Cloning of the human RHOB gene promoter: characterization of a VNTR sequence that affects transcriptional activity

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Genomics 81 (2003) 525–530

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Short Communication

Cloning of the human RHOB gene promoter: characterization of a VNTR sequence that affects transcriptional activity Daniel Tovar, Jean-Charles Faye, and Gilles Favre* De´partement Innovation The´rapeutique et Oncologie Mole´culaire-INSERM U563-CPTP, Institut Claudius Regaud, 20-24 rue du Pont Saint-Pierre, 31052 Toulouse Cedex, France Received 12 September 2002; accepted 3 February 2003

Abstract The RHOB gene is an immediate-early gene implicated in cell growth control, cytoskeletal organization, and neoplastic transformation. Although the mouse RHOB gene (Arhb) promoter has been described, the human promoter is unknown. We cloned the human RHOB gene (ARHB) 5⬘-flanking region from the human genome and characterized its promoter region. Unlike its mouse counterpart, the human gene shows a variable number of tandem repeats (VNTR) sequence with a 34-bp repetitive unit between positions ⫺1124 and ⫺821. We demonstrated that this VNTR sequence significantly decreases the transcriptional activity of ARHB and simian virus-40 (SV40) promoters. PCR amplification of the VNTR sequence using genomic DNA from many cell lines revealed the existence of at least four alleles containing a different number of the repetitive unit. Our data suggest a potential regulatory role for the VNTR sequence in ARHB expression. © 2003 Elsevier Science (USA). All rights reserved. Keywords: RHOB gene; 5⬘-Flanking region; VNTR; Transcriptional regulation; ARHB promoter

RHO GTPases are involved in the regulation of signalling pathways that control cytoskeletal organization, cell growth, differentiation, apoptosis, and neoplastic transformation [1–3]. Among the RHO GTPase family, RHOB differs in several features from the closely related RHOA and RHOC proteins. First, RHOB is associated with plasma membrane, early endosomes, and pre-lysosomal compartments, whereas the localization of RHOA and RHOC appears to be mainly cytoplasmic [4,5]. Second, RHOB was reported to be both famesylated and geranylgeranylated in vivo [4,6]. Third, unlike most of the small GTPases, RHOB is short-lived and belongs to the group of immediate-early gene-encoded proteins [7]. Expression of ARHB is rapidly induced by a variety of stimuli including, growth factors (EGF, PDGF, TGF-␤), cytoplasmic tyrosine kinases (v-Src, v-Fps), and DNA-damaging agents such as UV-C light or alkylating xenobiotics [7–9]. Moreover, recent results of our laboratory have established that RHOB is a potent inhibitor of malignant transformation as well as a suppressor of * Corresponding author. Fax: ⫹33-561424631. E-mail address: [email protected] (G. Favre).

tumor growth [10]. The 5⬘-flanking region of the RHOB gene was exclusively characterized for the mouse gene by Nakamura et al. [11] and Fritz and Kaina [9]. Arhb presents a unique structure among the known RHO genes, because its coding sequence contains a unique exon uninterrupted by any introns. Sequence analysis reveals the existence of two putative TATA box elements upstream of the translation start codon. By contrast, human RAC1, RAC2, RHOG, and mouse RAC2 genes lack both a TATA box and a CCAAT box; they display widely distributed GC boxes and a complex genomic organization [12–15]. We report here the cloning and characterization of the ARHB promoter, and reveal the existence of a VNTR sequence that influences the transcriptional activity of the promoter. To identify the ARHB promoter, we conducted a BLAST search with the 591-bp coding sequence (CDS) of ARHB as a virtual probe against the human genomic “nr” (non redundant) database at the NCBI web server (http://www.ncbi. nlm.nih.gov). We found an identical sequence in locus AC023137, a 72,963-bp piece of genomic DNA corresponding to human clone RP11-513P11, reported by R.H. Waterston (unpublished personal communication). This lo-

