title: Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation.
abstract: Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K(+), Na(+) or Li(+)), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.
authors.affiliation: Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada.
authors.forename: Bita
authors.initials: B
authors.lastname: Zamiri
authors.affiliation: Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada.
authors.forename: Mila
authors.initials: M
authors.lastname: Mirceta
authors.affiliation: UW Medicine South Lake Union, University of Washington, Seattle WA 98109, USA.
authors.forename: Karol
authors.initials: K
authors.lastname: Bomsztyk
authors.affiliation: Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Macgregor
authors.affiliation: Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada cepearson.sickkids@gmail.com.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Pearson
mesh_terms: D000073885:C9orf72 Protein; D018899:CpG Islands; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D006801:Humans; D011506:Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D012261:Ribonucleoproteins / Q000378:metabolism
chemical_list: D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D011506:Proteins; D012261:Ribonucleoproteins; C072074:HNRNPK protein, human; D006147:Guanine; D003596:Cytosine; D004247:DNA