GABAA α1 subunit gene
The α1 subunit gene is strongly expressed in all brain regions, being a component of the most abundant GABAA receptor subtype, α1β2γ2 . Kang et al  have experimentally isolated a 60 bp minimal promoter element in the 5' flanking DNA sequence of the human α1 subunit gene. It was demonstrated that reduction in transcription of the subunit gene following chronic benzodiazepine exposure is brought about by direct repression of the activity of this promoter. Bateson et al  have identified the TSS for the chicken GABAAα1 subunit gene, and reported a number of putative promoter elements, including a TATA box 30 bp upstream of the TSS, SP1 binding site and reverse CCAAT box. The promoter region is CG-rich. The TSS for the rat gene has also been experimentally determined, and lies 5 bp 3' of the chicken TSS (A.N. Bateson, personal communication, unpublished).
An alignment was produced from the 5' flanking genomic DNA sequence of the α1 receptor for four species – human, chicken, mouse and rat (Figure 2). This alignment reveals a highly conserved section, across all species, corresponding to the experimentally determined human promoter region, and the chicken and rat TSS. Sequence identity is almost 100% in the core promoter region. Sequence data for more evolutionarily distant species, with sufficient identity to reveal any crucially conserved motifs within more divergent regions of sequence, was unavailable for the α1 subunit gene. The chicken TSS AGC is unique in the alignment, replaced with CGC in the mammalian species. There exists cDNA sequence for the mouse α1 gene which extends at 137 nt upstream relative to the chicken TSS . This suggests the presence of a TSS upstream of that identified for the chicken, and the possibility of Multiple Start Sites for this gene. There is unpublished evidence that this is the case . However, a scan for promoter regions using the PromoterInspector program did not identify any putative promoter region upstream of the mouse cDNA sequence. The TATA box and SP1 site  are fully conserved. In addition, a DPE element GGACG is conserved in its precise position with respect to the chicken TSS (28 bp downstream). There is also a conserved cAMP Response Element (CREB) element upstream of the minimal promoter, detected by the P-MATCH program. An NFI motif CTCTGGCATGAAGTCACA lies between TATA box (partially overlapped) and TSS. An NRSE-like sequence TTCAGCAAAGGAGCACGCAGA in the human gene lying approximately 115 bp downstream of the TSS  is not conserved across species.
GABAA α2 subunit gene
The α2 subunit gene is strongly expressed only in the hippocampus and hypothalamus. Enhanced mRNA levels occur early in CNS development, and decline postnatally with a concurrent rise in α1 gene expression . Fuchs & Celepirovic  have investigated the rat α2 subunit gene, and identified six α2 subunit mRNA isoforms with distinct 5-end UTRs (the resulting protein sequence is not affected), generated from three alternative first exons by means of alternative splicing and alternate promoter usage. The isoforms are named AB8, SPL2, SPL3, SPL4, SPL5 and non-spliced variant nonSPL. About 70% of expressed mRNA is of the AB8 isoform. Multiple Starts Sites (MSS) for each isoform were identified within each of the three alternative first exons. The AB8 isoform putative promoter region is GC-rich, and contains putative INR elements and TF binding sites, including SP1 binding sites. Unusually, the AB8 promoter region is located downstream from the transcription initiation sites .
The human α2 subunit gene also exhibits a complex pattern of alternative splicing and alternative promoter use . There are four major isoforms composed using combinations of alternative exons. Two alternative 5' exons each lie downstream of two different promoter regions. The alternative first exons and their promoters all lie within a CpG island as detected by CPGPlot. MSS genes are characterised by a transcription initiation window of weak transcription start sites, and their promoters characteristically lack a TATA box. These characteristics are apparent from an analysis of the putative core elements in the identified rat and human promoter regions; the sequences are largely devoid of TATA boxes or other canonical core promoter motifs. An alignment was produced for the region encompassing the human exon 2a TSS and promoter region, and the homologous rat Exon 1A promoter region, for 3 species – human, mouse and rat (figure 3). The experimentally determined TSS sites for human and rat do not coincide. There are fully conserved IK2 (Ikaros) and SP1 sites within the region.
