Opportunity
Transcriptional regulation is fundamentally governed by epigenetic modifications, such as histone post-translational modifications (PTMs) like H3K4me3, H3K9me3, and H3K27me3, which critically influence chromatin structure and gene expression. Noncoding RNAs (ncRNAs), particularly long noncoding RNAs (lncRNAs), are increasingly recognized as pivotal epigenetic regulators that interact with histone-modifying enzymes to direct site-specific transcriptional control. Dysregulation of these lncRNAs is frequently induced various severe diseases, including cancer. However, the vast majority of lncRNAs in the human genome remain uncharacterized, significantly hindering their potential application as diagnostic biomarkers or therapeutic targets. Existing methods for identifying RNAs associated with specific chromatin modifications—such as ChRIP-seq, CARIP-seq, PIRCh-seq, and RT&Tag—rely heavily on antibody-based immunoprecipitation and often require covalent crosslinking steps. These dependencies introduce major limitations: antibody quality and specificity can vary considerably between lots, affecting data reliability and reproducibility, while crosslinking procedures may create artifacts or fail to capture transient or weak RNA-chromatin interactions efficiently. Consequently, there is a pressing need for a robust, crosslinking-free, and antibody-independent method to efficiently capture and identify RNAs associated with various chromatin epigenetic marks in living cells under near-physiological conditions.
Technology
The present invention provides a novel method and kit for real-time tagging of RNA molecules associated with epigenetically modified chromatin in living cells, eliminating the need for crosslinking and antibodies. The core innovation involves using an epigenetic "reader" module protein to specifically localize to chromatin regions bearing a target histone PTM (e.g., H3K27me3, H3K9me3, H3K4me3). This reader protein, such as the chromodomain from CBX7 for H3K27me3 or the PHD domain from TAF3 for H3K4me3, is engineered to recruit an engineered ascorbate peroxidase (e.g., APEX2). Upon introduction into living cells along with a biotin-aniline probe and hydrogen peroxide (H₂O₂), the localized APEX2 enzyme catalyzes the oxidation of the probe, generating highly reactive radicals that form covalent bonds with proximal RNA molecules at the target chromatin site. This results in biotin-tagged RNA, which can then be purified via streptavidin-based enrichment and analyzed by high-throughput sequencing. To enhance labeling efficiency, the system can incorporate a SunTag scaffold, where the reader is fused to multiple repetitive peptide epitopes (e.g., GCN4 polypeptide) that recruit ten times the number of APEX2 enzymes fused to a single-chain variable fragment (scFv), thereby amplifying the local enzyme concentration. This proximity-based biotinylation strategy occurs within minutes in living cells, capturing RNA-chromatin interactions under physiological conditions without external crosslinkers or immunoprecipitation.
Advantages
- Eliminates dependency on high-quality, lot-variable ChIP-grade antibodies, improving data consistency and reliability.
- Operates without covalent crosslinking (e.g., formaldehyde or UV), reducing experimental artifacts and preserving native RNA-protein-chromatin interactions.
- Functions in real-time within living cells under near-physiological conditions, capturing dynamic epigenetic interactions.
- Demonstrates higher sensitivity and specificity, identifying a significantly greater number of associated noncoding RNAs compared to existing antibody-based methods like PIRCh-seq.
- Requires substantially lower input material (e.g., ~0.5 million cells) and lower sequencing depth, reducing overall cost and time.
- Utilizes evolutionarily conserved, naturally occurring reader domains (e.g., chromodomains, PHD domains) which offer high affinity and specificity for target histone modifications.
- The platform is highly adaptable; reader domains can be engineered for higher affinity, to recognize novel modifications, or to simultaneously target multiple epigenetic marks.
Applications
- Genome-wide discovery and functional characterization of noncoding RNAs (lncRNAs, etc.) that regulate specific histone modifications.
- Epigenetic research for elucidating mechanisms of transcriptional regulation, chromatin dynamics, and cellular differentiation.
- Identification of novel epigenetic biomarkers for disease diagnosis and prognosis, particularly in cancer and developmental disorders.
- Drug discovery and development, enabling high-throughput screening for compounds that modulate specific RNA-epigenome interactions.
- Basic and translational studies in stem cell biology, neurobiology, and immunology where epigenetic regulation is crucial.
- Creation of research kits for efficient, cost-effective profiling of chromatin-associated RNAs across various cell types and conditions.
