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γ-aminobutyric acid (GABA)ρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5′-end of the GABAρ1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABAρ1 transcription and regulate its expression during retinal development.
γ-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the vertebrate brain and retina (Martin and Olsen 2000). GABA can activate two types of receptors: ionotropic GABAA and metabotropic GABAB receptors (Olsen and Sieghart 2008). These two receptors differ in their subunit composition and gating properties, and in their pharmacological and physiological profiles. GABAA receptors (GABAA-Rs) mediate fast inhibitory neurotransmission and allow the flow of chloride ions; they are assembled from five subunits encoded by a family of 19 genes including α (1–6), β (1–3), γ (1–3), δ, ε, θ, π, and ρ (1–3) subunits. GABAρ subunits assemble into functional, homomeric receptors (Martínez-Torres et al. 1998; Martínez-Torres and Miledi 1999; Watanabe et al. 2000; Simon et al. 2004; Olsen and Sieghart 2008) that do not desensitize during continuous application of GABA, are resistant to the GABAA receptor antagonist bicuculline and insensitive to the GABAB receptor agonist baclofen (Polenzani et al. 1991; Chebib 2004), and are activated specifically by the CACA and antagonized by the TPMPA (Ragozzino et al. 1996).
The expression profile of GABAA-Rs varies among brain regions in adult and neuronal types during development. GABAρ1 is found in several areas of the central nervous system (CNS), including the thalamus, hippocampus, pituitary, cortex, cerebellum, superior colliculus, spinal cord, amygdala, and neostriatum (Boue-Grabot et al. 1998; Wegelius et al. 1998; Enz and Cutting 1999; Rozzo et al. 2002; Mejia et al. 2008, Rosas-Arellano et al. 2011, 2012). GABAρ receptors have been widely studied as they serve as a major point of control along the visual pathway. In the GABAρ1 knock out mouse, the inhibition mediated by this receptor is totally abated in retina, demonstrating its role in proper neural transmission. GABAρ1 mRNA is very abundant in retina, and the receptor is located at the axonal terminus of bipolar cells, where it limits the release of glutamate onto ganglion cells (Qian and Dowling 1993; Pan and Lipton 1995; Lukasiewicz 1996; Koulen et al. 1997; Feigenspan and Bormann 1998; Fletcher et al. 1998; Ogurusu et al. 1999; McGillem et al. 2000; McCall et al. 2002; Sagdullaev et al. 2006).
GABAρ subunits are encoded by three genes; in mouse, GABAρ1 and GABAρ2 are arranged in tandem on chromosome 4, and separated by approximately 40 kb (Greka et al. 2000), and GABAρ3 is on chromosome 16. During mouse retinal development, GABAρ1 is highly expressed in ON bipolar cells after post-natal day 7 (Greka et al. 2000; Wu and Cutting 2001; Kim et al. 2008); nonetheless, transcriptional regulation of this gene is poorly understood. In silico studies indicate that a 5′-flanking region of the GABAρ1 gene lacks a TATA box and contains initiator elements (Inr) and several general transcription factor-binding sites (SP1 and AP1) that are well conserved in human, rat, and mouse.
Vertebrate GABAA-R genes are clustered and conserved with a basic structure with 9-coding exons (Tsang et al. 2007). Computational and experimental data revealed that GABAA-R promoters contain CpG islands, Inr elements, and a TATA-box. Core promoters for some GABAA genes have been located to 100- to 500-bp upstream of the translation start codon (For review see Steiger and Russek 2004; Joyce 2007). Nevertheless, there is no detailed information about the transcriptional regulation of GABAρ subunits.
Comprehension of the transcriptional regulation of GABAA genes can provide an understanding of the mechanisms that contribute to the etiology of neurological disorders and their responses to drug treatment, development, and maturing of the brain and retina. This first exploration of GABAρ1 subunit transcriptional regulation proposes that the mouse GABAρ1 receptor promoter is included within a 0.232-kb fragment upstream of this gene and that it activates transcription in the secretin tumor cells (STC-1) and cells in the inner nuclear layer (INL) of the retina, where bipolar cells are present.
