In silico comparative genomic analysis of GABAA receptor transcriptional regulation

Abstract

Background: Subtypes of the GABAA receptor subunit exhibit diverse temporal and spatial expression patterns. In silico comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABAA receptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABAA receptor gene clusters.

Results: Previously unreported putative promoters were identified for the beta2, gamma1, gamma3, epsilon, theta and pi subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABAA receptor gene cluster comprising the alpha1, beta2, gamma2 and alpha6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the alpha1 and beta2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs).

Conclusion: The putative regulatory features identified by genomic analysis of GABAA receptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the alpha1 and beta2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the alpha6 gene, which is proximal to a putative critical S/MAR.

Figure 1

(a) GABA_A receptor subunit composition (b) GABA_A receptor gene clusters. (a) Two α, two β and one γ subunit surround a central channel, with a GABA binding site located at each α-β interface. (b) Gene order, intergenic distance and the head-to-head orientation of the α and β subunit genes are conserved in each cluster. After Russek [8]

Figure 21

Model for S/MAR associations in GABA_A receptor subunit gene locus. (a) Gene locus anchored to nuclear matrix by flanking S/MARS (red) establishes open chromatin conformation. (b) Functional S/MAR mediates loop attachment to transcriptional machinery (pink). (c) Chromatin is reeled through transcriptional machinery in either direction to activate transcription of β2 or α1 gene (d) functional S/MAR dissociates upon elongation. (After Martins et el [15], Heng et al [17]).

Figure 2

α1 gene alignment, core promoter region. Experimentally determined chicken and rat TSS (green), and human promoter region (yellow) highlighted.

Figure 3

α2 gene promoter alignment. Experimentally determined rat and human promoter regions in human exon 2A and rat exon 1A (yellow), with TSS (green). ('N' in sequence indicates undetermined nucleotide).

Figure 4

α3 gene promoter alignment. Experimentally determined mouse GA-repeat promoter region and homologues (yellow), with TSS in green. Putative TFBS sites in bold. (CLUSTAL-X gap extend penalty = 1).

Figure 5

α4 gene alignment, core promoter region. Experimentally determined mouse CDS, TSS, EGR3 and SP1 sites (green), and minimal promoter region (yellow).

Figure 6

α5 gene alignment, exons 1A and 1B core promoter region. Experimentally determined human exons underlined.

Figure 7

α6 gene promoter alignment. Experimentally determined rat minimal promoter (yellow), with rat and mouse TSS and mouse NFI site in green. Putative sites in bold.

Figure 8

β1 gene alignment, core promoter region. Experimentally determined human minimum promoter (yellow), also INR, and GRE (positive) and CAAT and IK2 (negative) regulatory elements. Putative SP1 motif in bold.

Figure 9

β2 gene putative promoter alignment. Putative core promoter elements in bold

Figure 10

β3 gene alignment, core promoter region. Human alternate first exons underlined, pyramidine rich promoter (yellow). Putative SP1 elements in bold.

Figure 11

γ1 gene putative promoter alignment. Predicted human promoter (yellow) (NNPP) and GenBank TSS and CDS highlighted. Putative promoter elements in bold.

Figure 12

γ2 gene promoter alignment. Mouse exon underlined; TSS, GPE and NRSE (green). Putative promoter elements in bold.

Figure 13

γ3 gene promoter alignment. Predicted human CpG island (yellow) and GenBank CDS (green). Putative promoter elements in bold.

Figure 14

δ gene promoter alignment. Rat INR and downstream NRSE (green). Putative promoter elements in bold.

Figure 15

ε gene promoter alignment. Predicted human and mouse promoter regions (yellow) (MatInspector), and GenBank CDS (green). Putative promoter elements in bold.

Figure 16

θ gene putative promoter alignment. Predicted human CpG islands (yellow, CpGPlot) and GenBank CDS (green). Putative promoter elements in bold.

Figure 17

π gene promoter alignment. Predicted human promoter region (yellow, Promoter 2). Putative promoter elements in bold.

Figure 18

Intergenic conserved sequences in Chr5 GABA cluster. (a) Approximate location of conserved region. (b) Table showing Putative TFBSs. (Data from rVista).

Figure 19

Putative DNase I hypersensitive sites. Distribution of predicted HSs in Chromosome 5 GABA receptor gene cluster (data from Noble et al [41]), and their locations in gene cluster.

Figure 20

S/MAR predictions for human and mouse GABAA receptor gene cluster.