Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells

GENE-39429; No. of pages: 7; 4C: Gene xxx (2014) xxx–xxx

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Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells Sayonara Gonzalez a,b,⁎, Renata Binato a,⁎, Letícia Guida b, André Luiz Mencalha a, Eliana Abdelhay a

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Article history: Accepted 21 January 2014 Available online xxxx

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Keywords: ES cells Galanin promoter CREB HOX PAX SP1 Transcriptional activation

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Galanin and its receptors have been shown to be expressed in undifferentiated mouse embryonic stem (ES) cells through transcriptome and proteomic analyses. Although transcriptional regulation of galanin has been extensively studied, the regulatory proteins that mediate galanin expression in mouse ES cells have not yet been determined. Through sequence alignments, we have found a high degree of similarity between mouse and human galanin upstream sequences at − 146 bp/+ 69 bp (proximal region) and − 2408 bp/− 2186 bp (distal region). These regions could be recognized by ES cell nuclear proteins, and EMSA analysis suggests a specific functionality. Analysis of the proximal region (PR) using EMSA and ChIP assays showed that the CREB protein interacts with the galanin promoter both in vitro and in vivo. Additional EMSA analysis revealed that an SP1 consensus site mediated protein–DNA complex formation. Reporter assays showed that CREB is an activator of galanin expression and works cooperatively with SP1. Furthermore, analysis of the distal region (DR) using EMSA assays demonstrated that both HOX-F and PAX 4/6 consensus sites mediated protein–DNA complex formation, and both sites inhibited luciferase activity in reporter assays. These data together suggest that CRE and SP1 act as activators at the basal promoter, while HOX-F and PAX 4/6 act as silencers of transcription. The interplay of these transcription factors (TF) may drive regulated galanin expression in mouse ES cells. © 2014 Published by Elsevier B.V.

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Instituto Nacional de Câncer, Centro de Transplante de Medula Óssea, RJ, Brazil Departamento de Genética Médica, Instituto Fernandes Figueira, Fundação Oswaldo Cruz, RJ, Brazil

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1. Introduction

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Galanin is a highly conserved 29 (30 in human) amino acid peptide that plays a neuromodulatory role, in addition to an important trophic role, in neuronal tissues after injury and disease (Lang et al., 2007). Galanin has also been suggested to have a biological activity in progenitor or stem cells from both mesodermal and ectodermal origin (Louridas et al., 2009), and has been found through transcriptome and proteomic analyses in several lineages of undifferentiated human and mouse ES cells (Anisimov et al., 2002; Ramalho-Santos et al., 2002; Sato et al., 2003). To accomplish these functions, galanin signals through three receptors: GalR1 (Habert-Ortoli et al., 1994), GalR2 (Howard et al., 1997; Smith et al., 1997), and GalR3 (Wang et al., 1997).

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Abbreviations: Bp, Base pair; ChIP, Chromatin immunoprecipitation; DR, Distal region; EDTA, Ethylenediaminetetraacetic acid; EMSA, Electrophoretic mobility shift assay; ES, Embryonic stem cells; HEPES, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; kb, Kilobase; KCl, Potassium chloride; MEF, Mouse embryonic fibroblasts; mg, Miligram; MgCl2, Magnesium chloride; ml, Mililiter; mM, Milimolar; PBS, Phosphate-buffered saline; PCR, Polymerase chain reaction; PR, Proximal region; TF, Transcription factor; TBE, Tris/ borate/EDTA. ⁎ Corresponding authors at: Praça da Cruz Vermelha 23, 6°andar ALA C, Divisão de Laboratórios do CEMO, INCA, Rio de Janeiro, RJ CEP 20.230-130, Brazil. Tel.: + 55 21 3207 1874. E-mail addresses: sayonara@iff.fiocruz.br (S. Gonzalez), [email protected], [email protected] (R. Binato).