GABAA α3 subunit gene
The α3 subunit gene is expressed throughout the adult brain. Like the α2 gene, enhanced expression occurs early in CNS development and declines postnatally with an accompanying rise in α1 expression . The promoter region and major TSS of the mouse α3 subunit gene have been experimentally determined . The highly unusual promoter region contains a series of GA repeats about 40 bp upstream from the major TSS, which bind unspecified nuclear proteins and directly augment transcription, and in which lie several minor TSS sites. An adjacent series of three GC repeats, forming part of a putative E2F sequence motif, appear to also contribute to promoter activity.
An alignment was produced from the 5' flanking genomic DNA sequence of the α3 subunit gene for 3 species – human, mouse and rat (figure 4). The GA repeat regions, corresponding to the experimentally determined mouse promoter region, are of variable length across species . The major TSS is conserved in the alignment. The core promoter is apparently devoid of TATA boxes or INR-like sequences. There is, however, a conserved DPE motif GGAC [CT] located 29 bp downstream of the determined TSS. Searches for TFBSs in the larger region encompassing the GA repeat core promoter, using the TESS, P-MATCH and MatInspector programs did not reveal any conserved ubiquitous or neuronal TFBS motifs in the proximal region upstream of the TSS.
GABAA α4 subunit gene
The α4 subunit gene is expressed variably in different regions of the brain. Elevated expression levels are observed in animal models of temporal lobe epilepsy, and during withdrawal from alcohol and progesterone treatment, suggesting that the subunit may play a role in neuronal hyperexcitability . Ma et el  have determined the minimal promoter, major TSS and start codon ATG for the mouse α4 subunit gene. They also identified two SP1 sites within the proximal promoter region which are critical for high-level promoter activity in vivo, and bind to the TFs SP3 and SP4 to augment α4 subunit gene expression in neuronal cells. Other putative SP1, AP1, c-Myb, and E-box binding sites did not alter transcription levels when deleted. Roberts et al  have demonstrated increases in mRNA levels of α4, and in early growth response factor 3 (EGR3) expression, in response to induced epilepsy, accompanied by increased binding of EGR3 to α4 subunits in dentate granule cells. Also, EGR3 knockout mice exhibit reduced α4 hippocampal mRNA expression. This data demonstrates the role of EGR3 as a major regulator of α4 subunits, and implicates the factor in epileptogenesis.
The promoter appears to contain no canonical core promoter elements. A predicted CpG island (CPGPlot) envelops the TSS. Recruitment of the basal transcription factors to form the pre-initiation complex presumably involves the SP1 sites in combination with one or more unknown promoter elements. An alignment of the promoter region for four species – human, cow, mouse and rat – reveals that the TSS, and SP1 and EGR3 sites are conserved across all species (figure 5). A motif AGCGCGGGCGAGTGTG occurs only once immediately prior to the TSS in the human sequence, but is exactly repeated in other species. A conserved CREB element lies within the minimal promoter, upstream of the SP1 sites. There is also a conserved NFI motif; however this lies outside of the identified mouse minimal promoter region, immediately upstream of the CDS.
GABAA α5 subunit gene
Expression of α5 subunit receptors is restricted primarily to the hippocampus . Kim et al  have identified three isoforms of human α5 subunit mRNA, resulting from three alternative first exons. They have demonstrated that each exon appears to be regulated by a different promoter, located in intronic sequence regions immediately upstream. The differential transcriptional activation by alternative promoters may determine the alternative usage of the first exon isoform. No core promoter element motifs are observed in these promoter regions, however both regions are enveloped within a single CpG island and contain a large number of putative TF binding sites including SP-1, AP-1 and AP-2 sites.
An alignment was produced for human, rat and mouse sequences encompassing exons 1A, 1B, 1C and their associated promoter regions (figure 6). For the exon 1A promoter region, none of the putative binding sites identified  are conserved in the alignment; however there is a fully conserved NFI element within exon 1A, 124 bp downstream of the TSS. Within the Exon 1B promoter region lies a conserved sequence containing two overlapping reverse AP-2 motifs, and the exon itself contains IK2 and SP1 elements. Exon 1C and its promoter region are not well conserved across species, and none of the identified putative binding sites  are conserved in the alignment (data not show).
