rna-associated-chromatin-dna-dna-interaction-method

RNA-Associated Chromatin DNA-DNA Interaction Method

RNA-Associated Chromatin DNA-DNA Interaction Method (RDD) is a targeted strategy for mapping chromatin interactions anchored by a specific RNA. It ...

Southern University of Science and Technology

Epidermal growth factor receptor (EGFR) mutations are the strongest response predictors to EGFR tyrosine kinase inhibitors (TKI) therapy, but knowledge of the EGFR mutation frequency on lung adenocarcinoma is still limited to retrospective studies. The PIONEER study (NCT01185314) is a prospective molecular epidemiology study in Asian patients with newly diagnosed advanced lung adenocarcinoma, aiming to prospectively analyze EGFR mutation status in IIIB/IV treatment-naïve lung adenocarcinomas in Asia. We report the mainland China subset results. Eligible patients (≥20 yrs old, IIIB/IV adenocarcinoma and treatment-naïve) were registered in 17 hospitals in mainland China. EGFR was tested for mutations by amplification refractory mutation system using biopsy samples. Demographic and clinical characteristics were collected for subgroup analyses. A total of 747 patients were registered. Successful EGFR mutation analysis was performed in 741, with an overall mutation rate of 50.2%. The EGFR active mutation rate is 48.0% (with 1.3% of combined active and resistance mutations). Tobacco use (>30 pack-year vs. 0-10 pack-year, OR 0.27, 95%CI: 0.17-0.42) and regional lymph nodes involvement (N3 vs. N0, OR 0.47, 95%CI: 0.29-0.76) were independent predictors of EGFR mutation in multivariate analysis. However, even in regular smokers, the EGFR mutation frequency was 35.3%. The EGFR mutation frequency was similar between diverse biopsy sites and techniques. The overall EGFR mutation frequency of the mainland China subset was 50.2%, independently associated with the intensity of tobacco use and regional lymph nodes involvement. The relatively high frequency of EGFR mutations in the mainland China subset suggest that any effort to obtain tissue sample for EGFR mutation testing should be encouraged.

Molecular Epidemiology of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology - Mainland China Subset Analysis of the PIONEER study.

Interleukin6 (IL-6) is a key driver of hyperinflammation in COVID-19, and its level strongly correlates with disease progression. To investigate whether variability in COVID-19 severity partially results from differential  expression, functional single-nucleotide polymorphisms (SNPs) of  were determined in Chinese COVID-19 patients with mild or severe illness. An Asian-common  haplotype defined by promoter SNP rs1800796 and intronic SNPs rs1524107 and rs2066992 correlated with COVID-19 severity. Homozygote carriers of  variant haplotype were at lower risk of developing severe symptoms (odds ratio, 0.256; 95% confidence interval,  0.088 to 0.739; = 0.007). This protective haplotype was associated with lower levels of  and its antisense long noncoding RNA  by -expression quantitative trait loci analysis. The differences in expression resulted from the disturbance of stimulus-dependent bidirectional transcription of the / locus by the polymorphisms. The protective rs2066992- allele disrupted a conserved CTCF-binding locus at the enhancer elements of , which transcribed antisense to  and induces  expression in inflammatory responses. As a result, carriers of the protective allele had significantly reduced  expression and attenuated  induction in response to acute inflammatory stimuli and viral infection. Intriguingly, this low-producing variant that is endemic to present-day Asia was found in early humans who had inhabited mainland Asia since ∼40,000 years ago but not in other ancient humans, such as Neanderthals and Denisovans. The present study suggests that an individual's  genotype underlies COVID-19 outcome and may be used to guide IL-6 blockade therapy in Asian patients.  Overproduction of cytokine interleukin-6 (IL-6) is a hallmark of severe COVID-19 and is believed to play a critical role in exacerbating the excessive inflammatory response. Polymorphisms in  account for the variability of IL-6 expression and disparities in infectious diseases, but its contribution to the clinical presentation of COVID-19 has not been reported. Here, we investigated  polymorphisms in severe and mild cases of COVID-19 in a Chinese population. The variant haplotype , represented by rs1800796, rs1524107, and rs2066992 at the  locus, was reduced in patients with severe illness; in contrast, carriers of the wild-type haplotype -- had higher risk of severe illness. Mechanistically, the protective variant haplotype lost CTCF binding at the  intron and responded poorly to inflammatory stimuli, which may protect the carriers from hyperinflammation in response to acute SARS-CoV-2 infection. These results point out the possibility that  genotypes underlie the differential viral virulence during the outbreak of COVID-19. The risk loci we identified may serve as a genetic marker to screen high-risk COVID-19 patients.

A Low-Producing Haplotype of Interleukin-6 Disrupting CTCF Binding Is Protective against Severe COVID-19.

Pore-C simultaneously captures genome-wide multi-way chromatin interaction and associated DNA methylation status in Arabidopsis.

