Nucleophosmin C-terminal Leukemia-associated Domain Interacts with G-rich Quadruplex Forming DNA

Abstract

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling phosphoprotein, mainly localized at nucleoli, that plays a key role in ribogenesis, centrosome duplication, and response to stress stimuli. Mutations at the C-terminal domain of NPM1 are the most frequent genetic lesion in acute myeloid leukemia and cause the aberrant and stable translocation of the protein in the cytoplasm. The NPM1 C-terminal domain was previously shown to bind nucleic acids. Here we further investigate the DNA binding properties of the NPM1 C-terminal domain both at the protein and nucleic acid levels; we investigate the domain boundaries and identify key residues for high affinity recognition. Furthermore, we demonstrate that the NPM1 C-terminal domain has a preference for G-quadruplex forming DNA regions and induces the formation of G-quadruplex structures in vitro. Finally we show that a specific sequence found at the SOD2 gene promoter, which was previously shown to be a target of NPM1 in vivo, is indeed folded as a G-quadruplex in vitro under physiological conditions. Our data extend considerably present knowledge on the DNA binding properties of NPM1 and suggest a general role in the transcription of genes characterized by the presence of G-quadruplex forming regions at their promoters.

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

“…45 Importantly, it was shown that the SOD2 promoter sequence recognized by NPM1 also matches the sequence requirements for assuming a G-quadruplex fold and that this is the preferential structure, over the hairpin one, assumed by the oligonucleotide in vitro under physiological conditions. 45 …”

“…50 A well-characterized G-quadruplex sequence is present at the nuclease hypersensitive element III (NHE III ) of the c-MYC promoter, and regulates up to 90% of c-MYC gene transcription. 51,52 The extended C-terminal domain of NPM1 proved to bind this region with high affinity in vitro 45 and in vivo (unpublished results from Federici lab) and also to promote the G-quadruplex folding in the unstructured oligonucleotide. 45 These data suggest that NPM1 may be included in the constantly growing list of G-quadruplex-binding proteins.…”

“…51,52 The extended C-terminal domain of NPM1 proved to bind this region with high affinity in vitro 45 and in vivo (unpublished results from Federici lab) and also to promote the G-quadruplex folding in the unstructured oligonucleotide. 45 These data suggest that NPM1 may be included in the constantly growing list of G-quadruplex-binding proteins. 50 The structure of the NPM1 C-terminal extended domain in complex with the G-quadruplex oligonucleotide from the c-MYC promoter was recently investigated.…”

“…Conversely, basespecific interactions and interactions with nucleotides belonging to the G-quadruplex connecting loops appear marginal, explaining why NPM1 recognizes with similar affinities several G-quadruplexes with varying loop structures. 34,45 As mentioned above, a lysine-rich region that flanks the terminal three-helix bundle (residues 225-242) proved necessary for high-affinity recognition of all oligonucleotides tested, despite their structure. 45 The overall positive charge of this tail seems to be important for recognition, as shown by the loss of G-quadruplex affinity displayed by a double lysine to alanine mutant (K229A-K230A) in the tail.…”

“…34,45 As mentioned above, a lysine-rich region that flanks the terminal three-helix bundle (residues 225-242) proved necessary for high-affinity recognition of all oligonucleotides tested, despite their structure. 45 The overall positive charge of this tail seems to be important for recognition, as shown by the loss of G-quadruplex affinity displayed by a double lysine to alanine mutant (K229A-K230A) in the tail. 45 Interestingly, this region was shown to be unstructured before and also after binding, and does not NMR data).…”