GABAA α6 subunit gene
The α6 subunit gene is expressed exclusively in cerebellar granule cells , and as such provides a useful model for studying how neuron-specific gene expression is regulated. McLean et al  have identified several minor and one major TSS with a surrounding INR in homologous rat and mouse sequences. They have determined a 155 bp minimal promoter in the rat DNA, within which a 60 bp region, 70 bp upstream of the TSS, enhances expression in cerebellar granule cells only. This region is positionally dependent with respect to the TSS, and contains a conserved NF-1 (NFI, nuclear factor 1) motif. They have also identified a downstream negative regulatory region which is active in fibroblasts but inactive in cerebellar granule cells. Wang et al  have determined that NFI-A factor is abundant in cerebellar granule cells and binds to the α6 subunit gene promoter in vivo in the mouse gene, confirming its critical role in α6 subunit gene expression and the differentiation of cerebellar granule neurons. They have also identified the NFI motif TGCCAAAAC within the α6 gene promoter region which binds NFI-A proteins.
An alignment of the promoter region for four species – human, chicken, mouse and rat – reveals that the rat and mouse INR motif encompassing the TSS  is not conserved in the human and chicken (figure 7), however the human sequence contains an INR-like motif TCAGACT 9 bp upstream. The chicken sequence also contains a putative INR CTAATTC in the same region. A DPE element GGCTG at position +27 relative to mouse and rat TSS is conserved across species. For rat and mouse sequences, there exists a putative CCAAT box at position +10 relative to the TSS (not conserved in human and chicken). The NFI binding site  is fully conserved.
GABAA β1 subunit gene
The β1 subunit is present in a high proportion of the GABAA receptors from the hippocampus and cerebral cortex, and a very small proportion of cerebellar GABAA receptors . The human β1 subunit gene contains a TATA-less core promoter region of 270 bp . The core promoter is neural specific and contains an INR element at the major TSS which is critical to promoter activity. Upstream of the INR, A GRE consensus sequence has a neural-specific positive regulatory effect, and CCAAT (NF-Y) and IK2 motifs have negative regulatory effects. Persistent receptor activation by the GABA neurotransmitter induces down-regulation of β1 subunit mRNA expression. The exposure to GABA appears to directly reduce promoter activity by modulating the binding of sequence-specific basal TFs to the promoters INR element, thus regulating formation of the Pre-Initiation Complex (PIC) .
Each of the experimentally determined regulatory elements are conserved in an alignment of human and mouse sequences encompassing the core promoter region (figure 8). The negative regulatory sequence reported as an IK2 binding site could also be interpreted as an OCT1 (octamer binding protein) motif GGGATTGAAATCTG. A conserved SP1 motif is situated within the identified minimal promoter, 50 bp upstream of the major TSS.
GABAA β2 subunit gene
The β2 subunit is a component of the most abundant GABAA receptor subtype, α1β2γ2, and is strongly expressed in all brain regions, having an almost identical expression profile to the α1 receptor. Alternative splicing of the gene generates two transcript variants, differing by a 114 bp insertion . There is currently no experimental data available for transcription regulatory features in the β2 subunit gene. In order to search for a putative promoter region, GenBank DNA sequence for the human β2 subunit gene was searched using the programs PromoterInspector, NNPP, Promoter 2 and McPromoter, within a region extending from 2000 bp upstream of the mRNA start point to approximately 500 bp downstream. The results were compared and combined to derive a consensus predicted promoter region of 900 bp, 510 bp upstream of the GenBank CDS for further analysis. Homologous DNA sequences were then obtained for the β2 subunit gene in the mouse, rat and domestic dog species. This data was used to generate a multiple sequence alignment (figure 9), revealing a highly conserved region (78% for putative core promoter), containing putative TATA box, INR and DPE elements. Searches for TFBSs in the larger region encompassing the putative core promoter, using the TESS, P-MATCH and MatInspector programs did not reveal any fully conserved ubiquitous or neuronal TFBS motifs, however there is a partially conserved SP1 sequence and a CREB element upstream of the putative core promoter.