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Fig. 1. 5⬘-flanking region of human ARHB. (A) Standard PCR was conducted using human peripheral blood lymphocyte genomic DNA as template. DNA sequences corresponding to the primers used for amplification of the 1.9-kb fragment (gray) and the tandem-repeat region (white) are boxed. Restriction sites used for deletion constructs are italicized and underlined. The TATA boxes and the transcription initiation site (⫹1, G), are in boldface and underlined. The tandem repeats are in boldface. RHOB gene coding sequence is represented in uppercase letters with the ATG start codon in boldface. (B) Mouse and human RHOB genes 5⬘-flanking regions alignment in the TATA box-containing region. The TATA boxes and the transcription initiation site are in boldface. Differing nucleotides between the two sequences are indicated (*).

cus contains the coding sequence for ARHB (from 37,917 to 38,507), and thus revealed 37,916 bp in the 5⬘ region and 34,456 bp in the 3⬘ region of the gene. Analysis of the 38-kb genomic fragment corresponding to the 5⬘-flanking region of the ARHB with a promoter search program at the Sanger Centre Web Server confirmed that, as described for Arhb

[9,11], it presents classical features of a promoter region. Moreover, the coding region of human RHOB gene does not contain intron sequences. We designed primers based on the sequence of the bacterial artificial chromosome (BAC) AC023137, so as to amplify part of the 5⬘-flanking region (Fig. 1A). Standard PCR experiments using human periph-

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Fig. 2. Promoting activity of several deletion constructs of the human ARHB promoter. (A) The 1,876-bp Mlul/Xhol fragment (hRB), the 1,330-bp Nhel/Xhol fragment (hRB1), the 792-bp HindIII/Xhol fragment (hRB2), the 306-bp Asp718/Xhol fragment (hRB3), and the 156-bp Sacl/Xhol fragment (hRB4) were inserted into pGL3-Basic (Promega) upstream of the Firefly luciferase gene. Each reporter construct was verified by sequencing. Human MCF7 and A549 and murine NIH3T3 cells were co-transfected with 0.1 ␮g of the tested construct and 5 ng of pRL-CMV (Promega) as an internal control (Renilla luciferase under the control of a CMV promoter), using polyethylenimine (PEI) (Sigma). Murine promoter subcloned in to pGL3-Basic was used as control for promoter activity in NIH3T3 cells (data not shown). Cell lysates were assayed 48 h after transfection for Firefly and Renilla luciferases in a MicroLumat Plus microplate luminometer (EG&G Berthold) using Dual-Luciferase Assay Reporter system (Promega) according to the manufacturer’s protocol. Results are expressed as the ratio of activity of Firefly to Renilla luciferase. The average mean ⫾ SD values from triplicate transfections are shown, representative of two independent experiments. (B) The 5⬘-located TATA box (TATATT) of the hRB3 construct (panel A) was mutated into a BamHI restriction site (GGATCC) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Each reporter construct was verified by sequencing. Human MCF7 cells were transfected and assayed for Firefly and Renilla luciferases as described in (A). Results are expressed as the ratio of activity of Firefly to Renilla luciferase. The average mean ⫾ SD values and the induction factor, relative to the positive control, from triplicate transfections are shown, representative of three independent experiments.

eral blood lymphocytes (PBL) genomic DNA as template, resulted in amplification of a 1.9-kb fragment corresponding to part of the 5⬘-flanking region of ARHB. We cloned the 1.9-kb putative promoter region into pGL3-Basic (Promega) and subjected the construct to double-strand sequencing (Fig. 1A). Analysis of the sequenced fragment showed a full identity with the sequence of BAC AC023137. We compared the 1.9-kb fragment of the 5⬘-flanking region of human ARHB with the mouse Arhb promoter using the SIM Alignment Tool for Nucleic Acid Sequences at http://www.expasy.ch. The sequence ranging from ⫺426 to ⫹430 is highly conserved in both species, especially in

the TATA box-containing region (93% base identity in region ⫺65 to ⫹60). Both 5⬘-flanking regions share five short blocks of 60 – 80% identity. The search for consensus transcription factor binding sites in the human sequence was conducted with MatInspector version 2.2 software and revealed the presence of numerous sites including CCAAT/ NFY, AP-2, AP-4, SP1, NF␬B, c-REL, SRF, and PPAR. An in silico approach to determine CpG island distribution indicated the existence of two CpG-rich methylation-sensitive regions spanning from ⫺415 to ⫹430 and from ⫺814 to ⫺507, unlike the mouse gene, which presents a unique CpG island. A striking feature of the ARHB promoter is the