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Transcriptional in vivo and in vitro studies of the galanin gene from different organisms have made it possible to identify a plethora of regulatory elements at the basal promoter, such as NRE, AP1, STAT, CRE, SP1 and ERE, that activate galanin expression in several cell types or tissues (Kofler et al., 1996; Rökaeus et al., 1998). Corness et al. (1997) found a CRE element at the proximal promoter and a repressor element localized at −2.2 kb and −1.4 kb of the rat galanin gene that is active in primary sensory neurons in culture. In another study, Bacon et al. (2007) found ETS, STAT and Bicoid consensus sites localized at − 1.9 kb of the mouse galanin gene that direct galanin expression after axotomy. Additionally, bovine galanin promoter sequences spanning from 5 kb or 131 bp were studied in human neuroblastoma SH-SY5Y cells and in transgenic mice, and the presence of silencer and enhancer sequences was revealed (Rökaeus et al., 1998). However, despite the large amount of data addressing galanin activation, functional transcriptional regulators of mouse galanin expression in ES cells have not yet been determined. In the present study, we have found, through sequence alignment, a high degree of conservation between mouse and human galanin upstream sequences, located at − 146 bp/+ 69 bp of the proximal region (PR) and − 2408 bp/− 2186 bp of the distal region (DR). In both regions, there was conservation of transcription factor (TF) binding sites including HOX-F and PAX 4/6 in the DR, and SP1 and CRE in the PR. By analyzing the proximal region, we showed through EMSA and ChIP

0378-1119/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gene.2014.01.059

Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059

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2. Materials and methods

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2.1. Computer analysis of the promoter region

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Approximately 3 kb of the upstream sequence of the mouse (L11144.1) and human (L38575.1) galanin genes was obtained from GenBank (www.ncbi.nlm.nih.gov.br). Conservation between both species was assessed through the sequence alignment program FASTA VIRGINIA (http://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml), and conservation of TF binding sites was assessed using online databanks MatInspector (www.genomatix.de), rVista 2.0 (http://rvista.dcode.org) and IFTI (www.ifti.org).

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2.2. Cells and culture conditions

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USP-1, a murine ES cell line (Sukoyan et al., 2002) was kindly provided by Dr. Lygia Pereira (Universidade de São Paulo) and cultured as follows: ES cells were maintained on a feeder layer of irradiated MEFs (murine embryonic fibroblast) in DMEM high glucose media (Invitrogen) supplemented with 15% fetal bovine serum (Hyclone), 100 mM nonessential amino acids (Invitrogen), 2 mM glutamine (Invitrogen), 100 U of penicillin/ml (Invitrogen), 100 mg/ml of streptomycin (Invitrogen), 0.55 μM β-mercaptoethanol (Sigma) and 1000 U/ml of leukemia inhibitory factor (LIF; Chemicon) on 0.2% gelatin-coated plates, at 37 °C in a humidified chamber with 5% CO2. The medium was changed 3 h before splitting, and the cells were rinsed twice with 1X phosphate-buffered saline (PBS), treated with 0.25% trypsin/0.5 mM EDTA for 5 min and split 1:3 to be maintained in culture or used for preparation of protein nuclear extracts or transfection assays after removal of feeder cells. Feeders were removed by replating into a new gelatin-coated dish for propagation. These ES cells without MEF cells were expanded 1–2 passages in 50% MEF conditioned media and 50% fresh ES cell media.

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2.3. Preparation of nuclear extract

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For preparation of nuclear protein extracts, 1 × 107 ES cells without MEF cells were rinsed twice with PBS 1X and treated with 0.25% trypsin/ 0.5 mM EDTA. The material was centrifuged and the nuclear proteins were extracted as described by Dignam et al. (1983). Protein concentrations were determined by the Bradford method (Bradford, 1976).