GABAA β3 subunit gene
The β3 subunit is the most abundant β isoform in GABAA receptors of the hippocampus, and is also widely expressed in the cerebral cortex and cerebellum. It is frequently co-localised with either a β1 or β2 subunit in the same GABAA receptor, in proportions which vary with brain region . The human β3 subunit gene contains two alternate first exons, 1 and 1A, which are expressed differentially during development and between different brain regions, and encode two dissimilar signal peptide-like sequences. Exon 1A lies upstream of exon 1 and encodes a peptide signal sequence. Transcription of exon 1A is initiated by multiple start sites in a pyrimidine-rich promoter region, which lies in between the alternative first exons, and binds SP1 and other nuclear factors. Nuclear factor binding sites overlap each of these transcription start sites, which may be involved in differential expression of the alternative first exons. The human and rat coding and promoter regions are highly conserved .
An alignment was produced of the human and mouse 5-end gene sequences, encompassing the upstream regions of the alternate first exons (figure 10). There are three conserved SP1 sequences upstream of exon 1, and a partially conserved TATA box. The exon 1A promoter region contains no canonical core promoter elements. This is not surprising given that it is composed almost entirely of pyramidines. Several conserved SP1-like sequences are located upstream from the pyrimidine-rich promoter region.
GABAA γ1 subunit gene
Expression of the γ1 subunit is more restricted than that of the γ2 subunit, occurring at high levels predominantly in certain amygdaloid, hypothalamic and premammillary nuclei . The γ1 subunit gene promoter region has not been characterised. Analysis of the 2000 bp region upstream of the GenBank computationally-derived TSS for the human sequence by PromoterInspector and McPromoter predicted no promoter in this region; however the NNPP and Promoter 2 programs predicted promoter regions, 251–301 bp and 489–589 bp upstream of the GenBank TSS respectively. Homologous rat and mouse sequences for the human region encompassing the predicted promoter regions and GenBank TSS were obtained and aligned (figure 11). The GenBank putative TSS and CDS for each species is conserved in the alignment. An INR consensus sequence CCACACT is located 16 bp upstream of the human GenBank TSS. The region also contains an IK2 binding sequence and two conserved reverse CCAAT boxes, which could also be interpreted as NF1 binding sites.
GABAA γ2 subunit gene
The γ2 subunit gene is expressed widely throughout the brain, occurring at high levels early on in CNS development . Mu and Burt  have found two major transcription start sites in a TATA-less promoter region of the mouse γ2 subunit gene. There are two subtypes of the subunit, which are formed by alternate splicing which is regionally and developmentally regulated. The first intron of both the mouse and human γ2 genes contains a conserved neuron-restrictive silencer element (NRSE) site, which was shown to bind to NRSF and direct expression in neuronal cells, and to repress expression in non-neuronal cells. Another sequence element, the "Gamma Promoter Element" (GPE1), which also promotes expression in neuron-like cells, lies 70 bp downstream of the second major TSS. Mu and Burt  report that "probably only a portion of the 24 bp sequence is involved". The first 12 bp matches the cAMP-responsive element binding protein (CREB) consensus sequence (program P-MATCH).
An alignment of the promoter region for four species – human, cow, mouse and rat – reveals that the identified region maintains a very high level of conservation across all species downstream of the TSS (figure 12). Sequence identity is 88% in this region, but is not so high at the 5-end of the first major TSS. A number of putative TFBS sites, including NF1, AP2, and several SP1 sequences, are in conserved in the alignment downstream of the TSS sites. The experimentally determined NRSF and GPE elements  are also conserved in all species. The promoter appears to be without TATA box or INR; there exists however a conserved DPE sequence AGC [TC]G located at +27 bp with respect to the second the mouse major TSS.
