011-22 – Exploration of the potential for G4 structure formation in a polymorphic regulatory domain of the CFAP410 gene and its ability to modify expression

A variable number tandem repeat (VNTR) located within a regulatory region of the amyotrophic lateral sclerosis (ALS) risk gene CFAP410 has a sequence containing a GGGG repeat; altered expression of CFAP410 is hypothesised to contribute to an ALS phenotype. A G-quadruplex (G4) DNA formation is a complex secondary structure that can form in genes where there are at least three guanines in sequence with that sequence repeated four times. This structure has been suggested to act as a genetic switch to modify the expression of certain genes that are under their regulation. Therefore, we theorised that the VNTR could have potential for G4 formation which would suggest a mechanism for modified gene expression in different physiological conditions. One method of determining G4 structure involves identifying the repertoire of proteins capable of binding to suspected G4 sites. Therefore, to explore whether the CFAP410 VNTR has G4 potential we established the optimum binding conditions of HEK293 nuclear proteins to our VNTR sequence using electrophoretic mobility shift assays (EMSAs). We then analysed these proteins using mass spectrometry. We discovered that two G4 related proteins, hnRNP K and CNBP were capable of binding, which suggests that G4 structures may form at this locus. We also determined that other proteins involved in neurodegenerative conditions – PCBP1/2, hnRNP D and several of the S100A family – could also bind, which increases the potential regulatory properties of the CFAP410 VNTR.