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- Materials and methods
- Supporting Information
In this study, we analyzed the minimal promoter region of the GABAρ1 gene. The GABAρ1 receptor regulates rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, controlling the excitatory input to ganglion neurons. GABAρ1 mRNA is highly expressed in retina, and the receptor is present in the terminals of the ON bipolar cells (Enz et al. 1996; Lukasiewicz 1996; Koulen et al. 1997; Boue-Grabot et al. 1998; Feigenspan and Bormann 1998; Fletcher et al. 1998; Wässle et al. 1998).
Initially, in silico analysis revealed that the GABAρ1 promoter lacks a TATA box, contains putative AP1, AP2, Ik-2, SP1, SRY, and E2F-myc transcription factor-binding sites, and two Inr elements that are well conserved in human, rat, and mouse. Indeed, the 5′-flanking region of the GABAρ1 gene is conserved among species of mammals and birds, but is quite dispersed when compared with amphibian and fish (Figures S1 and S2). Similar to many other genes, including genes coding for some GABAA-Rs, such as the human and mouse α3 (Mu and Burt 1999a), human α5 (Kim et al. 1997), human α6 (McLean et al. 2000), rat α6 (Jones et al. 1996; Bahn et al. 1997), mouse α6 (Jones et al. 1996; McLean et al. 2000), human β1 (Russek et al. 2000), human β3 (Kirkness and Fraser 1993), mouse γ2 (Mu and Burt 1999a, b), rat δ (Motejlek et al. 1994), and mouse δ (Sommer et al. 1990), the GABAρ1 gene promoter lacks a TATA box and contains multiple TSSs. Interestingly, the most abundant TSS detected by 5′ RACE contains the dinucleotide AC, which is a common transcription start point of RNA polymerase II transcripts (Breathnach and Chambon 1981). TATA-less promoters are not exclusive to ligand-gated ion channels. Genome-wide analyses of promoter regions have made it clear that TATA-driven, pre-initiation complex assembly is the exception in eukaryotic transcription, and only 10–20% of promoters contain a functional TATA box (Carninci et al. 2006; Yang et al. 2007). It has also been proposed that TATA-less promoters are commonly subject to evolutionary change in mammals (for review, see Sandelin et al. 2007).
Promoters cannot accurately be identified from sequence information alone, but they can be functionally classified as proximal or distal promoters. The proximal promoter is responsible for the correct positioning of the RNA polymerase II complex with respect to TSS, and its function is mediated by TATA and/or Inr elements that recruit the basal transcriptional machinery. On the other hand, the distal promoter contains multiple transcription factor-binding sites that confer greater specificity to transcription by stabilizing the pre-initiation complex (Lemon and Tjian 2000). After deleting the 5′ region of the GABAρ1 promoter, we observed that the minimal promoter, a 0.232-kb fragment, was enough to activate transcription in four different cell lines and in retina. Indeed, other GABAA subunits have minimal promoters with a comparable size; for example the rat α6 subunit gene, a 0.155-kb region (McLean et al. 2000); the human β1 subunit gene, a 0.279-kb fragment (Russek et al. 2000); and the human β3 subunit gene, a 0.143-kb minimal promoter region (Kirkness and Fraser 1993). This fact is noteworthy because it has been suggested that the GABAA-R clusters arose from an ancestral α-β subunit gene pair giving rise to the present GABAA-R subunits (Tsang et al. 2007). Remarkably, a model based on the selective action of putative scaffold/matrix attachment regions was proposed for the coordinate control and parallel expression of the α1 and β2 subunits, which are present in the mammalian GABAA-R gene cluster that comprises the α1, β2, γ2, and α6 subunits (Joyce 2007).
The mouse GABAρ1 minimal promoter contains a crucial Inr element, which is important for activating transcription, and is well conserved in the species of mammalian analyzed in our study. These elements are also included and required for functional activity of GABAA-R subunit promoters, such as the rat α5 (Kim et al. 1997); human, rat, and mouse α6 (Jones et al. 1996, 1996; Bahn et al. 1997; McLean et al. 2000, 2000), human β1 (Russek et al. 2000), mouse γ2 (Mu and Burt 1999a, b), and rat and mouse δ (Sommer et al. 1990; Motejlek et al. 1994). Deleting or mutating the second Inr element significantly decreased the reporter gene activity in transfected STC-1 cells and in electroporated retinal explants (Figs 3, 4 and 7). In TATA-less promoters, Inr elements regulate core promoter strength, determine the position of the TSS, and the interaction between Inr-binding proteins and components of the basal transcriptional machinery recruits RNA polymerase II to the transcription initiation complex (Weis and Reinberg 1992). Indeed, one Inr element is present in the 270-bp core promoter of the human GABAA β1 subunit gene, and it mediates down-regulation of this gene (Russek et al. 2000).