GABAA γ3 subunit gene
The γ3 subunit gene is expressed at low level in the cerebellum and hippocampus . The gene promoter has not yet been experimentally determined. Analysis of the 2000 bp region upstream of the GenBank computationally-derived TSS for the human sequence by the PromoterInspector, McPromoter, NNPP and Promoter 2 programs gives a consensus promoter prediction within a region approximately 550–100 bp upstream of the GenBank putative TSS and CDS. This region is GC rich and contains no TATA sequences. The program CPGPlot predicts a 554 bp CpG island which matches closely the putative promoter region. The γ3 subunit gene promoter may therefore lie within a CpG island, in common with a number of other GABAAreceptor subunit genes such as α2, α4, α5 and δ. Although such 5-end CpG islands commonly extend downstream of the promoter into the transcription unit , this does not appear to be the case for any of the GABAA subunit genes.
An alignment of the region was generated using human and homologous mouse sequence (figure 13). The region contains numerous putative regulatory elements such as SP1, AP2 and NRSE, detectable by MatInspector etc; however these are generally not conserved in the alignment. An INR element TCAAC located 70 bp downstream of the start of the predicted CpG island is conserved.
GABAA δ subunit gene
Expression of the GABAA receptor δ subunit gene is most abundant in granule cells of the cerebellum and dentate gyrus. The 5-end of the murine δ subunit gene contains a region with CpG island features, and lacks canonical promoter elements such as the TATA box . The rat δ subunit gene and promoter has been characterised by Motejlek et al  who have mapped the major TSS, which is enclosed within an INR element CCACTCT. The promoter region lies within a CpG island. They have also identified a 22-bp purine-rich element, present in seven partially-overlapping copies, approximately 1800 nucleotides upstream of the TSS. The element is bound by a novel "brain-specific factor" (BSF1) that is present predominantly in cerebellar granule cells, correlating with GABAAδ subunit expression, however it is not clear if the factor plays any role in transcription regulation of the δ subunit gene.
An alignment was produced using the genomic sequence encompassing the rat promoter region with upstream purine-rich BSF1 element repeats, and homologous human and mouse sequences. Whilst there is homologous sequence between species for the promoter region (figure 14), no sequence similar to the BSF1 element repeats is observable for the human and mouse. A functional negative strand NRSF binding site  exists downstream of the TSS in the first exon, TCCGCCGTCCTCGGTGCTGAA. This is conserved, with a single nucleotide deletion in the human sequence. The INR element enclosing the major TSS is not conserved in the human sequence. A number of putative SP1 sites are detectable in the region but generally these are not fully conserved in the alignment.
GABAA ε subunit gene
Alternative splicing of the ε subunit gene yields a number of different transcript isoforms. Expression of the full protein sequence occurs abundantly only in discrete areas of the adult brain, such as the hypothalamus and locus ceruleus. In other tissues, truncated subunit protein sequences are transcribed . The ε subunit gene promoter has not been characterised. The programs McPromoter, NNPP Cister and Promoter 2 predicted no promoter in the 2000 bp region upstream of the GenBank computationally-derived CDS for the human sequence GABAAε subunit gene; however the program PromoterInspector predicted a 324 bp promoter within a region approximately 400 bp upstream of the GenBank computationally-derived CDS. For the mouse sequence, PromoterInspector also predicts a 200 bp promoter in a 180 bp upstream of the GenBank CDS.
The human predicted promoter region encompasses that of the mouse in a sequence alignment (figure 15). However, the use of cross-species sequence alignments to search for conserved transcription regulatory features is particularly problematic for the ε and θ subunits, because the genes from mouse and rat have been shown to display an unusually high level of divergence from their human homologues, and appear to be evolving at a faster rate than other GABAA receptor subunits . The ε and θ subunit expression patterns and the functional properties of receptors containing these units may differ significantly between species. The transcription regulatory features will therefore probably be more variable between species than for the other subunits.
The putative region appears to contain no fully conserved canonical core promoter features, however the mouse and rat sequence have an INR motif CCAGACC 45 bp upstream of the GenBank CDS, whereas the human sequence contains an INR motif at the beginning of the putative promoter region. For all species, the region has the features of a CpG island (CPGPlot), and contains conversed SP1 binding site sequences.