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Fig. 3. Polymorphic VNTR and its negative regulatory effect on transcription. (A) Examples of the polymorphic variable number of tandem repeats (VNTR). Genomic DNA from several cell lines and human peripheral blood lymphocytes served as template for standard PCR using primers flanking the tandem repeat region (Fig. 1A). Genomic DNA extracted from NIH3T3 cells was used as negative control (data not shown). ARHB coding sequence was amplified, as a control for the quality of genomic DNA. The amplification products were separated on 2% agarose gel and stained with ethidium bromide. MCF7, Human breast adenocarcinoma; A549, human lung adenocarcinoma; T24, human bladder carcinoma; HBL100, normal human breast epithelium; PBL, human peripheral blood lymphocytes. Cell lines HT29 (human colorectal adenocarcinoma), MCF10A, and HMEC (normal human breast epithelium) showed the same profile as MCF7 cells (data not shown). (B) Effect of the VNTR on SV40 heterologous promoter. The Mlul/Xhol fragments TR8, TR9, and TR11 obtained by standard PCR with primers flanking the tandem-repeat region (Fig. 1A) were inserted into pGL2-Promoter (Promega), thus linking the tandem repeat region to the SV40 promoter upstream of the Firefly luciferase gene. Each reporter construct was verified by sequencing. Human MCF7 cells were transfected and assayed for Firefly and Renilla luciferases as described in Fig. 1A. Results are expressed as the ratio of activity of Firefly to Renilla luciferase. The average mean ⫾ SD values and the induction factor, relative to the pGL2-Promoter construct, from triplicate transfections are shown, representative of two independent experiments.

presence of nine tandem repeats (TR) of a 34-bp sequence (5⬘-TTCTATTGTACAATAGTGTATATTCTATTGTACA-3⬘) spanning from ⫺1124 to ⫺821 bp upstream of the transcription initiation site. The 34-bp repeating unit consists of two identical 12-mer segments (TTCTATTGTACA) separated by the 10-mer ATAGTGTATA (repeat number 7 lacks a TA dinucleotide). Interestingly, the 34-bp TR are solely present in the human promoter as compared with the mouse counterpart, as well as the other described members of the RHO family genes. We constructed a series of deletion mutants of the human 5⬘-flanking region upstream of the luciferase gene in the promoterless reporter vector pGL3-Basic (Promega) so as to

characterize the promoter region important for basal transcription. Moreover, we investigated the role of the TR sequence, because evidence has emerged that some TR sequences located near promoter regions may play important roles in transcriptional regulation [16,17]. We transiently transfected the resulting deletion constructs into human adenocarcinoma cell lines MCF7 and A549 and mouse NIH3T3 cells using the polyethylenimine (PEI) method [18] (Fig. 2A). The largest deletion, hRB4 (from ⫺1766 to ⫺45), completely abolished transcriptional activity, suggesting that the sequence between ⫺195 and ⫺45 bp contains elements that contribute to basal transcriptional activity of human ARHB. The ARHB promoter contains two

