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.

Figure 1.

CD spectra of indicated C9orf72 DNAs. All experiments were done at 5 μM strand concentration, in 10 μM Tris (pH 7.5), and 100 μM of the indicated salt.

Figure 2.

UV melting curves of indicated C9orf72 DNAs. All experiments were done at 5 μM strand concentration, in 10 μM Tris (pH 7.5), and 100 μM of the indicated salt. Melting temperature units are °C.

Figure 3.

Methylation type and salt influence electrophoretic migration patterns. Migration of [γ-³²P] ATP end-labeled DNA (frozen and thawed at room temperature) in native 8% polyacrylamide gel. [γ-³²P] ATP end-labeled (GGGGCC)₈ and (CCCCGG)₈ containing the indicated modifications were dissolved in 50 mM Tris–HCl (pH 7.4) with the indicated salt, incubated for 20 min and then electrophoresed for 2 h at 120 V (9 V/cm).

Figure 4.

Methylation type influences DNA-protein interactions. Human recombinant hnRNPK band-shift with the [γ-³²P] ATP end-labeled d(GGGGCC)₈ or d(GGCCCC)₈ DNAs (∼80 fmol) and 0, 50, 200 or 400 ng of purified recombinant hnRNPK. DNAs are either not CpG methylated, or contain 5-methylcytosine or 5-hydroxymethylcytosine bases at each CpG within the sequence. Samples were pre-incubated with the protein in a buffer solution containing 10 mM Tris (pH 7.4), 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl₂, and 0.05% Non-idet P40. Samples were electrophoresed on a 4% native polyacrylamide gel for 1 hour at 200 volts (15V/cm). Lanes were densimetrically assessed using ImageQuant software to quantify the percent bound (%).

Figure 5.

Proposed quadruplex structure in the C9orf72 8-repeat sequence and biological implications regarding R-loop formation. (A) Model of quadruplex structure forming in the (GGGGCC)₈ and (GGCCCC)₈ strands with various tetrad assemblies. Green colored boxes represent methylatable cytosines within the repeat sequence. (B) Intramolecular quadruplexes can form by either the G-rich or C-rich DNA strands of the C9orf72 repeat and may stabilized R-loops. They can also reflect the ‘pearls’ in a ‘pearls-on-a-string’ model of slipped-quadruplexes. See text.