GABAA θ subunit gene
GABAA receptor θ subunits show very similar distributions to ε subunits throughout the brain, and may also vary significantly between species . Novel α3θε GABAA receptors with unique electrophysiological and pharmacological properties may form in monoaminergic neuronal cell-groups . There is no experimental data for the θ subunit promoter. Promoter prediction programs were used to search both mouse and human sequences. No promoters were predicted by the MatInspector or McPromoter programs in either sequence. The program NNPP predicts two core promoters in the human sequence approximately 1500–1600 bp upstream of the GenBank TSS. Each putative promoter has a TATA motif. No promoter was predicted by NNPP in the homologous mouse sequence, and the TATA sequences are not conserved.
Further downstream, CpG islands are predicted for both human and mouse (figure 16), 100–200 bp upstream of the GenBank TSS. This region contains a number of putative TFBS elements such as an negative CCAAT box and SP1 consensus sequences which are conserved in the alignment. There are also conserved consensus sequences for two INR elements, one of which has a downstream DPE sequence. Genes for ε and θ subunits from rodent species display a high level of divergence from their human homologues (. Despite the expectation that the transcription regulatory features may therefore be more variable between species than for the other subunit genes, a high level of sequence identity was observed in the aligned putative promoter regions for these genes, with conservation of putative core promoter elements.
GABAA π subunit gene
The subunit encoded by this gene is expressed in several non-neuronal tissues, and most abundantly in the uterus .
The π subunit gene promoter has not yet been experimentally determined. The programs PromoterInspector, McPromoter and NNPP predicted no promoters in the 2000 bp region upstream of the GenBank start point for the human GABAAπ subunit gene sequence. Program Promoter 2 makes a marginal prediction (score 0.613) of a promoter between 200–300 bp upstream. There are several INR motifs in the human sequence 450 bp upstream of the gene start point, one of which with a DPE element at +28 from the putative TSS. However when an alignment was made with homologous sequence from the cow gene these motifs were not conserved (figure 17). Putative AP1 and SP1 sites found by the Cister program in the vicinity of this region of sequence were not conserved.
The 5-end of this gene displays some characteristics of a multiple start site promoter region such as that observed in the α2 subunit gene. Here also, there is no TATA sequence or well-defined TSS, but a number of INR motifs form a transcription initiation window of weak transcription start sites, which again are not fully conserved across species. Sequence identity in this region is lower than in the promoters of GABAA receptor genes with a single TSS, and individual start sites are not as well conserved. This appears to be characteristic of MSS promoters, and is presumably a result of relaxed selected pressure due to a degree of redundancy introduced by MSS transcription. It is quite possible also that the computationally derived GenBank gene origin is incorrect, and the TSS and promoter may lie outside this region of sequence.
Transcriptional regulation of the GABAA receptor subunit gene cluster on chromosome 5
The human α1, β2, γ2 and α6 subunit gene cluster is located on chromosome 5q33 and spans 500–700 kb. The genes lie in the order β 2 - α 6 - α 1 - γ 2, with β2 and α6 in a head-to-head orientation. α6 high-level expression is restricted to cerebellar granule cells, and is probably regulated independently from the other genes in the cluster . Wang et al  have determined that NFI-A factor is abundant in cerebellar granule cells and binds the α6 subunit promoter, playing a critical role in α6 gene expression. The γ2 subunit gene's expression profile overlaps with that of α1 and β2, but is yet more widespread, combining with several subtype variants in addition to the most common 2α 1 - 2β 2 - 1γ 2 configuration. Transcriptional control of γ2 is therefore at least partially independent from that of α1 and β2. Its promoter is known to contain an NRSF binding site and a "Gamma Promoter Element", both of which direct expression in neuronal cells .
The α1 and β2 genes are widely expressed in neural tissue, and have almost identical expression profiles. The question therefore arises as to whether they have the same expression profiles by virtue of each possessing common transcriptional regulatory features in individual promoters, or whether coordinate regulation is achieved by the action of shared long-range transcriptional regulatory elements such as enhancers, silencers or an LCR. The α1 subunit gene core promoter is well characterised, and contains a canonical promoter , with TATA box, INR and DPE motifs, and an upstream SP1 site, typical of ubiquitously expressed housekeeper genes. There are also putative sites for NFI and NRSE in the α1 subunit gene, but there is no experimental evidence that they are required for tissue-specific expression (indeed, the NRSE motif is not conserved in cross-species alignments). Although the β2 subunit gene core promoter is undetermined, computational analysis here provides evidence that this subunit is under the control of a very similar promoter, with TATA box, INR, DPE core elements, also with an upstream SP1 site. Based upon this predicted promoter, the β2 subunit expression profile may closely matches that of the α1 subunit at least in part by possessing the same core promoter characteristics.
