analyzing-telomeric-protein-dna-interactions-using-single-molecule

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

Nankai University

A telomere carrying repetitive sequences ends with a single-stranded overhang. The G-rich overhang could fold back and bind in the major groove of its upstream duplex, forming an antiparallel triplex structure. The telomeric triplex has been proposed to function in protecting chromosome ends. However, we lack strategies to mechanically probe the dynamics of a telomeric triplex. Here, we show that the topological dynamics of a telomeric triplex involves 3' overhang binding at the ds/ssDNA junction inferred by DNA mechanics. Assisted by click chemistry and branched polymerase chain reaction, we developed a rescue-rope-strategy for mechanically manipulating an artificial telomeric DNA with a free end. Using single-molecule magnetic tweezers, we identified a rarely forming (5%) telomeric triplex which pauses at an intermediate state upon unzipping the Watson-Crick paired duplex. Our findings revealed that a mechanically stable triplex formed in a telomeric DNA can resist a force of 20 pN for a few seconds in a physiological buffer. We also demonstrated that the rescue-rope-strategy assisted mechanical manipulation can directly rupture the interactions between the third strand and its targeting duplex in a DNA triplex. Our single-molecule rescue-rope-strategy will serve as a general tool to investigate telomere dynamics and further develop triplex-based biotechnologies.

The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics.

Single-molecule force spectroscopy is a powerful tool to directly measure protein-protein interactions (PPI). The high specificity and precision of PPI measurements made it possible to reveal detailed mechanisms of intermolecular interactions. However, protein aggregation due to specific or nonspecific interactions is among the most challenging problems in PPI examination. Here, we propose a strategy of a parallel DNA circuit to probe PPI using single-molecule magnetic tweezers. In contrast to PPI examination using atomic force microscopy, microspheres as probes used in magnetic tweezers avoided the single-probe issue of a cantilever. Negatively charged DNA as a linker circumvented the severe aggregation in the PPI construct with a protein linker. The unnatural amino acid encoded in proteins of interest expanded the choices of biorthogonal conjugation. We demonstrated how to apply our strategy to probe the PPI between the PHD3-Bromo and the histone H3 methylated at K4, a critical epigenetic event in leukemia development. We found a rupture force of 12 pN for breaking the PPI, which is much higher than that required to peel DNA off from a nucleosome, 3 pN. We expect that our methods will make PPI measurements of mechanics and kinetics with great precision, facilitating PPI-related research, e.g., PPI-targeted drug discovery.

Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit.

Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 kBT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2-9 s at a critical force of F1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.

Dynamics of TRF1 organizing a single human telomere.

Epigenetics, such as the dynamic interplay between DNA methylation and demethylation, play diverse roles in critical cellular events. Enzymatic activity at CpG sites, where cytosines are methylated or demethylated, is known to be influenced by the density of CpGs, methylation states, and the flanking sequences of a CpG site. However, how the relevant enzymes are recruited to and recognize their target DNA is less clear. Moreover, although DNA-binding epigenetic enzymes are ideal targets for therapeutic intervention, these targets have been rarely exploited. Single-molecule techniques offer excellent capabilities to probe site-specific protein-DNA interactions and unravel the dynamics. Here, we develop a single-molecule approach that allows multiplexed profiling of protein-DNA complexes using magnetic tweezers. When a DNA hairpin with multiple binding sites is unzipping, strand separation pauses at the positions bound by a protein. We can thus measure site-specific binding probabilities and dissociation time directly. Taking the TET1 CXXC domain as an example, we show that TET1 CXXC binds multiple CpG motifs with various flanking nucleotides or different methylation patterns in an AT-rich DNA. We are able to establish for the first time, at nanometer resolution, that TET1 CXXC prefers G/C flanked CpG motif over C/G, A/T, or T/A flanked ones. CpG methylation strengthens TET1 CXXC recruitment but has little effect on dissociation time. Finally, we demonstrate that TET1 CXXC can distinguish five CpG clusters in a CpG island with crowded binding motifs. We anticipate that the feasibility of single-molecule multiplexed profiling assays will contribute to the understanding of protein-DNA interactions.

Single-molecule multiplexed profiling of protein-DNA complexes using magnetic tweezers.

CpG islands recruit MLL1 via the CXXC domain to modulate chromatin structure and regulate gene expression. The amino acid motif of CXXC also plays a pivotal role in MLL1's structure and function and serves as a target for drug design. In addition, the CpG pattern in an island governs spatially dependent collaboration among CpGs in recruiting epigenetic enzymes. However, current studies using short DNA fragments cannot probe the dynamics of CXXC on long DNA with crowded CpG motifs. Here, we used single-molecule magnetic tweezers to examine the binding dynamics of MLL1's CXXC domain on a long DNA with a CpG island. The mechanical strand separation assay allows profiling of protein-DNA complexes and reports force-dependent unfolding times. Further design of a hairpin detector reveals the unfolding time of individual CXXC-CpG complexes. Finally, in a proof of concept we demonstrate the inhibiting effect of dimethyl fumarate on the CXXC-DNA complexes by measuring the dose response curve of the unfolding time. This demonstrates the potential feasibility of using single-molecule strand separation as a label-free detector in drug discovery and chemical biology.

