Insertion of G-quadruplex DNA Structures Induces Long-Range Chromatin Activation and Gene Expression

Core Concepts

Insertion of an array of G-quadruplex (G4) DNA structures into chromatin directly induces long-range chromosomal interactions and enhancer-like epigenetic changes, leading to activation of genes up to 5 Mb away.

Abstract

The study aimed to directly test the role of G-quadruplex (G4) DNA structures in altering long-range chromatin organization and gene regulation. The researchers inserted an array of G4-forming sequences or a control sequence lacking G4 capability at an isolated genomic locus in HEK293T cells using CRISPR-Cas9 genome editing. Key findings: The G4 array insertion led to a significant increase in the number of long-range chromosomal interactions across the genome, compared to the control insertion, as revealed by Hi-C analysis. The G4 array induced enhancer-like epigenetic changes, with increased recruitment of H3K4Me1 and H3K27Ac histone marks and the transcriptional coactivator p300, at the insertion site. This resulted in increased expression of genes up to 5 Mb away from the insertion site, which was reversed upon disruption of the inserted G4 structures using LNA probes. The increased long-range interactions were predominantly observed in the downstream region of the insertion site, likely due to the proximity of the G4 array to a topologically associated domain (TAD) boundary. Together, these results provide direct evidence that G4 DNA structures can induce long-range chromatin interactions and enhancer-like functions, leading to activation of distant genes. This sheds new light on how DNA secondary structures can directly influence 3D genome organization and gene regulation.

Stats

The number of genome-wide raw Hi-C contacts originating from a ±10 kb window with the insertion site at the middle was 6390 in the G4-array insert cells, compared to 3133 in the G4-mutated insert (control) cells. The number of normalized Hi-C contacts originating from the same ±10 kb window was 1.61 in the G4-array insert cells, compared to 0.79 in the G4-mutated insert (control) cells and 1.00 in the HEK293T control cells.

Quotes

"Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array." "Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site." "Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions."

Key Insights Distilled From

Artificially inserted G-quadruplex DNA secondary structures induce long-distance chromatin activation

by Roy,S. S., B... at www.biorxiv.org 11-28-2023

https://www.biorxiv.org/content/10.1101/2023.11.27.568900v3

Deeper Inquiries

How might the specific sequence context and structural features of different G4 motifs influence their ability to induce long-range chromatin interactions and gene regulation?

The specific sequence context and structural features of different G4 motifs can significantly impact their ability to induce long-range chromatin interactions and regulate gene expression. The sequence context of a G4 motif refers to the surrounding nucleotides that can influence the stability and formation of the G4 structure. Certain sequences may be more prone to forming stable G4 structures due to the presence of runs of guanines and appropriate flanking sequences that facilitate G4 formation. The structural features of G4 motifs, such as loop size, number of G-tetrads, and overall stability, can also affect their functional properties. In terms of inducing long-range chromatin interactions, G4 motifs with higher stability and specific loop configurations may have a greater propensity to interact with other genomic regions through DNA looping. The presence of G4 motifs near gene promoters or enhancer regions can facilitate long-range interactions with distal regulatory elements, leading to changes in gene expression. Additionally, the orientation and distribution of G4 motifs within the genome can influence the formation of chromatin loops and the establishment of topologically associated domains (TADs). Regarding gene regulation, the specific sequence context of G4 motifs can determine their interaction with transcription factors, chromatin remodeling complexes, and epigenetic modifiers. G4 motifs located in gene promoters or enhancers can act as regulatory elements by modulating the accessibility of DNA to transcriptional machinery. The stability and dynamics of G4 structures can also impact the recruitment of proteins involved in gene expression, ultimately influencing transcriptional activity. In summary, the sequence context and structural features of G4 motifs play a crucial role in their ability to induce long-range chromatin interactions and regulate gene expression by influencing G4 stability, interactions with regulatory proteins, and overall chromatin architecture.

What other DNA secondary structures, besides G4s, might also play a direct role in shaping the 3D genome organization and gene expression programs?

In addition to G4 structures, several other DNA secondary structures can play a direct role in shaping the 3D genome organization and gene expression programs. Some of the key DNA secondary structures that have been implicated in these processes include: Holliday Junctions: Holliday junctions are four-way DNA structures that form during genetic recombination and DNA repair processes. These structures can impact chromatin looping and higher-order chromatin organization by mediating DNA strand exchange and crossover events. Cruciform DNA: Cruciform DNA structures arise from inverted repeat sequences that can form hairpin loops, leading to DNA bending and structural changes. Cruciform DNA can influence gene expression by affecting transcription factor binding and nucleosome positioning. R-loops: R-loops are three-stranded nucleic acid structures formed by the hybridization of RNA with the DNA template strand, leaving the non-template DNA single-stranded. R-loops have been associated with transcriptional regulation, DNA replication, and chromatin organization. Slippery DNA: Slippery DNA structures can form due to repetitive sequences or microsatellites, leading to DNA slippage and the formation of non-B DNA conformations. These structures can impact gene expression by affecting DNA replication, repair, and recombination processes. Z-DNA: Z-DNA is a left-handed helical DNA structure that forms under specific sequence contexts. Z-DNA can influence gene expression by modulating chromatin accessibility, transcription factor binding, and epigenetic modifications. Each of these DNA secondary structures can interact with chromatin-associated proteins, transcription factors, and epigenetic modifiers to regulate gene expression and contribute to the spatial organization of the genome in three dimensions.

Could the G4-induced long-range chromatin interactions and enhancer functions be exploited for targeted gene regulation in therapeutic applications?

The G4-induced long-range chromatin interactions and enhancer functions hold significant potential for targeted gene regulation in therapeutic applications. By leveraging the ability of G4 motifs to modulate chromatin architecture and gene expression over long distances, novel therapeutic strategies can be developed to selectively regulate specific genes or pathways. One approach could involve designing small molecules or oligonucleotides that specifically target G4 motifs associated with enhancer regions or regulatory elements of disease-related genes. These molecules could stabilize or disrupt G4 structures to modulate the interactions between enhancers and gene promoters, leading to precise control over gene expression. Furthermore, the exploitation of G4-induced chromatin looping could enable the development of gene editing technologies that target specific genomic loci for therapeutic interventions. By utilizing the natural ability of G4 motifs to influence long-range interactions, gene editing tools could be engineered to target and modify disease-associated genes with high precision and efficiency. Overall, the unique properties of G4-induced long-range chromatin interactions and enhancer functions provide a promising avenue for the development of targeted gene regulation strategies in therapeutic settings. Further research and technological advancements in this area could lead to innovative therapeutic approaches for treating various genetic disorders and diseases.