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putative TATA box elements (TATATTAA and TTTAAA) in the region ⫺466 to ⫺394 bp upstream of the ATG start codon (Fig. 1B). This region shares 96% base identity with the mouse counterpart. On the basis of the results of the primer extension experiments described for mouse Arhb [9], we hypothesized that the functional TATA box is the 5⬘located (TATATTAA). We confirmed this hypothesis by directed mutagenesis. Figure 2B shows that the mutation of the 5⬘-located TATA box (TATATT) into GGATCC decreased the promoter activity up to 80% in MCF7 cells (Fig. 2B). Deletion from ⫺1766 to ⫺1219 (hRB1) did not substantially modify the promoter activity in human cell lines as compared with the full-length promoter. By contrast, the deletion of the TR sequence (hRB2), increased the promoter activity in the three cell lines tested (from 1.5- to 2.5-fold) relative to the hRB construct (Fig. 2A), thus suggesting that the TR sequence may negatively regulate the promoter activity. To test whether the TR had suppressive activity on a heterologous promoter, we synthesized primers corresponding to DNA sequences flanking the TR region (Fig. 1A) and generated pGL2p-TR9, a construct that linked the PCR-amplified fragment containing the nine TR sequence, immediately upstream of the SV40 promoter in the reporter vector pGL2-Promoter (Promega). In transient transfection assays using MCF7 cells, the nine TR-containing construct substantially decreased promoter activity as compared with the activity of the reporter vector used as control (Fig. 3B). These results confirm that the TR sequence acts as a negative regulatory element for the ARHB promoter, as well as for a heterologous promoter. The mechanism by which the TR influences the transcriptional activity is still unknown, but could play an important role in the regulation of human RHOB gene expression. To detect the 34-bp TR sequence elsewhere in the human genome, we conducted a BLAST search using the former BAC AC023137 as a probe, against the HTGS database (unfinished High-Throughput Genomic Sequences) at the web server of the NCBI. We found a homologous region in BAC AC019034 that revealed the presence of 12 repeats of the TR sequence within the 5⬘-flanking region of ARHB, suggesting the existence of a VNTR of the 34-bp sequence. We carried out PCR experiments with primers corresponding to DNA sequences flanking the VNTR region (Fig. 1A) using genomic DNA extracted from human PBL and several cell lines, and found four different-sized fragments corresponding to alleles carrying 8 (TR8) and 11 (TR11) tandem repeats (Fig. 3A). We cloned the 8- and 11-TR-containing alleles into the pGL2-Promoter and obtained pGL2p-TR8 and pGL2p-TR11, respectively. In transient transfection assays using MCF7 cells, both TR-containing constructs significantly decreased SV-40 promoter activity (Fig. 3B). Nevertheless, the negative regulatory effect on transcription seems to be independent of the number of TR found, in that the inhibitory effect is not significantly different with TR8, TR9, and TR11 alleles.

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We demonstrated here the presence of a VNTR sequence in the human ARHB promoter. Association of certain VNTR with the risk of diseases [19] and the influence of the polymorphic VNTR sequence in human HRAS on the risk or on the penetrance of several cancers have been reported [20,21]. We [10] and others [3] have strongly suggested that the RHOB gene is a tumor suppressor gene, thus emphasizing the importance of investigating a link between the VNTR polymorphism and cancer development. Although there is a strict amino acid identity between human and mouse RHOB, the present work highlights important differences between promoter structures of human and mouse RHOB genes. This suggests differences in the regulation of gene expression between the two species. Of particular interest is the existence of a regulatory VNTR sequence in the ARHB promoter. These data open the way to future investigations concerning the regulation of ARHB expression as well as the influence of VNTR polymorphism in the human ARHB promoter on the biological functions of RHOB.

Acknowledgments We are grateful to Philippe Mulsant (Laboratoire de Ge´ ne´ tique Cellulaire-INRA, Toulouse), Loı¨c Ponger (Laboratoire de Biome´ trie et Biologie Evolutive-UMR CNRS 5558, Lyon), and Robert Dante (Laboratoire de Ge´ ne´ tiqueUMR CNRS 5641, Lyon) for the excellent cooperation in in silico studies. This work was supported by the “Institut National de la Sante´ et de la Recherche Medicale,” the French “Ministe`re de l’Enseignement Supe´ rieur et de la Recherche,” and grants from the “Groupe de Recherche de l’Institut Claudius Regaud” and the “Ligue Nationale de Lutte contre le Cancer” (e´ quipe labe´ lise´ e La Ligue).

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