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2.4. Electrophoretic mobility shift assays (EMSA)

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For EMSA assays, we used probes from PCR synthesized fragments and double-stranded (ds) DNA oligonucleotides, both end-labeled with [γ-32P] ATP (Amersham, Pharmacia) using T4 polynucleotide kinase (Invitrogen). The primer sequences used to generate PCR fragment probes corresponding to the PR and DR were as follows: PRsense 5′-AGACTGTGGGTGATC CTCTC-3′, PRantisense 5′-CTGGATGGTCGCTT ACTG-3′, DR sense 5′-GCTTTGTGTGCTGTGTCCATTACT-3′, DRantisense 5′-CCCATA TCACTGACAGATTCGCTCC-3′. The following primers (and their complements) were annealed to generate dsDNA oligonucleotide probes: SP1-sense 5′-CAGGAGGC GGCGCTGAGCGG-3′, CREB-sense 5′-

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The ChIP assays with anti-CREB antibody (Santa Cruz technologies) were performed according to the online protocol provided by Abcam Company (http://www.abcam.com/ps/pdf/protocols/N-ChIP. pdf). DNA extractions from bound fractions were performed following the Abcam (www.abcam.com) protocol. The immunoprecipitated DNA was amplified for sequences containing binding sites by using the following CRE element sequence primers present in the mouse galanin gene promoter: ChIP-sense 5′-GGTCTGAGAC TGTGGGTGAT-3′; ChIP-antisense 5′-CTGCTGCCGCTATTTATG-3′. Quantification was evaluated by RT-qPCR analysis. Immunoprecipitation of anti-tubulin antibody (Santa Cruz Technologies) was used as a negative control.

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2.6. Reporter vector design

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The galanin firefly luciferase reporter constructs were made by cloning fragments corresponding to the PR and DR of the mouse galanin promoter into the firefly PGL3-Basic vector (Promega) upstream of the luciferase reporter gene. The constructs p80, p60 and p60mut were obtained by cloning PCR-amplified fragments corresponding to −78 bp/+357 bp and −60 bp/+357 bp into XhoI and HindIII restriction sites of pGL3-Basic. The primer sequences used for the PCR reactions were: p80-sense: 5′-ATATCTCGAGTCGCAGGAGGCGGCGCT-3′; p60-sense: 5′ATC-TCTCGAGAGCCGGTGACGCGGCAG-3′. The p60mut construct contained, in the forward primer, a C to A base substitution (underlined) at the CRE binding site to generate a mutant version of this element: p60mut-sense: 5′-ATCTCTCGAGAGCCGGTGAAGCGGCAG3′. Reverse primer pPR-antisense 5′-GCCCGAAGCTTCATCTGGAAGGA AAAGTGG-3′ was used to generate all the products. Forward primers have a XhoI restriction site and reverse primers have a HindIII restriction site. To generate the p2400 and the p2300 constructs corresponding to the DR, we first digested the p80 luciferase reporter construct with SacI and XhoI restriction enzymes. This digested construct was used for subsequent cloning of −2408 bp/−2186 bp and −2365 bp/−2186 bp PCR fragments from the mouse galanin gene separately. The primer sequences used for the PCR reactions were: p2400 sense: 5′GGAATGAGCTCCTTTGTGTGCTGTGTCC-3′ and p2300 sense: 5′TGAATGAGC TCTAGCTCCACGCTGGGCTG-3′. Reverse primer pDRantisense: 5′-GGCTGCTC GAGCCCATATCACTGACAGAT-3′ was used to generate both products. Forward and reverse primers have SacI and XhoI restriction sites, respectively. All reporter pGL3 vector sequences were confirmed by DNA sequencing at the Automated DNA Sequencing Facility of Instituto de Biofísica Carlos Chagas Filho-UFRJ.