Prediction of long-range regulatory features using comparative analysis
Both proximal and long-range regulatory features are more highly conserved between species by evolutionary selective pressure than is the surrounding non-regulatory DNA. Functional long-range regulatory features in a gene cluster should therefore stand out as relatively short regions of intergenic sequence which are more highly conserved than the surrounding background DNA. In order to investigate whether the α1 and β2 subunit genes share common long-range regulatory elements, the rVista program ( was used to perform cross-species, comparative analysis of the human chromosome 5 GABAA cluster locus, and intergenic homologous regions were isolated for further analysis.
Firstly the human and chicken DNA sequences were compared. A cluster of three conserved regions with sequence identity greater than 70% were identified between 3–5 kb from the end of the β2 gene (figure 18). The first of these regions (numbered 1 in figure 18), 458 bps with 81.2% identity, is particularly rich in putative sites for ubiquitous or neural tissue-associated TFBSs. It includes motifs for NFI, a regulator of the α6 subunit , OCT1, FOX (fork-head box) and EGR3, which is linked with α4 subunit promoter activity . Given the location of the three human-chicken homologous intergenic sequences, 3–5 kb beyond the final β2 subunit gene exon, it is also possible that the high sequence identity indicates the presence of a degenerate or un-annotated alternative exon. For the third sequence, for example, a possible ORF extends from base 73 to 234 on the reverse (β2 gene) strand. However, as the region is rich in regulatory motifs it probably merits further analysis as a possible locus for regulation of one or more genes in the cluster. Several other regions of unknown function in the cluster were locally conserved between human and chicken, but were not enriched with TFBS motifs.
The rVista program was also used to compare human and mouse DNA in the GABAA cluster, to identify potential regulatory features limited to mammalian species. This revealed a number of intergenic homologous regions with sequence identity greater than 75%, not conserved in the chicken species. These were further analysed for putative neuronal or ubiquitous TFBS motifs. In addition to the motifs found in the conserved chicken homolog, these regions contain motifs for STAT (signal transducer and activator of transcription), CEBP (CCAAT/enhancer binding protein), CDP (CCAAT displacement protein), NF (Nuclear Factor) Kappa B, activator protein 4 (AP4), upstream stimulating factor (USF), activating transcription factor (ATF), enhancer factor 4 (E4F). An analysis of conserved intergenic regions is summarised in figure 18. Each of these conserved regions represent potential distal regulatory elements for one or more of the genes in the cluster, which could be inactivated by the neomycin gene insert  which was observed to cause disruption of α1 and β2 expression.
Potential DNase I hypersensitive sites in the gene cluster
Regions of DNase I hypersensitivity within a gene locus, which are not associated with core promoters, are indicators of distal cis-regulatory elements such as enhancers or silencers . Clusters of HSs provide markers for possible LCRs controlling transcription in the locus. As there is currently no HS prediction tool available, the locus of the GABAA chromosome 5 cluster was analysed based upon published locations of predicted HSs  (Coordinates are from the April 2003 human genome assembly). Although this data is based upon analysis of erythroid sequences, it was hypothesised that a proportion of the predicted sites would also be DNase I hypersensitive in other cell lines [41, 42], including neuronal cells.
