1. The PCSK6 region associated with handedness controls expression of a shorter novel isoform
Robert Shore, Rebeca Diaz, Yiwang Xu, Silvia Paracchini
School of Medicine, University of St Andrews
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Introduction
Handedness presents a weak but consistent heritability across various studies, estimated to be 25%. Recently, we have identified PCSK6 as the first gene associated with a handedness-related measure at a statistically significant level (P<0.5 *10-8)(1)(2)
The same locus within PCSK6 has been found to be associated with the degree of handedness (rather than direction) in an independent general population cohort (3). This study reported association with a 33bp variable-number tandem repeat (VNTR)
PCSK6 is known to control the activation of the NODAL pathway required for left-right axis determination in very early embryonic development. This raises the possibility that the same biological pathways controlling structural laterality may partly be implicated in contributing to behavioural laterality.
In this research we aim to take forward these genetic findings and understand the molecular mechanisms underlying the association between PCSK6 and handedness
Figure 1 PCSK6 locus associated with relative hand skills Top associated markers from previous studies shown in proximity to a secondary bidirectional promoter within PCSK6. Our research examines the effect of these variants on promoter activity and PCSK6 expression
Methods and Materials
Results
In silico analysis
TRANSFAC was queried for transcription factor bind sites (TFBSs) centred on each SNP allele in a 12.7kbp LD block of SNPs (Fig1). This LD block consists of 15 SNPs in LD with the highest associated marker in our previous GWAS study, rs (>0.4 R2, CEU HapMap3). Only rs was predicted to lose/gain a TFBS on allelic variation (nr vertebrate min false positive TRANSFAC profile). Although the junction of the VNTR does create TFBSs (Tel-2, c-Ets-1), no evidence in the literature suggests these transcription factors work in multiplex, therefore only rs and the highest associated marker in our previous GWAS study (2), rs , were considered for further functional analysis.
Electrophoretic Mobility Shift Assay
EMSAs were used to analyse if either rs or rs SNPs were associated with altered binding of nuclear transcription factor proteins to biotinylated probes in an allele or tissue-specific manner.
Luciferase Reporters
A Promega Dual Luciferase assay was used to establish if the promoter was bidirectional in its transcription as predicted by the epigenetic markings in the UCSC Genome Browser (Track 3, Figure 1). The assay was also used to determine how promoter activity might be affected by allelic variation of the VNTR and rs variants and if this level of activity was cell-type specific.
Gene Expression
PCR was used to confirm expression of the shorter novel PCSK6 isoform across a range of cell lines (Figure4). Primers span from the first exon of the novel PCSK6 isoform to the 3’ UTR.
EMSAs were conducted using nuclear extract isolated from a range of cell lines (HEK293, hNSC, Neuronal, SH-SY5Y and K562). No difference in binding affinity was observed for rs , while a clear allele difference was observed for rs across all cell lines - allele T indicated protein banding which was absent for allele A (circled Fig 2). A
Figure 2 EMSA displays the binding of hNSC nuclear extract to probes containing the rs SNP T versus A allele
The promoter drives transcription in a bidirectional manner with a 6.4-fold bias towards producing a short PCSK6 isoform in the sense direction and a lncRNA in the antisense direction (Fig3A). A similar bias in transcription direction was observed in the SH-SY5Y and HEK293 cell lines though at greatly reduced levels in comparison to the K562 cell line, suggesting the secondary promoter activity to be tissue specific. The 6,9 and 10 VNTR alleles showed no significant difference in luminescence (Fig3B). B
Figure 3 Luciferase Assay preliminary data. Bars indicate transcription in a Forward and Reverse direction (A), the 6,9 and 10 VNTR alleles (B) and the rs A and T alleles in a Forward direction. Graphs display K562 cell line relative luminescence. Error bars indicate SEM.
The shorter PCSK6 isoform transcribed in the sense direction from the bidirectional promoter is not a 4 exon transcript as predicted by UCSC Genome Browser (Track 2, Fig1) but a much longer novel isoform that spans from the bidirectional promoter to the canonical PCSK6 3’UTR (Track 1, Fig1); PCR indicates this shorter isoform to be present across a range of cell lines (Figure 4).
Figure 4 PCR indicating expression of the novel PCSK6 shorter isoform in different cell lines; K562 (2), HEK293 (3), hNSC (4), Neuron (5), SH-SY5Y (6). Ladder box indicates 1500bp. Expected size is 1247bp.
Conclusions
We provide data from an ongoing functional analysis of a PCSK6 locus associated with handedness. While EMSA data showed the rs SNP to affect the binding of nuclear transcription factors at this region, analysis of the SNP using an allele-specific luciferase assay remains inconclusive with further research being required to investigate whether or not the rs SNP affects functionality of the bidirectional promoter. We could not find any evidence supporting a significant VNTR role. Our data also shows a large bias towards transcription in the forward rather than the reverse direction and we hope to establish the biological function of the shorter PCSK6 isoform and the effect, if any, the ncRNA has on PCSK6 expression.
Key References
(1) Scerri TS, Brandler WM, Paracchini S, et al (2011) PCSK6 is associated with handedness in individuals with dyslexia. Hum Mol Genet, 20, (2) Brandler WM, et al (2013) Common Variants in Left/Right Asymmetry Genes and Pathways are Assoicated with Relative Hand Skill. PLoS genetics, 9, e (3) Arning L, et al (2013) VNTR Polymorphism Is Associated with Degree of Handedness but Not Direction of Handedness. PLoS One, 8, e67251