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Allelic differences in nuclear protein binding at a genome-wide significant risk variant for schizophrenia in ZNF804A

Molecular Psychiatry volume 16, pages 787–789 (2011)Cite this article

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The single-nucleotide polymorphism rs1344706 within the second intron of ZNF804A shows robust association with schizophrenia,1, 2, 3, 4, 5 but does not have an obvious effect on protein coding sequence. Although this could reflect linkage disequilibrium with the actual causal variant(s), re-sequencing and detailed association analyses of ZNF804A in the largest sample to date have not uncovered a variant that is more strongly associated.2 Given its intronic location, any functional effects of this single-nucleotide polymorphism are likely to be at the level of transcription or splicing, a hypothesis supported by reports of association between rs13344706 and ZNF804A RNA expression.2, 3 However, additional tests of ZNF804A allelic expression carried out in one of these studies2 suggest that the association with ZNF804A expression may not be directly attributable to this single-nucleotide polymorphism, or that other variants are also involved.

As gene expression is fundamentally controlled by binding of nuclear proteins to regulatory regions in genomic DNA, we investigated the functional potential of rs13344706 by assessing its binding to nuclear proteins derived from human neural cell lines using electromobility shift assays (EMSA). Complementary 50 base biotinylated DNA oligonucleotides were synthesised along with unlabelled competitor oligonucleotides for each allele of rs1344706 (sequences in Supplementary Table S1). Equimolar amounts of complementary oligonucleotides were annealed in a thermocycler to form double-stranded DNA. Nuclear extracts were prepared from undifferentiated SH-SY5Y cells and two human neural progenitor cell lines, one derived from foetal cortical neuroepithelium and one from foetal hippocampus (described in references 6 and 7,6, 7 respectively). Nuclear extract binding reactions were carried out using the LightShift EMSA Optimisation and Control Kit (Thermo Scientific, Waltham, MA, USA). Binding specificity was assessed by addition of a 200-fold excess (4 pmols) of unlabelled oligonucleotide. Electrophoresis was carried out using 6% Novex DNA retardation gels (Invitrogen, Paisley, UK). Gels were transferred to positively charged nylon membranes and cross-linked using a UV transilluminator. Membranes were visualised using the LightShift Chemiluminescent EMSA kit (Thermo Scientific), and image acquisition and analysis performed using a ChemiDoc-It camera system (UVP, Upland, CA, USA). DNA-protein bands were quantified as percentage intensity of total lane intensity. For comparison of binding between sequences containing alternative alleles of rs1344706, two separate incubation reactions were performed for each allele using the same volume (3 μl) of the same nuclear extract for all four reactions, which were then assayed on a single gel. This was repeated on a further two occasions, using separate nuclear extracts, to yield six measures of binding intensity for each allele.

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Acknowledgements

This work was supported by the Medical Research Council, UK (Grant ID: G0802166).

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  1. Department of Neuroscience, Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King's College London, London, UK

    M J Hill & N J Bray

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  1. M J Hill
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Correspondence to M J Hill.

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Hill, M., Bray, N. Allelic differences in nuclear protein binding at a genome-wide significant risk variant for schizophrenia in ZNF804A. Mol Psychiatry 16, 787–789 (2011). https://doi.org/10.1038/mp.2011.21

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