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AGCCGGTGACGCGGCAGCTCC-3′, CREB -Mut-sense 5′-AGCCGGTGAA GCGGCAGCTCC-3′, HOX-F-sense 5′-ATTACTCTA ATGGTGACCCATTTTG3′, PAX-4/6-sense 5′-CTCCAGGCTGGGCTGCCTT-3′. Gel shift reactions were performed in a binding buffer [25 mM HEPES (pH-7.6), 30 mM KCl, 5 mM MgCl2, 5% glycerol], containing 1 μg of poly (dIdC), 7–10 μg of nuclear extract proteins and 80,000 cpm of [γ-32P]-labeled PCR dsDNA probes. The reactions occurred at 25 °C for 40 min and were subsequently resolved on a 4% or 4.5% native polyacrylamide gel when PCR fragments or oligonucleotides were utilized as probes, respectively; in 0.5 × TBE buffer (22.5 mM Tris/Borate/1 mM EDTA) for 4 h at 140 V. For competition reactions, a 100 molar excess of unlabeled competitor oligonucleotide or a mutated version of the specific consensus binding site was added 20 min before addition of the probe. For supershift experiments, 2 μg of anti-CREB1 (C-21) antibody (Santa Cruz Biotechnology Inc.) was incubated with a nuclear protein extract 60 min before addition of the specific probe. After addition of the specific probe, the incubation was continued for 40 min at 25 °C. The dried gels were exposed on Kodak MS films on intensifying screens at −70 °C for 48 h.

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assays that CREB proteins interact with the galanin gene promoter in vitro and in vivo. Further EMSA analysis using SP1, HOX-F and PAX 4/6 consensus sites showed that all of these sequences also have the ability to bind the nuclear proteins of ES cells. When the cooperative role of these sites was investigated through transfection assays, we observed that SP1 and CRE, using proximal constructs, activated luciferase signal cooperatively, while using distal constructs, HOX-F and PAX 4/6 exerted an inhibitory action on the luciferase signal. These data together suggest that CRE and SP1 at the basal promoter act as activators, while HOX-F and PAX 4/6 act as silencers of galanin expression in mouse ES cells.

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Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059

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3. Results

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ES cells (5 × 104) were plated into 24-well plates pretreated with 0.2% gelatin and incubated for 4 h in ES cell media as described previously. Then, the cells were transfected with 1.0 μg of either pGL3 reporter vectors containing galanin promoter inserts or pGL3-basic vector. To normalize transfection efficiency, we co-transfected 0.01 μg of Renilla luciferase phRL-TK plasmid into each well. Transfection was carried out using Lipofectamine LTX 2000 (Invitrogen) according to the manufacturer's protocol. Firefly and Renilla luciferase activities were measured in cell lysates 48 h after transfection using the DualGlo Luciferase Assay System (Promega) on a Veritas TM Microplate Luminometer (Turner Biosystems), following the manufacturer's protocol. All experiments were performed in triplicate. Ratios of Renilla luciferase readings to firefly luciferase readings were measured for each experiment, and triplicates were averaged. The average values of the tested constructs were normalized to the activity of the empty pGL3-basic vector, which was arbitrarily set at a value of 1.

3.1. In silico analysis of conserved regions within the galanin gene promoter 204

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Evolutionary conservation of sequences can be analyzed through sequence alignments of distantly related species, such as human and mouse. Because noncoding DNA has diverged between species, a conserved region in a noncoding sequence suggests the biological activity of regulatory elements that have been under selective pressure. This computational approach, supplemented with experimental analysis, is required for the elucidation of cis-acting transcriptional regulatory elements. Initially, we performed a computational comparative analysis of 3 kb (upstream from the translation codon) of the human (GenBank accession number L38575.1) and mouse (GenBank accession number L11144.1) galanin genes. The alignment of both sequences demonstrated a high degree of similarity and conservation at position −146 bp/+69 bp and at −2408 bp/−2186 bp (Figs. 1A and B). Subsequent in silico analysis

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Fig. 1. Alignment of the mouse (M) and human (H) galanin promoter. (A) and (B) Distal region (DR) and proximal region (PR), respectively. Boxes demonstrate conserved transcription factor binding sites. (C) Schematic representation depicting the conserved binding sites of the promoter region of the human and mouse galanin gene. Positions are relative to the transcription start site. The dashed line indicates a 1.0 kb sequence insertion in the human promoter. The arrows indicate the primer positions that generated the PCR fragment probe. (D) and (E) EMSA assay of binding reactions using PCR probes (P), corresponding to the PR and DR, and ES cell nuclear extracts (NE). Competition reactions were performed using 100-fold excess of unlabeled wild type (Cwt) oligonucleotides. The arrows indicate the resulting DNA–protein complexes. DR—distal region, PR—proximal region.

Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059

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To investigate the functionality of the conserved binding sites of the proximal and distal regions of the galanin promoter, we carried out EMSA assays using PCR-amplified fragments covering the PR and DR, containing conserved putative TF binding sites and nuclear proteins from ES cells. We observed the formation of three specific complexes (a, b, and c) and one nonspecific complex (d) on the PR (Fig. 1D). Competition analysis using 100-fold excess of unlabeled oligonucleotide as a competitor showed that specific binding was abolished (Fig. 1D). EMSA assays of the DR showed the formation of one specific complex (Fig. 1E),

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and this binding was abolished when we used 100-fold excess of the unlabeled oligonucleotide as a competitor. These results reinforce the assumption of a functional role for conserved regions. Within the PR, the CRE binding site has been found to be conserved through evolution in the rat, bovine, human and mouse galanin promoters. The bovine CRE binding site has been shown to be functional in human SH-SY5Y cells and rat pheochromocytoma-derived PC12 cells (Rökaeus et al., 1998). In addition, CREB protein has been shown to be highly expressed at the inner cell mass (ICM), a region of the blastocyst from which ES cells are derived (Bleckmann et al., 2002). Because most of the findings addressing the regulation of galanin expression were generated in other species, it is not possible to predict whether the regulatory network of galanin transcription, as currently defined, also applies to mouse ES cells. To address this issue, we examined the consensus sequences located at the PR. EMSA assays using an oligonucleotide containing a CRE consensus binding site and ES cell nuclear proteins resulted in the formation of complexes. When we used 100-fold molar excess of the unlabeled DNA-oligo containing the CRE consensus binding site, the complex was completely abolished. Conversely, the complex was maintained when we used an unlabeled mutated analog as a competitor, confirming its specificity. Supershift assays were performed using anti-CREB antibody and this assay demonstrated that CREB was present in the protein complex formed with the CRE consensus sequence (Fig. 2A). To confirm the EMSA results, we performed ChIP assays. The ChIP assay allows in vivo analysis of nuclear protein–DNA interactions. Chromatin fractions bound to the CREB antibody in ES cells were quantified by RT-qPCR using primers to amplify the promoter region that contains the CRE consensus site. The results revealed that CREB was bound to this site in vivo (Fig. 2B). We next investigated whether the SP1 site was also shifted in EMSA analysis. This SP1 site, similar to the CRE site, is also evolutionarily conserved (Rökaeus et al., 1998). In our assays, nuclear proteins from

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of these two overlapping sequences revealed the conservation of putative binding sites at the proximal region (PR) as reported previously (Kofler et al., 1996; Rökaeus et al., 1998). A CRE site, the cAMP response element that is recognized by CREB (CRE-binding protein), was found at −55 bp/−47 bp. CREB is a cellular transcription factor (TF) that is highly expressed in ES cells and regulates the transcription of numerous target genes (Bleckmann et al., 2002). We also found a binding site for SP1 at −75 bp/−66 bp, which is a TF implicated in the activation of a variety of genes involved in proliferation, differentiation and apoptosis (Black et al., 2001). At the distal region (DR), our analysis found HOX-F consensus sequence at −2389 bp/−2366 bp and PAX 4/6 consensus sequence at −2360 bp/−2342 bp that are recognized by the products of developmental genes belonging to PAX and HOX family members (Gehring and Hiromi, 1986; Krumlauf, 1994; Wehr and Gruss, 1996). It is interesting to note that between the PR and the DR, there is a 1.0 kb sequence in the human promoter with no similarity to the mouse gene, suggesting that there was a sequence insertion in the human genome during evolution. With the exception of this sequence insertion, the binding sites were conserved at the PR and DR of both species, suggesting a selective pressure on these regulatory sequences and possibly a functional role (Fig. 1C).