Deletions and mutations of the AP1-binding site decreased significantly the GABAρ1 promoter activity. The AP1 transcription factors are a group of proteins that recognize and bind to specific AP1 DNA motifs in the promoter regions of genes; however, several other transcription factors recognize the AP1-binding site. The AP1 transcription factor is a dimer, and its complexity begins with the transcription factor itself (Morgan and Curran 1991). It is composed of many different combinations of hetero or homodimers, and the composition of AP1 determines which genes it regulates. Nevertheless, until now, it is unknown whether any member of the GABAA-R family interacts with the AP1 transcription factor and whether these proteins are important for transcriptional activity.
Several genes, such as the mouse metabotropic glutamate receptor type 6 (mGluR6), the Purkinje cell protein 2 (Pcp-2), and the Purkinje cell-specific L7 protein (L7) are expressed in retinal bipolar cells (Nordquist et al. 1988; Oberdick et al. 1988; Nawy and Jahr 1990; Shiells and Falk 1990; Berrebi et al. 1991; Nakajima et al. 1993; Nomura et al. 1994), but little is known about the transcriptional mechanisms that induce or repress their expression. Ueda et al. (1997) determined the spatial and temporal expression pattern of the mGluR6 using a transgenic mouse, which expresses the lacZ reporter gene under the regulation of a 9.5-kb fragment located at the 5′-flanking region of the mGluR6 gene. This 9.5-kb fragment triggers cell-specific and developmentally regulated expression of the mGluR6 gene in ON bipolar cells. Remarkably, the reporter gene (β-gal) and the endogenous mGluR6 gene are expressed in temporal coordination with the differentiation pattern of bipolar cells, indicating that this DNA fragment responds to a genetic program of retinal bipolar cell differentiation (Ueda et al. 1997). As far as we know, a detailed molecular dissection of the mGluR6 promoter and identification of putative transcription factor-binding sites have not been performed.
Pcp-2 is also expressed in retinal bipolar and cerebellar Purkinje neurons (Oberdick et al. 1990). The analysis of the upstream DNA sequence of the gene revealed the presence of general transcription factor-binding sites such as AP1, CRE, and Oct. Interestingly, a construct carrying 0.4-kb of upstream and 0.3-kb of downstream Pcp-2-flanking DNA to LacZ activated the β-galactosidase expression in a large number of neurons, including Purkinje neurons and bipolar cells. In contrast, a second construct carrying an additional 3.1-kb fragment of Pcp-2 upstream sequences limited the β-gal expression to Purkinje cells, whereas bipolar cells of the retina did not show activity of the reporter gene. These results suggest that the 3.1-kb fragment includes elements that suppress the transcription in bipolar cells, that is to say, additional components are involved in the negative regulation of the Pcp-2 gene within the retina (Vandaele et al. 1991).
For studying the L7 gene expression pattern, a transgenic mouse driving the β-gal expression was employed. This model contains 4-kb upstream of the start of the TSS and 2-kb downstream of the polyadenylation signal. High levels of the reporter gene were detected in retina and cerebellum; however, the essential sequences that drive the expression in bipolar neurons were not explored in this study (Oberdick et al. 1990).
When we tried to electroporate a construct carrying the 0.232-kb fragment in P6 cerebellum, we could not detect significant levels of GFP after 7 days in vitro (Figure S3). It is possible that other regulatory sequences are required for GABAρ1 expression in neurons and glia of the cerebellum, where this subunit has been found to be expressed (Harvey et al. 2006; Mejía et al. 2008, Reyes-Haro et al. submitted).
In conclusion, on the basis of all of these results, we suggest that a 0.232-kb fragment located upstream of the GABAρ1 gene is functionally important for activating transcription, and that the second Inr element (Inr-B) and the AP1 site are critical for promoter activity in transfected STC-1 cells and in vitro electroporated retinae. As the GABAρ1 receptor acts as a major point of control along the visual pathway, further studies should be considered to identify the specific transcription factors that participate in GABAρ1 gene transcriptional regulation during retinal development.