As a triple-stranded nucleic acid structure composed of DNA-RNA hybrid and single-stranded DNA, the R-loop plays a dual role in gene expression regulation, DNA replication, and genome stability maintenance, and its abnormality is closely related to cancer, neurodegenerative diseases, and so on. Analyzing the functional mechanism of R-loop needs to break through the limitation of simple positioning, and DRIPc-seq technology provides a key tool to achieve this goal with its chain specificity and high resolution.

This technology can not only accurately draw the whole genome map of R-loop, but also reveal the internal relationship between R-loop and transcription dynamics and chromatin state through the integrated analysis with transcriptomics (RNA-seq), epigenomics (ChIP-seq), and other omics data. This paper focuses on the application of DRIPc-seq and the Multiomics integration strategy in functional research, and discusses how to analyze the mechanism of R-loop in physiological and pathological processes from the molecular level, so as to provide a new perspective for understanding the genome regulatory network and the occurrence and development of diseases.

This article explores how DRIPc-seq, with its strand specificity and high resolution, integrates with multi-omics (RNA-seq, ChIP-seq) to analyze R-loop's links to transcription, replication conflicts, DNA repair, re-evaluates classic loci, and highlights its role in disease research.

Correlating R-loops with Transcription and Chromatin

R-loop a special nucleic acid structure, the loop plays a key role in gene expression regulation. Its association with the transcription process and chromatin state is an important starting point for analyzing genome function. In the past, technology was difficult to correlate accurately, and the appearance of DRIPc-seq made it possible to reveal how R-loops influenced biological processes by regulating transcription and chromatin state.

Limitations of DRIP-seq in Mechanism Research

DRIP-seq provides basic location information for research by locating the distribution of R-loops in the whole genome, but it has obvious limitations in mechanism research. This technology can't distinguish the chain specificity of R-loop, which makes it impossible to determine the relationship between R-loop and transcription direction, and it is difficult to directly associate it with specific transcription events.

At the same time, due to the limited resolution, the relationship between R-loop and chromatin marker can not be accurately located, which makes the correlation analysis between R-loop and chromatin state lack accuracy. For example, when studying the R-loop in the promoter region of a gene, DRIP-seq can't make clear the specific relationship between it and the transcriptional activation or inhibition state at the promoter, and can only give roughly overlapping information, so it can't deeply analyze its mechanism of action in gene regulation.

Outstanding Advantages of DRIPc-seq in Association Analysis

DRIPc-seq can accurately correlate R-loop with transcription events and chromatin markers by virtue of its chain specificity, which provides a powerful tool for revealing the gene regulation mechanism of R-loop.

The chain specificity of DRIPc-seq enables it to directly associate the R-loop with transcription events in a specific direction. Through the joint analysis with RNA-seq data, we can make clear the corresponding relationship between the chain where the R-loop is located and the transcript, and determine whether the R-loop is related to sense transcription or antisense transcription.

In addition, DRIPc-seq can be accurately associated with specific chromatin markers. For the H3K4me3 marker in the promoter region, DRIPc-seq can accurately locate the positional relationship between R-loop and the marker, and analyze whether R-loop affects the modification state of H3K4me3, thus affecting the transcription initiation of the gene.

In the genome region, the role of R-loop in transcription extension can be explored through the association analysis with the H3K36me3 marker. With the help of ChIP-seq verification, the dynamic regulation relationship between R-loop and chromatin state can be revealed more accurately.

Senataxin removes DSB-induced RNA:DNA hybrids in active loci (Cohen et al., 2018)

DRIPc-seq in Transcription-Replication Collision

The genome conflict between transcription and replication (TRC) is the key cause of genome instability, and the R-loop often plays an important role in this process. With the advantage of chain specificity, DRIPc-seq can accurately locate the chain of the R-loop, provide key information for analyzing the interaction between the replication fork and transcription machine in TRC, and become a powerful tool to explore this conflict mechanism.

A. Combination of DRIPc-seq and Replication Time Series Data

a) Combining DRIPc-seq data with replication time series data can provide more comprehensive information for the study of transcription-replication conflicts and help to deeply understand the regions and potential mechanisms of conflicts. DRIPc-seq can accurately locate the position and chain information of R-loop in the genome, and the replication time series data can reflect the replication time of each region of the genome. By integrating them, we can determine whether there is a correlation between the R-loop enrichment area and the elapsed time of the replication fork.

b) The specific combination method includes:

• Overlapping the position information of R-loop obtained by DRIPc-seq with the replication time sequence map by bioinformatics method, and counting the distribution of R-loop in different replication time regions.
• Analyze the relationship between the chain specificity of R-loop and the moving direction of replication fork, and judge whether there is the possibility of direction conflict between them.
• This combination lays a foundation for further study on the mechanism of transcription-replication conflict.

