB1 gene cluster, and (v) to compare Keywords: the common TFs in the early lesions to those identified in advanced atherosclerotic lesions and in various cancers. Transforming growth factor-beta 1 Atherosclerosis Our results show that EGR1, SP1 and KLF6 could be responsible for TGFB1 basal expression, KLF6 appearing Vascular smooth muscle cells specific to atherosclerotic lesions. Among the TFs co-expressed with the gene cluster, transcriptional activators Transcriptome (SLC2A4RG, MAZ) and repressors (ZBTB7A, PATZ1, ZNF263) could be involved in the fine-tuning of TGFB1 Gene co-expression expression in atherosclerosis. Microarray © 2014 Elsevier Inc. All rights reserved. 1. Introduction migration and by promoting vSMC differentiation. Indeed, long-term abrogation of TGF-ß signaling aggravated the extent and/or the inflam- It is widely accepted that co-expressed genes are likely to share matory phenotype of the lesions in atherosclerosis-prone mice [9–12]. similar regulatory mechanisms. This hypothesis is the basis for almost Conversely, overexpression of TGF-ß limited plaque growth and stabi- all attempts to use mRNA expression data from microarray experiments lized plaque structure in ApoE-deficient mice [13,14]. Taken together, to discover regulatory networks [1–4]. Genes with strongly correlated the available evidence suggests that upregulating TGF-β activity in ath- mRNA expression profiles are more likely to have their promoter erosclerosis is a viable therapeutic option, either to prevent or reduce regions bound by a common transcription factor (TF). This effect is atherosclerotic plaque formation, or more likely to induce a transition present only at relatively high levels of correlation of expression. In from a macrophage-rich unstable plaque phenotype to a more stable, order for two genes to have a greater than 50% chance of sharing a fibrous lesion [8]. However, due to its ubiquitous expression and pleio- common TF binder, it has been suggested that the correlation between tropic role, systemic upregulation of TGF-ß activity might be detrimen- their expression profiles must be greater than 0.84 [3]. tal. This is exemplified by the dual role of TGF-ß in cancer [15].Hence, Our study focuses on the transcriptional regulation of the TGF-ß signaling elicits protective or tumor suppressive effects during transforming growth factor-beta (TGF-ß) in human atherosclerosis. the early growth-sensitive stages of tumorigenesis. However, later in TGF-ß is a ubiquitous cytokine known to have pleiotropic, but context- tumor development when carcinoma cells become refractory to TGF- dependent, effects on differentiation, proliferation and apoptosis in ß-mediated growth inhibition, the tumor cell responds by stimulating physiological as well pathological situations [5] and to play a major role pathways with tumor progressing effects. At late stages of malignancy, in the regulation of inflammation and immunity [6]. The “protective tumor progression is driven by TGF-ß overload that induces cytokine hypothesis” [7,8] attributes to TGF-ß a beneficial role in the epithelial–mesenchymal transition of the tumor cells, neoangiogenesis prevention of atherosclerosis on the assumption that TGF-ß plays an and loss of immunosurveillance in the stroma [16]. important role in maintaining the normal structure of the vessel wall by By far, the vSMCs are the predominant cell type in the arterial wall inhibiting vascular smooth muscle cell (vSMC) proliferation and and the control of TGF-ß expression in the vSMCs should be pivotal in the understanding of the protective role exerted by the cytokine in ath- erosclerosis. The transcriptional control of the TGFB1 promoter has been ⁎ Corresponding author at: Université Lyon 1, EA4173 Génomique Fonctionnelle de the subject of numerous studies [17–32]. However, to our knowledge, l'Hypertension Artérielle, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France. Fax: +33 478 777 517. no dedicated study to the transcriptional control of TGF-ß expression E-mail address: [email protected] (C. Cerutti). in the vSMCs has ever been published yet. http://dx.doi.org/10.1016/j.ygeno.2014.05.001 0888-7543/© 2014 Elsevier Inc. All rights reserved. 