Effects on promoter activity of common SNPs in 5' region of GABRB3 exon 1A

Abstract

Purpose: The β3 subunit of the γ-aminobutyric acid type A receptors (GABA(A) -Rs) is an essential component of GABA(A) -Rs in fetal, perinatal, and adult mammalian brain. Various transcripts of the β3 subunit gene (GABRB3) produce various proteins with different N-termini. Rare variants in this N-terminus (exon 1A and exon 2) of GABRB3 protein segregate in affected family members of two multigeneration-multiplex families with remitting childhood absence epilepsy (rCAE), suggesting GABRB3 is a major Mendelian epilepsy gene for rare families with CAE. Therefore, the N-terminus of GABRB3 could be important for GABRB3 regulation in development, and its alteration could produce rCAE. Herein we determine if single nucleotide polymorphisms (SNPs) within the 1,148-bp region upstream from exon 1A influence the expression of GABRB3.

Methods: We studied luciferase reporter expression for promoter activity, 1,148-bp upstream from exon 1A, using human embryonic kidney 293 cells. We generated constructs of the promoter region and compared different SNP haplotypes in 48 patients with rCAE. Next, we compared frequencies of rs20317, located in the core promoter region, and rs4906902, located in the enhancer region between 48 patients with rCAE and >500 healthy controls matched for ethnicity and ancestral origin.

Key findings: Highest luciferase expression occurred 230-bp upstream of exon 1A. The construct that excluded this region lost luciferase activity. Therefore, this region contains the core promoter of exon 1A. Allele C but not allele G (rs20317) significantly increased luciferase expression activity. Allele C creates binding motifs for cMYB and EGR-3. Longer constructs overlapping this region have a binding motif for REST (RE1-silencing transcription factor), a critical epigenetic modulator for neuronal genes. REST represses expression of neuronal genes in nonneuronal tissues, resulting in reduced luciferase expression activity. Even in the suppressed condition, the longer construct enhanced luciferase expression activity of the shorter construct, which excluded the distal end containing rs4906902. However, allele frequencies of rs20317 and rs4906902 were not significantly associated with 48 rCAE patients in comparison to >500 controls matched for ethnicity and ancestral origin.

Significance: Common SNPs in the promoter region increase luciferase expression activity. An epigenetic modulator, REST, specifically alters expression of GABRB3 exon 1A transcripts, suggesting epigenetic regulation by REST dominantly controls the expression of GABRB3 variant 2 transcript in early life GABA(A) signaling. Abnormal epigenetic regulation could be involved in absence seizures.

Fig 1. Promoter constructs and relative luciferase activity in HEK 293 cells

Five deletion constructs of 5′ region are employed in the analyses. They were generated as described under Methods. Data are presented as average-fold difference of luciferase activity versus control vector (pGL3 enhancer in bottom of Fig 2) ± SE of six independent experiments. Luciferase activity in P4 is 11-fold higher compared to controls. All constructs containing RE1 (P1, P2, P3, P5) have decreased activities.

Figure 2. Promoter activity of each SNP combination in HEK 293 cells

Graph shows luciferase activity in HEK 293 cells for each construct with the SNP haplotype and the length of the construct in the description on the left. Data are presented as average-fold difference of luciferase activity versus negative control (pGL3 enhancer) vector ± SE of three independent experiments. Luciferase activity of the short construct with the C allele of db SNP ID rs20317 was significantly higher than the G allele; however, all longer constructs have no significant difference in activity.

Figure 3

The 5′ region of human and mice β3 subunit genes: Predicted transcriptional motifs in SNP locations and predicted conserved transcriptional motifs between human and mice The nucleotide sequences of human and mice β3 subunit genes were aligned by inserting horizontal dashed lines (- - - -) to maximize homology, and conserved nucleotides are indicated by asterisks. Each exon 1A is surrounded by a yellow colored rectangle ( ) and each translational start codon is shadowed by green. The arrow ( ) shows the transcriptional start site of exon 1A. Location of SNPs are painted white against a black background ( ) and each accession number is shown above each nucleotide. The black triangles (▼) represent the border of each segment for the deletion study. The gray larger triangle ( ) represents the common end nucleotide for each segment of the deletion study except P5. The region which was predicted to contain transcriptional motifs associated with neuronal cells and several ubiquitous major motifs are shown by shadowed letters ( ), and each annotation is presented under each motif. The REST binding motif (RE1) is also surrounded by a rectangle. Common predicted matrixes between human and mice have over 0.89 of matrix similarity in MatInspector version 8.01. In region A, SNP rs20317 is located in the potential core promoter region, which is over 75 % identical between mouse chromosome 7 and human chromosome 15. Three specificity protein 1 (SP1) binding sites which overlap three EGR motifs and one E2F motif are predicted in the region from −124 bp to −77 bp upstream from the translational start codon in region A. These three sorts of motifs are also predicted in the mouse genome. GpC island clustering is found in the flanking region −140 bp upstream from the start codon ATG. The matrix of the TATA box lies in the region of SNP rs4906901, but three SP1 binding sites reside downstream of this motif. The allele G of rs4906901 makes SOX 5 binding motif but the allele T does not. SOX5 has been shown the involvement in neurogenesis, neuronal differentiation and neocortical neuron diversity (Lai et al., 2008). In region B, FAST1 and SMAD protein plays a critical role in early embryonic development as well as REST (Silvestri, 2008).