Comparison of FoxA1 ChlP-Seq and ChlP-chip (Zhang et al., 2008)

B. The Key Role of Chain-specific Information in Analyzing TRC

a) Chain-specific information is the key to understanding whether the stagnant replication fork and the transcription machine collide on the same template chain in transcription-replication conflict, and it is very important to clarify the mechanism of TRC-induced genomic instability.

b) In the transcription-replication conflict, it is essential to understand the essence of the conflict to know whether the stagnant replication fork and the transcription machine are on the same template chain. The chain-specific information of DRIPc-seq can accurately determine the chain where the R-loop is located, and the position of the R-loop is often related to the position of the transcription machine. By analyzing the chain information of R-loop and the moving direction of replication fork, it can be determined whether transcription and replication are carried out on the same chain, and then it can be judged whether the same direction or reverse conflict occurs.

c) When transcription and replication are carried out in the same direction on the same chain, it may happen that the replication fork catches up with the transcription machine, leading to conflicts. The reverse conflict may be more intense and more likely to cause genomic instability. Chain-specific information can clearly distinguish the two situations, help researchers to deeply analyze the mechanism of genomic instability, such as DNA damage and chromosome abnormality induced by TRC, and provide important clues for understanding the occurrence and development of related diseases.

Integrating DRIPc-seq with DNA Repair

Abnormal accumulation of R-loops often induces DNA damage, and the DNA repair mechanism is very important to maintain genome stability. With its chain specificity and high resolution, DRIPc-seq can accurately locate the R-loop region and associate it with the distribution of DNA damage markers and repair factors, which provides a unique perspective for analyzing the mechanism of R-loop-mediated damage repair and promotes the study of their interaction.

A. Accuracy of DRIPc-seq in Locating R-loop Enrichment Region

a) DRIPc-seq can accurately determine the enrichment region of R-loop, which provides a reliable location basis for studying the relationship between R-loop and the DNA damage and repair process.

b) DRIPc-seq can accurately identify the enrichment region of R-loop in the genome, including its specific boundary and chain, through the advantages of chain specificity and high resolution. Compared with other technologies, its positioning accuracy is higher, which can be accurate to the single base level, enabling researchers to more accurately correspond R-loop with the regions related to DNA damage and repair.

c) In some cancer cells, DRIPc-seq can accurately locate the R-loop enrichment in a specific chromosome region, which provides a clear goal for the follow-up study of DNA damage in this region and helps to further explore the role of R-loop in DNA damage.

B. Overlapping Analysis with DNA Damage Markers

a) The close relationship between R-loop and the DNA repair process can be revealed by superimposing the rich region of R-loop located by DRIPc-seq with the genome long-range map of DNA damage markers and repair factors.

b) By superimposing the R-loop data obtained by DRIPc-seq with γH2AX ChIP-seq (DNA damage marker) data, we can analyze whether the R-loop enrichment region overlaps with the DNA damage region, and the degree of overlap, to judge whether the R-loop is more likely to cause DNA damage.

c) At the same time, overlapping with the ChIP-seq map of BRCA1, RAD51, and other repair factors can explore whether these repair factors gather in the R-loop enrichment region and reveal whether the DNA damage induced by R-loop has initiated the corresponding repair mechanism.

d) For example, it is found that R-loop is enriched in a certain genomic region, and the γH2AX signal in this region is strong, and BRCA1 and RAD51 gather here, which suggests that R-loop may cause DNA damage here and activate related repair pathways.

Different repair pathways for DSB lesions (San et al., 2021)

C. The Role of Chain Information in Inferring the Damage Mechanism

a) The chain information provided by DRIPc-seq is helpful to infer the mechanism of DNA damage mediated by R-loop, and provides a deeper perspective for understanding the role of R-loop in maintaining genome stability.

b) The chain information of the R-loop can reflect its relationship with transcription, and then help to infer the damage mechanism. If an R-loop is located in the template chain, it may affect the transcription, lead to transcription stagnation, and then lead to DNA damage; However, the R-loop located in the non-template chain may affect the DNA structure in other ways, leading to damage.

c) Combining the chain information with the distribution of DNA damage and repair factors, we can further infer the specific mechanism of damage. For example, the R-loop located in the template chain leads to transcription stagnation, which in turn leads to replication fork stagnation, and finally leads to DNA double-strand breaks, and repair factors will gather here for repair. Chain information makes the inference of these mechanisms more accurate and targeted.

