G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type–specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.

including the URL of the record and the reason for the withdrawal request.

To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.

An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase–hexahistidine–2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.

Strigolactones positively regulate defense against infection by root-knot nematode in tomato through a process that is dependent on MYC2 integrating SL, ABA, and JA signaling