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Fig. 2. Characterization of CREB binding to the galanin promoter. (A) EMSA analysis of binding reactions using a CREB oligonucleotide as probe (P) and ES cell nuclear extracts (NE). Competition was performed using 100-fold excess of unlabeled wild type (Cwt) or mutated (CMut) oligonucleotides. The arrows indicate the resulting DNA–protein complexes. Supershift assays were performed using CREB-1 antibody. Control reactions were prepared using nonspecific antibodies, anti c-Myc and anti cyclin-D. (B) ChIP analysis confirms CREB interaction with the galanin promoter in vivo. Q-PCR quantification of the CRE consensus sequence in ES cells. DNA amplification was quantified in bound and unbound fractions after normalization with protein A nonspecific amplification. Normalized fractions were used to calculate the bound/input ratio.

Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059

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3.3. Transcriptional activity of the proximal region of the galanin gene promoter

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To clarify the regulatory role of TF binding sites found by EMSA assays on mouse galanin gene expression, we created reporter constructs and performed transfection assays in ES cells. Initially, we created a P80 construct that contained CRE and SP1 consensus sequences and cloned it into a pGL3 reporter vector upstream of the firefly luciferase gene. This construct demonstrated high transcriptional activity in the reporter assay when compared with pGL3 (15-fold induction). When we deleted the SP1 binding site (P60 construct), we observed a 3.5-fold reduction of luciferase expression, suggesting a stimulatory role for this sequence on the

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The galanin gene has been isolated from several species of vertebrates, including mammals, birds, reptiles and fish, and has been described to have wide-ranging biological effects, particularly within the neuroendocrine and central or peripheral nervous system. Studies of the bovine galanin promoter in human neuroblastoma SH-SY5Y cells demonstrated that 0.1 kb of the promoter conferred high basal expression of the gene. In this region, a highly conserved cyclic AMP response element (CRE)-like sequence between −66 and −44 bp of the galanin promoter was found (Rökaeus et al., 1998). The same group showed that 131 bp of the bovine galanin promoter, including the CRE consensus binding site, is sufficient to induce high basal activity in SH-SY5Y cells. However, the presence of upstream silencers was detected in the region between 451 bp and 5 kb. The same was observed in human choriocarcinoma cells and rat pheochromocytoma cells, but not in human breast cancer cells, indicating that the repressor element present in the galanin gene promoter could act in a tissue-specific manner

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4. Discussion

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galanin promoter. When the plasmid containing a sequence alteration in the CRE consensus site (P60mut construct) was transfected into ES cells, we observed a greater reduction of luciferase expression, similar to the control levels (Fig. 4A). To evaluate the regulatory role of DR on basal galanin transcription, we cloned a PCR fragment containing HOX-F and PAX4/6 binding sites into the P80 construct upstream of the CRE and SP1 sites. When the resulting construct (P2400) was transfected into ES cells, the promoter activity was reduced by 1.5 × compared to basal promoter activity (P80) When we removed the HOX-F binding site and retained only the PAX4-6 upstream PR (P2300 construct), we observed a slight reduction in luciferase activity (Fig. 4B).

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ES cells were able to bind to the SP1 consensus sequence (Fig. 3). Competition analysis using 100-fold excess of unlabeled oligonucleotides confirmed the specificity of binding. To investigate the consensus sequences located at the DR, we used two [γ-32P]-labeled double-stranded DNA oligonucleotides, one containing the consensus sequence for HOX-F and the other containing the consensus sequence for PAX 4/6, and ES cell nuclear extracts. When the HOX-F consensus sequence was used, we observed six specific complexes. (Fig. 3B, complexes a, b, c, d, e and f). These seemed to display different binding affinities because shifted bands were of different intensities. After competition with 100-fold excess of unlabeled oligonucleotide, complexes a, b, c, d and e disappeared completely, while complex f showed a significant reduction in intensity. When PAX 4/6 consensus sites were subjected to EMSA analysis, two complexes were formed, as indicated in a and b (Fig. 3C). Only complex a was completely abolished by the competition reaction with 100-fold excess of the unlabeled oligonucleotide.