An analysis of the gene cluster reveals that there are 18 predicted HSs within the 862 kb locus. The positions of the β2, α6 and α1 gene promoters in the cluster closely coincide with HS predictions, illustrating the potential value of this approach. Nine of the 18 predicted HSs conspicuously fall into a 2.5 kp region, which is contiguous apart from three 125 bp segments. This putative HS cluster, approximately 1.5 kb downstream from the β2 gene (on the anti-sense strand in figure 19), is a candidate site for a transcriptional regulatory region controlling one or more genes in the cluster. The proximity to the β2 promoter suggests a regulatory feature specific to this gene. Or, based upon the beta globin and other LCR models, these could be constitutive HSs clustered with other neuronal-specific HSs forming an LCR, augmenting transcription of both β2 and α1. HSs, like other transcriptional control sequences, tend to be conserved across species , and the organisation of the β-globin LCR, for example, is highly conserved in the human and the mouse gene locus . There is no cross-species data available for HSs in the GABAA receptor gene cluster locus with which to perform comparative analysis. However, it is of interest to note that the predicted cluster of HSs corresponds closely with a cluster of four intergenic homologous regions identified by comparative analysis of human and mouse DNA (figure 18, numbered 4–7).
Prediction of S/MARS and a model for coordinate regulation of β2 and α1 subunit genes
The program MAR-Wiz was used to predict S/MARS within and around the human chromosome 5 GABAA receptor gene cluster, and in the homologous mouse cluster on chromosome 11. Results with a cut-off score of 0.7 were considered.
For both species, S/MARs are predicted in the vicinity of the α6 gene promoter region, and flanking the gene cluster (figure 20). This configuration, with a centrally located S/MAR and two S/MARs flanking the cluster, suggests the model similar to that proposed by Heng et al , in which S/MARs are selectively utilised to provide two functions: as structural anchor points for chromosomal attachment to the nuclear matrix, and as dynamic features which draw potentiated chromatin loops into transcriptional machinery in the proximity of the nuclear matrix. The model proposed here (figure 21) is also based partially upon that proposed for the multigenic human protamine chromatin domain, where in absence of a well-defined LCR, S/MARS bounding the domain act synergistically to regulate the expression of the gene cluster :
(a) Firstly, the region surrounding the gene cluster is anchored to the nuclear matrix by flanking S/MARS, which establish a tissue-specific chromatin domain and define the boundaries of transcriptional activity by preventing the propagation of chromatin condensation, thus shielding the locus from the silencing effects of neighbouring chromatin. Tethering of the domain to the nuclear matrix may also bring it into proximity with chromatin modifying processes which participate in opening of the chromatin loop domain to potentiate transcription.
(b) A central, functional S/MAR in the vicinity of the α6 subunit gene promoter migrates to the nuclear matrix, under the control of accessory transcriptional regulator factors. This causes the potentiated chromatin to associate with transcriptional machinery proximal to the nuclear matrix. The location is approximately 100 kb from a region containing a cluster of predicted for HSs, which was also observed to be rich in conserved putative TFBSs upon comparative analysis of the human and mouse genomes. The central S/MAR may also play a role in the demethylation of DNA regions to trigger further chromatin conformational changes.
(c) The transcriptional machinery reels in the open chromatin in either direction until a promoter is encountered, at which point gene transcription initiates. Whilst the α6 and γ2 genes in the cluster are regulated independently by other cell-specific TFs, a shared complement of TFs and corresponding cis-regulatory elements presumably accounts for the identical expression profile of the β2 and α1 subunit genes. Distal enhancer elements, some of which are shared by both β2 and α1 subunit genes, may loop back into the region of the transcriptional machinery to provide tissue-specific transcriptional augmentation.
(d) Transcriptional elongation causes the functional S/MAR to dissociate from the vicinity of the nuclear matrix, so that once the transcription is complete, a new cycle of transcription can be initiated. Selection of either the β2 or α1 gene for transcription may be random, or there may be an unknown mechanism of alternating the expression of β2 and α1 to ensure parallel expression levels.
The model provides an explanation for number of observations – the head-to-head configuration of the β2 and α1 subunit genes, their parallel expression profiles, and the parallel down-regulation of expression observed upon insertion of the neomycin gene insert at exon 8 of the α6 subunit gene , which is the vicinity of the proposed functional S/MAR, and is presumably close enough to interact and to reduce its effectiveness. The basic model could also incorporate expression of the α6 subunit, assuming that additional gene-specific factors contribute to produce a different tissue-specific expression profile. However it is probable that the functional S/MAR would not be critical for γ 2 gene transcription, since its expression is unaffected by the neomycin gene insert and subsequent S/MAR disruption.