The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies not only offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for multi-way chromatin interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell-cycle-associated genes. Taken together, MCIBox represents an invaluable tool for the study of multiple chromatin interactions and inaugurates a previously unappreciated view of 3D genome structure.

MCIBox: a toolkit for single-molecule multi-way chromatin interaction visualization and micro-domains identification.

Chromatin structural domains, or topologically associated domains (TADs), are a general organizing principle in chromatin biology. RNA polymerase II (RNAPII) mediates multiple chromatin interactive loops, tethering together as RNAPII-associated chromatin interaction domains (RAIDs) to offer a framework for gene regulation. RAID and TAD alterations have been found to be associated with diseases. They can be further dissected as micro-domains (micro-TADs and micro-RAIDs) by clustering single-molecule chromatin-interactive complexes from next-generation three-dimensional (3D) genome techniques, such as ChIA-Drop. Currently, there are few tools available for micro-domain boundary identification. In this work, we developed the MCI-frcnn deep learning method to train a Faster Region-based Convolutional Neural Network (Faster R-CNN) for micro-domain boundary detection. At the training phase in MCI-frcnn, 50 images of RAIDs from  RNAPII ChIA-Drop data, containing 261 micro-RAIDs with ground truth boundaries, were trained for 7 days. Using this well-trained MCI-frcnn, we detected micro-RAID boundaries for the input new images, with a fast speed (5.26 fps), high recognition accuracy (AUROC = 0.85, mAP = 0.69), and high boundary region quantification (genomic IoU = 76%). We further applied MCI-frcnn to detect human micro-TADs boundaries using human GM12878 SPRITE data and obtained a high region quantification score (mean gIoU = 85%). In all, the MCI-frcnn deep learning method which we developed in this work is a general tool for micro-domain boundary detection.

MCI-frcnn: A deep learning method for topological micro-domain boundary detection.

Single-cell multiomics techniques have been widely applied to detect the key signature of cells. These methods have achieved a single-molecule resolution and can even reveal spatial localization. These emerging methods provide insights elucidating the features of genomic, epigenomic and transcriptomic heterogeneity in individual cells. However, they have given rise to new computational challenges in data processing. Here, we describe Single-cell Single-molecule multiple Omics Pipeline (ScSmOP), a universal pipeline for barcode-indexed single-cell single-molecule multiomics data analysis. Essentially, the C language is utilized in ScSmOP to set up spaced-seed hash table-based algorithms for barcode identification according to ligation-based barcoding data and synthesis-based barcoding data, followed by data mapping and deconvolution. We demonstrate high reproducibility of data processing between ScSmOP and published pipelines in comprehensive analyses of single-cell omics data (scRNA-seq, scATAC-seq, scARC-seq), single-molecule chromatin interaction data (ChIA-Drop, SPRITE, RD-SPRITE), single-cell single-molecule chromatin interaction data (scSPRITE) and spatial transcriptomic data from various cell types and species. Additionally, ScSmOP shows more rapid performance and is a versatile, efficient, easy-to-use and robust pipeline for single-cell single-molecule multiomics data analysis.

ScSmOP: a universal computational pipeline for single-cell single-molecule multiomics data analysis.

The three-dimensional (3D) organization of chromatin within the nucleus is crucial for gene regulation. However, the 3D architectural features that coordinate the activation of an entire chromosome remain largely unknown. We introduce an omics method, RNA-associated chromatin DNA-DNA interactions, that integrates RNA polymerase II (RNAPII)-mediated regulome with stochastic optical reconstruction microscopy to investigate the landscape of noncoding RNA -associated chromatin topology for gene equalization to achieve dosage compensation. Our findings reveal that  anchors to the target gene transcription end sites (TESs) and spreads in a distinctive boot-shaped configuration, promoting a more open chromatin state for hyperactivation. Furthermore,  arches TES to transcription start sites to enhance transcriptional loops, potentially facilitating RNAPII convoying and connecting proximal promoter-promoter transcriptional hubs for synergistic gene regulation. These TESs cluster as  compartments, surrounded by inactive domains for coactivation of multiple genes within the  territory. In addition,  structures gradually form and scaffold for stepwise coactivation in dosage compensation.

3D chromatin structures associated with ncRNA  for hyperactivation and coactivation across the entire X chromosome.

CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.

Specific multivalent molecules boost CRISPR-mediated transcriptional activation.

Three-dimensional genome organization reveals that gene regulatory elements, which are linearly distant on the genome, can spatially interact with target genes to regulate their expression. DNA fluorescence in situ hybridization (DNA-FISH) is an efficient method for studying the spatial proximity of genomic loci. In this study, we developed an optimized Tn5 transposome-based DNA-FISH method, termed Tn5-labeled DNA-FISH. This approach amplifies the target region and uses a self-assembled Tn5 transposome to simultaneously fragment the DNA into ~100 bp segments and label it with fluorescent oligonucleotides in a single step. This method enables the preparation of probes for regions as small as 4 kb and visualizes both endogenous and exogenous genomic loci at kb resolution. Tn5-labeled DNA-FISH provides a streamlined and cost-effective tool for probe generation, facilitating the investigation of chromatin spatial conformations, gene interactions, and genome architecture.