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Fig. 3. Interactions of nuclear proteins from ES cells with the galanin gene promoter. EMSA analysis of binding reactions using (A) SP1, (B) HOX-F and (C) PAX4-6 oligonucleotides as probes (P) and ES cell nuclear extracts (NE). Competition reactions were performed using 100-fold excess of unlabeled wild type (Cwt) oligonucleotides. The arrows indicate the resulting DNA–protein complexes.

Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059

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(Rökaeus et al., 1998). Although described as a neuropeptide, galanin expression was also identified in mouse embryonic stem (ES) cells through transcriptome analysis as one of the most abundant transcripts, indicating that galanin may play an important role in these cells. Tarasov et al. have suggested that galanin could affect pathways involved in regulating cell number, and this effect may be modulated by LIF (Anisimov et al., 2002; Tarasov et al., 2002). In light of this, understanding how galanin is regulated in ES cells becomes important, as this gene has an effect on the growth of these cells. To identify the putative cis-acting elements responsible for mouse galanin gene expression, we compared approximately 3 kb of upstream sequences from mouse and human galanin genes. This comparison showed two regions of similarities between these two species, located at 146 bp/+ 69 bp (proximal region) and at − 2408 bp/− 2186 bp (distal region). Further in silico analysis revealed the conservation of several transcription factor binding sites, including SP1 and CRE, at the proximal region, and HOX-F and PAX4/6 at the distal region. The CRE binding site has been found to be evolutionarily conserved. The bovine CRE binding site has been shown to bind PMA (phorbol-12-myristate-13-acetate)-inducible human proteins from SH-SY5Y cells and also proteins from bovine chromaffin cells. Additionally, in unstimulated conditions, without PMA administration, it has been demonstrated that CRE on the bovine promoter confers high basal galanin expression in SH-SY5Y cells and rat pheochromocytoma-derived PC12 cells (Rökaeus et al., 1998).

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Fig. 4. Functional analysis of the mouse galanin promoter. (A) Reporter activity of P80, P60 and P60Mut constructs in transiently transfected ES cells. The histogram shows induction of luciferase activity when we used the construct p80 (with SP1 and CRE consensus binding sites) and the levels of luciferase activity decreased when we used the construction without the SP1 consensus binding site, indicating cooperative activity among the 2 binding sites to increase transcriptional activation. (B) Reporter activity of p2400 and p2300 constructs in transiently transfected ES cells. The histogram shows reduction of luciferase activity when we used both constructions p2400 and p2300 (the first containing PAX4-6 and HOX consensus binding sites and the second with only the PAX4-6 consensus binding site) in the presence of the basal promoter. Luciferase activity was measured and normalized to Renilla levels. P80—construct containing CRE and SP1 consensus sequences. P60—construct containing the CRE consensus sequence. P60Mut—construct containing an alteration of the CRE consensus sequence. P2400—construct containing HOX and PAX4-6 consensus sequences. P2300—construct containing PAX4-6 consensus sequence. LUC—empty vector.

As the galanin gene has been described to play an important role in the biological activity of stem cells, we investigated whether the conserved regions of the galanin gene were functional in mouse ES cells. First, we explored whether nuclear proteins could bind to the proximal region through EMSA analysis. We found that this region was bound by ES cell nuclear proteins in a specific manner, reinforcing the possibility of their functionality. To further dissect the proximal region, we explored protein binding to CRE consensus sites in EMSA assays. We found that nuclear proteins isolated from ES cells could bind to this sequence, and this binding disappeared when using an unlabeled probe containing CRE sequence. Conversely, when we used a mutated CRE site in competition assays, the protein binding could not be competed away, confirming the specificity of the complex. The presence of CREB protein in the complex was verified by supershift assays. An antibody against CREB-1 protein inhibited the formation of these complexes, indicating that CREB-1 binds to the mouse galanin promoter. Correspondingly, when we performed a ChIP assay using ES cells, immunoprecipitation with anti-CREB antibody, followed by the amplification of genomic DNA surrounding the CRE site in the galanin proximal promoter, revealed that CREB was also bound to the galanin promoter in vivo. Additional EMSA assays showed that an SP1 site was also shifted in EMSA analysis, and this binding disappeared when we competed with unlabeled probe containing SP1 sequence. The SP1 site, similar to the CRE site, is also evolutionarily conserved and is arranged in an

Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059

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This work was supported by grants from Convênio INCA/FIOCRUZ.

