2025-08-27 パデュー大学
<関連情報>
- https://www.purdue.edu/newsroom/2025/Q3/putting-the-brakes-on-cancer/
- https://www.science.org/doi/10.1126/science.adr1752
MYCプロモーターG四重鎖のヌクレオリン認識における構造基盤 Structural basis for nucleolin recognition of MYC promoter G-quadruplex
Luying Chen, Jonathan Dickerhoff, Ke-wei Zheng, Satchal Erramilli, […] , and Danzhou Yang
Science Published:18 Apr 2025
DOI:https://doi.org/10.1126/science.adr1752
Editor’s summary
G-quadruplexes (G4s) are DNA structures important for gene regulation. Chen et al. determined high-resolution structures showing how the protein nucleolin uses multiple binding motifs to bind to the G4 in the promoter region of MYC gene, a key driver in many cancers, thus influencing MYC expression. This result suggested that G4s are nucleolin’s primary cellular targets, providing insights into gene regulation through G4-epigenetic mechanisms. This research advances our understanding of molecular recognition between G4s and proteins and offers potential directions for drug discovery. —Di Jiang
Structured Abstract
INTRODUCTION
G-quadruplexes (G4s) are DNA or RNA secondary structures formed in guanine (G)–rich sequences with significant functional roles. These globular structures form under physiological conditions and are stabilized by cellular potassium or sodium ions (K+ or Na+). G4s in oncogene promoters regulate gene transcription and are promising anticancer drug targets. The MYC oncogene, particularly, contains a G4-forming region in its proximal promoter NHE III1, which controls 75 to 85% of transcription. In K+ solution, the MYC NHE III1 forms two parallel-stranded G4s, Myc121 and Myc161. Parallel G4 structures are robust and commonly found in promoter regions and RNA G4s.
RATIONALE
Numerous proteins have been reported to interact with G4s, but only a few structures of G4-protein complexes have been resolved. They all involve processive helicases that unfold G4, generating full access to external G tetrads for stacking interactions. However, genomic G4 recognition cannot be solely attributed to G tetrad–stacking interactions, as flanking regions within continuous duplex DNA hinder access to external G-tetrads.
Nucleolin was identified in 2009 as a major binding protein of the MYC promoter G4 (M3ycG4) and functions as a transcriptional repressor. This modular protein features a central DNA or RNA binding domain consisting of four RNA binding domains [RBDs or RNA-recognition motifs (RRMs)] interconnected by three linkers. Notably, nucleolin has a higher affinity for MycG4 than for its well-established substrate, the stem-loop nucleoin recognition element (NRE) RNA. However, structural information of the G4-nucleolin complex is unavailable.
RESULTS
We resolved the crystal structure of the 1:1 nucleolin-MycG4 complex at 2.6 Å, facilitated by a de novo NCL-MycG4 complex-specific fragment antigen-binding region (Fab) and an optimized Myc161 sequence. All four RBDs of nucleolin are essential for high-affinity binding to MycG4, unlike its interaction with NRE RNA, which involves only two RBDs. Nucleolin prefers the Myc161 G4 (dissociation constant Kd ~3 nM). In the complex structure, the Myc161 G4 adopts a folded parallel-stranded topology with two K+ ions between three G tetrads, closely resembling the nuclear magnetic resonance (NMR) solution structure of free Myc161, including a similar 5’ DNA triad capping structure.
The four RBDs of nucleolin wrap around MycG4 to form multivalent interactions with its three loops and 5′ flanking region for specific and high-affinity recognition. RBD1, RBD2, and Linker12 extensively interact with the 6–nucleotide (nt) central loop and the 5′ flanking region of Myc161, with Linker12 adopting a highly structured 9–amino acid α helix. RBD3 and RBD4 bind the 1-nt loops of MycG4, as demonstrated by NMR. The complex structure reveals distinct nucleolin-specific nucleobase-recognition motifs located outside the canonical RNP motifs, allowing multivalent binding to multiple individual nucleobases spatially dispersed on the globular G4 substrate. Nucleolin binding to MycG4 inhibits polymerase extension. Cleavage under targets and tagmentation sequencing (CUT&Tag) demonstrates nucleolin binding to the MYC promoter G4-forming region in cells. Notably, nucleolin’s genome-wide binding sites align closely with G4 loci, suggesting that G4s are nucleolin’s primary cellular substrates.
CONCLUSION
We report the first high-resolution structure of a G4 in complex with a regulating protein. Our structure reveals the first G4 conformation–based protein recognition. The globular G4 structure, with its multiple loops and flanking regions, provides an ideal platform for multivalent interactions with the modular nucleolin protein. The specific and high-affinity recognition of MycG4 requires all four RBDs of nucleolin. Nucleolin shows little interactions with the buried G tetrad core but recognizes the exposed loop and flanking regions of the globular G4, which may be a general feature of G4 interactions for nonhelicase proteins. Our findings suggest that G4s are nucleolin’s primary cellular substrates and indicate a G4-based epigenetic transcriptional regulation. The specific and high-affinity binding of the MycG4 by nucleolin can block the binding of other transcription factors to the same promoter region or recruit regulatory proteins. Further, the nucleolin-MycG4 complex structure will help G4-targeted drug discovery.
G4 conformation–based protein recognition for G4 epigenetic transcription control.
Modular nucleolin (NCL) protein binds the globular MycG4 through G4 conformation–based recognition through multivalent interactions with MycG4 loops and flankings, enabling specific and high-affinity binding. Nucleolin binds the MYC promoter G4-forming region in cells, and this high-affinity binding suggests a MycG4-epigenetic transcription control by blocking the binding of transcription factors or recruiting regulatory proteins.
Abstract
The MYC oncogene promoter G-quadruplex (MycG4) regulates transcription and is a prevalent G4 locus in immortal cells. Nucleolin, a major MycG4-binding protein, exhibits greater affinity for MycG4 than for nucleolin recognition element (NRE) RNA. Nucleolin’s four RNA binding domains (RBDs) are essential for high-affinity MycG4 binding. We present the 2.6-angstrom crystal structure of the nucleolin-MycG4 complex, revealing a folded parallel three-tetrad G-quadruplex with two coordinating potassium ions (K+), interacting with RBD1, RBD2, and Linker12 through its 6–nucleotide (nt) central loop and 5′ flanking region. RBD3 and RBD4 bind MycG4’s 1-nt loops as demonstrated by nuclear magnetic resonance (NMR). Cleavage under targets and tagmentation sequencing confirmed nucleolin’s binding to MycG4 in cells. Our results revealed a G4 conformation-based recognition by a regulating protein through multivalent interactions, suggesting that G4s are nucleolin’s primary cellular substrates, indicating G4 epigenetic transcriptional regulation and helping G4-targeted drug discovery.