The principle of single-cell sequencing (Han et al., 2019)

Case Study: Re-evaluating a Classic Locus with DRIPc-seq

R-loop research of classical gene loci often has cognitive bias due to technical limitations. Although DRIP-seq reveals some distribution characteristics, it is difficult to analyze the real picture of complex regions due to the lack of chain specificity. Through the reanalysis of classical loci by DRIPc-seq, we can use its chain specificity and high resolution to correct past misjudgments, explore hidden biological mechanisms, and provide a new perspective for understanding the function of R-loops.

Reanalysis of the rDNA Region

The rDNA region is a classic site for studying R-loops, and its reanalysis with DRIP-seq and DRIP-seq can show the advantages of DRIP-seq.

The rDNA region contains a large number of repetitive sequences, which are active in transcription and easily form R-loops. When analyzing this region, DRIP-seq is unable to clarify the specific relationship between R-loop and rDNA transcription due to the lack of chain specificity, and the obtained signal is vague, so it is difficult to distinguish R-loops on different chains.

However, the chain specificity of DRIPc-seq can clearly reveal the chain where the R-loop of the rDNA region is located. It is found that during the transcription of rDNA, the R-loop is mainly located in the template chain, and it is highly related to the transcription activity. By combining with RNA-seq data, it is confirmed that these R-loops are closely related to the transcription process of rRNA, and may be involved in the transcription regulation of rDNA. The analysis results of DRIPc-seq corrected the previous vague understanding of the R-loop in DRIP-seq and provided a new basis for understanding the transcriptional regulation mechanism of the rDNA region.

In-depth Analysis of the C9orf72 Locus

The C9orf72 locus is related to amyotrophic lateral sclerosis. The analysis of this locus by DRIPc-seq reveals new biological phenomena and provides new clues for the study of disease mechanisms.

When analyzing the C9orf72 locus, DRIP-seq found that there was an R-loop in this region, but due to the mixed signals, it was impossible to determine its specific source and its association with diseases. The chain specificity of DRIPc-seq reveals that there is an antisense transcript-related R-loop at this site, which has not been found before.

The R-loop related to these antisense transcripts may be involved in the occurrence and development of amyotrophic lateral sclerosis by affecting the expression of the C9orf72 gene or causing genomic instability. DRIPc-seq accurately analyzed the previously vague mixed signals at this locus, which provided a new biological explanation for further understanding the molecular mechanism of the disease, and also highlighted the advantages of DRIPc-seq in the study of complex gene loci.

Sequenced RAD marker mapping (Baird et al., 2008)

Conclusion

DRIPc-seq has become the key technology to analyze the function of R-loop by virtue of its core advantages of chain specificity and high resolution. It can not only accurately map the genome distribution of R-loop, but also provide a three-dimensional perspective for revealing the biological function of R-loop through the integration with multiple omics data.

• Combined with RNA-seq, the bidirectional regulatory relationship between R-loop and transcription dynamics was clarified, such as the activation or inhibition of transcription initiation by R-loop in the promoter region, and the cooperation or conflict between R-loop in the gene body region and transcription extension.
• Integration with ChIP-seq revealed the interaction network between R-loop and chromatin modification, such as the maintenance of epigenetic status by the promoter R-loop labeled with H3K4me3.
• Combined with replication time sequence and DNA damage data, the dual role of R-loop in transcription-replication conflict and genome stability regulation was clarified.

In the future, with the development of single-cell DRIPc-seq technology, combined with multidimensional data such as spatial transcriptome and protein interaction group, the dynamic changes of R-loop in cell heterogeneity and its association with disease phenotype will be further analyzed. The deep integration of DRIPc-seq and Multiomics not only promotes the understanding of the basic biological function of R-loop, but also provides a new idea for the mechanism research and therapeutic target discovery of cancer, neurodegenerative diseases, and other related diseases, highlighting the powerful force of technological innovation driving scientific discovery.

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