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may function to downregulate galanin gene expression. Binding of spe- 454 cific combinations of these trans-activating factors is likely necessary for 455 appropriate galanin regulation in mouse ES cells. 456

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overlapping manner with the NGF responsive element (NRE) at the galanin promoter. It has been shown that administration of NGF can induce the activation of the bovine galanin gene in PC12 cells through this NRE site. Interestingly, this sequence can also bind nuclear proteins in unstimulated basal conditions (Rökaeus et al., 1998). The study of the distal region identified protein binding to an HOX-F consensus sequence, resulting in six specific complexes that seemed to display different binding affinities because shifted bands showed different intensities. After competition with the unlabeled oligonucleotide, 5 of the 6 complexes disappeared completely, while one complex showed a significant reduction in intensity but did not disappear. Differences in binding activities may be a result of diverse complexes forming at the HOX-F site. This site is the general recognition site for HOX genes, a set of homeodomain TFs that in the vertebrate genome are organized in four clusters composed of 13 subgroups, called paralog groups (Sharkey et al., 1997). The HOX-F consensus site is recognized by HOX paralogs 1–8, belonging to the four HOX clusters (matrix family assignment—www.genomatix.de). When the PAX 4/6 consensus sites were subjected to EMSA analysis, a specific complex was formed. To understand the regulatory role of elements in the proximal region on galanin expression, deletional and mutational analysis of galanin reporter constructs was performed in ES cells. The reporter vector bearing CRE and SP1 consensus sequences demonstrated high transcriptional activity. When we removed the SP1 binding site, we observed a 3.5fold reduction of luciferase expression, suggesting a stimulatory role for this sequence on the galanin promoter. When the plasmid containing a sequence alteration to the CRE consensus site was transfected into ES cells, we observed a greater reduction in luciferase expression, similar to the control levels (Fig. 4). The importance of SP1 and CRE binding sites to galanin gene expression has been shown for the bovine galanin gene in induced and non-induced processes, as discussed earlier. Their relevance is also supported by the observation that interactions among SP1 and CRE binding sites have been reported in the literature for other promoters and may be a common signature that drives the expression of several genes (Piera-Velazquez et al., 2007; Xia et al., 2008). It is possible that the same mechanism may operate to regulate mouse galanin in ES cells. To evaluate the regulatory role of DR on basal galanin transcription, we cloned a PCR fragment containing HOX-F and PAX4/6 binding sites into the construct containing CRE and SP1 sites. When the resulting construct was transfected into ES cells, the promoter activity was reduced by 1.5 ×. When we removed the HOX-F binding site and retained only the PAX4-6 site, we observed a slight reduction in luciferase activity. Pax and Hox genes are developmentally regulated and can activate or repress several genes, mainly regulating cell differentiation. It has also been postulated that Hox genes are crucial to tissue-specific stem cells, specifically for self-renewal, tissue specificity and quiescence (Shah and Sukumar, 2010). Expression profiles of ES cells have been rigorously investigated because of the therapeutic possibilities of these cells. Expression of Pax and Hox genes has been documented (Baumann et al., 2003; Ramalho-Santos et al., 2002; Sharkey et al., 1997), but their roles have not yet been elucidated. Because galanin has been suggested to be involved in the maintenance of ES cells in an undifferentiated and proliferative state (Tarasov et al., 2002), we postulate that Pax and Hox genes may suppress galanin expression in mouse ES cells and, as a result, these cells can follow the appropriate path of differentiation. In the present work, our in silico analysis, together with the molecular studies of the galanin gene in mouse ES cells, suggested that both SP1 and CREB proteins may serve as important transcription factors to activate galanin basal promoter activity. However, HOX-F and PAX4-6 binding sites, which are conserved in the distal region, may interact with homeobox and paired box family members and

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Please cite this article as: Gonzalez, S., et al., Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.059