358 N. Dhaouadi et al. / Genomics 103 (2014) 357–370 In the present study our aim was to understand the transcriptional (mean correlation coefficient at 0.89, clustering coefficient of 0.93 at control of TGFB1 expression in the vSMCs of human atherosclerotic p b 10−8). Therefore this set of 11 co-expressed genes was called the carotid samples. Starting from microarray transcriptional data obtained TGFB1 cluster. in the laboratory, we selected the genes whose expression was tightly Fig. 2B shows that all of these genes were highly expressed. Suppl. correlated [33] with that of TGFB1 and analyzed the promoters of Table 1 gives information about these genes and the functional role of these genes with various bioinformatics approaches to identify the encoded proteins. In particular, it is worth noting that the expres- common transcription factor binding sites (TFBS) or transcription factor sions of AKT1 (atheroprotective [36,37] and modulating TGF-ß signaling cis-regulatory modules. Our analysis shows that, in addition to TFs [38–40]), and of ENG and BAG6 (modulators of TGF-ß signaling [41,42]) and modules already known to be involved in the transcriptional were correlated with that of TGFB1. control of TGFB1, unidentified TFs might be responsible for the tissue- Our approach was based on the double hypothesis that (i) high cor- specificity and the fine-tuning of TGFB1 expression in the arterial wall. relation between two transcripts results from common transcriptional regulation of the corresponding genes, and (ii) over-representation of 2. Results potential transcription factor binding site (TFBS) motifs in a set of co- expressed gene promoters may indicate common regulation. Therefore We studied human carotid endarterectomy samples that were we used the TGFB1 cluster to identify TFs potentially involved in the divided in two parts: the atheroma plaque (ATH) and the nearby TGFB1 transcription in the carotid MIT. macroscopically intact tissue (MIT). The MIT samples corresponded to type I or type II lesions [34] with very few foam cells and very limited extracellular lipid deposits in contrast to type III and IV lesions [34]. 2.3. Common TFBS in the promoter sequences of the TGFB1 cluster The neointima of the MIT contained both myofilament-rich (contrac- tile) and rough endoplasmic reticulum-rich (synthetic) vSMCs [35] Using the Genomatix Software Suite, we first identified 18 TF that produce an abundant extracellular matrix. No vasa vasorum was families and 32 matrices of individual TFs showing TFBS in the proximal observed in the intima of the MIT and the only endothelial cells present promoter sequences of at least 10 genes of the TGFB1 cluster (Table 1). were those lining the lumen of the carotid. Due to the level of the surgi- Three individual matrices from the Genomatix database were excluded: cal plane of eversion at endarterectomy, MIT as well as ATH samples the V$SP1F/V$GC.01 matrix that did not correspond to a defined TF and included several layers of media that comprised contractile vSMCs both the V$ZF02/V$ZF9.01 and V$KLFS/V$KLF6.01 matrices. Indeed, al- only. In short, the main cellular contingent in the MIT was, by far, though ZF9 is the same TF as KLF6, Genomatix attributed two matrices contractile and synthetic vSMCs. Thus transcriptomic data from MIT to the same TF and these two matrices were very dissimilar. We decided were a reliable reflection of gene expression in the vSMCs. Total to create a new one called U$KLF6 from 20 genomic sequences that had mRNA from MIT and ATH paired samples obtained in 32 patients was been experimentally verified by other authors (see Suppl. data Part 2). analyzed with Affymetrix HuGene 1.0ST microarray. The data have As a result and given that EGR1, SP1 and PLAG1 each had 2 or 3 individ- been deposited in the Gene Expression Omnibus (GEO) public database ual matrices in the Genomatix database, we identified the potential (accession GSE43292). binding of 24 distinct TFs on the 11 promoters of the TGFB1 cluster. 2.1. Expression analysis of TGF-ß isoforms 2.3.1. Statistical validation Expression data obtained from microarrays in 32 patients showed In order to test the robustness of the identified TFs, we performed a that the 3 isoforms of the TGF-ß (TGFB1, TGFB2, and TGFB3) were highly similar analysis on the promoters of larger sets of genes correlated with expressed in MIT and ATH: TGFB1 8.51 ± 0.37 in MIT and 8.67 ± 0.35 in TGFB1, namely 15 and 50 genes.