Elsevier

Gene

Volume 538, Issue 2, 1 April 2014, Pages 228-234
Gene

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

https://doi.org/10.1016/j.gene.2014.01.059Get rights and content

Highlights

  • ES cell proteins bind to conserved regions in mouse and human galanin promoter

  • CRE and SP1 at the basal promoter act as activators of transcription in mouse ES cells.

  • HOX-F and PAX 4/6 act as silencers of transcription in mouse ES cells.

Abstract

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.

Introduction

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).

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 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.

Section snippets

Computer analysis of the promoter region

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).

Cells and culture conditions

USP-1, a

In silico analysis of conserved regions within the galanin gene promoter

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

Discussion

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

Conflict of interest

There is no conflict of interest.

Acknowledgment

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

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