Dynamics and inhibition of MLL1 CXXC domain on DNA revealed by single-molecule quantification.

The transformation from one compound to another involves the breaking and formation of chemical bonds at the single-bond level, especially during catalytic reactions that are of great significance in broad fields such as energy conversion, environmental science, life science and chemical synthesis. The study of the reaction process at the single-bond limit is the key to understanding the catalytic reaction mechanism and further rationally designing catalysts. Here, we develop a method to monitor the catalytic process from the perspective of the single-bond energy using high-resolution scanning tunneling microscopy single-molecule junctions. Experimental and theoretical studies consistently reveal that the attack of a halogen atom on an Au atom can reduce the breaking energy of Au-S bonds, thereby accelerating the bond cleavage reaction and shortening the plateau length during the single-molecule junction breaking. Furthermore, the distinction in catalytic activity between different halogen atoms can be compared as well. This study establishes the intrinsic relationship among the reaction activation energy, the chemical bond breaking energy and the single-molecule junction breaking process, strengthening our mastery of catalytic reactions towards precise chemistry.

The role of halogens in Au-S bond cleavage for energy-differentiated catalysis at the single-bond limit.

Synchronized ferroptosis contributes to nephron loss in acute kidney injury (AKI). However, the propagation signals and the underlying mechanisms of the synchronized ferroptosis for renal tubular injury remain unresolved. Here we report that platelet-activating factor (PAF) and PAF-like phospholipids (PAF-LPLs) mediated synchronized ferroptosis and contributed to AKI. The emergence of PAF and PAF-LPLs in ferroptosis caused the instability of biomembranes and signaled the cell death of neighboring cells. This cascade could be suppressed by PAF-acetylhydrolase (II) (PAFAH2) or by addition of antibodies against PAF. Genetic knockout or pharmacological inhibition of PAFAH2 increased PAF production, augmented synchronized ferroptosis and exacerbated ischemia/reperfusion (I/R)-induced AKI. Notably, intravenous administration of wild-type PAFAH2 protein, but not its enzymatically inactive mutants, prevented synchronized tubular cell death, nephron loss and AKI. Our findings offer an insight into the mechanisms of synchronized ferroptosis and suggest a possibility for the preventive intervention of AKI.

PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury.

Author Correction: PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury.

Erk signaling is indispensable for the self-renewal and differentiation of mouse embryonic stem cells (ESCs), as well as telomere homeostasis. But how Erk regulates these biological processes remains unclear. We identified 132 Erk2 interacting proteins by co-immunoprecipitation and mass spectrometric analysis, and focused on Ddx39 as a potential Erk2 substrate. We demonstrated that Erk2 phosphorylates Ddx39 on Y132 and Y138. Ddx39 knockout (KO) ESCs are defective in differentiation, due to reduced H3K27ac level upon differentiation. Phosphorylation of Ddx39 promotes the recruitment of Hat1 to acetylate H3K27 and activate differentiation genes. In addition, Ddx39 KO leads to telomere elongation in ESCs. Ddx39 is recruited to telomeres by the telomere-binding protein Trf1, consequently disrupting the DNA loop formed by Trf1 and suppressing the alternative lengthening of telomeres (ALT). Phosphorylation of Ddx39 weakens its interaction with Trf1, releasing it from telomeres. Thus, ALT activity is enhanced, and telomeres are elongated. Altogether, our studies reveal an essential role of Ddx39 in the differentiation and telomere homeostasis of ESCs.

Article	Year
The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics. Nucleic acids research\| PubMed ID: 31114915	2019
Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit. Bioconjugate chemistry\| PubMed ID: 31714753	2019
Dynamics of TRF1 organizing a single human telomere. Nucleic acids research\| PubMed ID: 33347580	2021
Single-molecule multiplexed profiling of protein-DNA complexes using magnetic tweezers. The Journal of biological chemistry\| PubMed ID: 33493518	2021
Dynamics and inhibition of MLL1 CXXC domain on DNA revealed by single-molecule quantification. Biophysical journal\| PubMed ID: 34280370	2021
The role of halogens in Au-S bond cleavage for energy-differentiated catalysis at the single-bond limit. Nature communications\| PubMed ID: 38001141	2023
PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury. Nature chemical biology\| PubMed ID: 38287154	2024
Author Correction: PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury. Nature chemical biology\| PubMed ID: 38316917	2024
Novel role for Ddx39 in differentiation and telomere length regulation of embryonic stem cells. Cell death and differentiation\| PubMed ID: 39107495	2024

Zhongbo Yu

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