Tn5-Labeled DNA-FISH: An Optimized Probe Preparation Method for Probing Genome Architecture.

Transcription factors (TFs) play a crucial role in the regulation of gene expression and the structural organization of chromatin. They interact with proteins, RNA, and chromatin DNA to exert their functions. Therefore, an efficient and straightforward experimental approach that simultaneously captures the interactions of transcription factors with DNA, RNA, and proteins is essential for studying these regulatory proteins. In this study, we developed a novel method, TF-chRDP (Transcription Factor binding Chromatin-associated RNA, DNA, and Protein), which allows for the concurrent capture of these biomolecules in a single experiment. We enriched chromatin complexes using specific antibodies and divided the chromatin into three fractions: one for DNA library preparation to analyze the genomic binding sites of transcription factors, another for RNA library preparation to investigate the RNA associated with transcription factor binding, and the third for proteomic analysis to identify protein cofactors interacting with transcription factors. We applied this method to study the transcription factor p53 and its associated chromatin complexes. The results demonstrated high specificity in the enrichment of DNA, RNA and proteins. This method provides an efficient tool for simultaneously capturing chromatin-associated RNA, DNA and protein bound to specific TF, making it particularly useful for analyzing the role of protein-DNA-RNA complexes in transcriptional regulation.

TF-chRDP: a method for simultaneously capturing transcription factor binding chromatin-associated RNA, DNA and protein.

The three-dimensional (3D) chromatin structure of Epstein-Barr virus (EBV) within host cells and the underlying mechanisms of chromatin interaction and gene regulation, particularly those involving EBV's noncoding RNAs (ncRNAs), have remained incompletely characterized. In this study, we employed state-of-the-art techniques of 3D genome mapping, including protein-associated chromatin interaction analysis with paired-end tag sequencing (ChIA-PET), RNA-associated chromatin interaction technique (RDD), and super-resolution microscopy, to delineate the spatial architecture of EBV in human lymphoblastoid cells. We systematically analyzed EBV-to-EBV (E-E), EBV-to-host (E-H), and host-to-host (H-H) interactions linked to host proteins and EBV RNAs. Our findings reveal that EBV utilizes host CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) to form distinct chromatin contact domains (CCDs) and RNAPII-associated interaction domains (RAIDs). The anchors of these chromatin domains serve as platforms for extensive interactions with host chromatin, thus modulating host gene expression. Notably, EBV ncRNAs, especially Epstein-Barr-encoded RNAs (EBERs), target and interact with less accessible regions of host chromatin to repress a subset of genes via the inhibition of RNAPII-associated chromatin loops. This process involves the cofactor nucleolin (NCL) and its RNA recognition motifs, and depletion of either NCL or EBERs alters expression of genes crucial for host infection control, immune response, and cell cycle regulation. These findings unveil a sophisticated interplay between EBV and host chromatin.

Article	Year
Molecular Epidemiology of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology - Mainland China Subset Analysis of the PIONEER study. PloS one\| PubMed ID: 26599344	2015
A Low-Producing Haplotype of Interleukin-6 Disrupting CTCF Binding Is Protective against Severe COVID-19. mBio\| PubMed ID: 34634929	2021
Pore-C simultaneously captures genome-wide multi-way chromatin interaction and associated DNA methylation status in Arabidopsis. Plant biotechnology journal\| PubMed ID: 35313066	2022
MCIBox: a toolkit for single-molecule multi-way chromatin interaction visualization and micro-domains identification. Briefings in bioinformatics\| PubMed ID: 36094071	2022
MCI-frcnn: A deep learning method for topological micro-domain boundary detection. Frontiers in cell and developmental biology\| PubMed ID: 36531953	2022
ScSmOP: a universal computational pipeline for single-cell single-molecule multiomics data analysis. Briefings in bioinformatics\| PubMed ID: 37779245	2023
3D chromatin structures associated with ncRNA for hyperactivation and coactivation across the entire X chromosome. Science advances\| PubMed ID: 39058769	2024
Specific multivalent molecules boost CRISPR-mediated transcriptional activation. Nature communications\| PubMed ID: 39174527	2024
Tn5-Labeled DNA-FISH: An Optimized Probe Preparation Method for Probing Genome Architecture. International journal of molecular sciences\| PubMed ID: 40076846	2025
TF-chRDP: a method for simultaneously capturing transcription factor binding chromatin-associated RNA, DNA and protein. Frontiers in cell and developmental biology\| PubMed ID: 40123855	2025
Landscape of the Epstein-Barr virus-host chromatin interactome and gene regulation. The EMBO journal\| PubMed ID: 40425856	2025

Meizhen Zheng

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