We successfully generated the G4-CUT&Tag libraries with pre-assembled Tn5 transposome efficiently ( 1 d) using 1 105 HEK293T cells (Supplemental Fig. 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. G-quadruplexes (G4s) are four-stranded intramolecular structures that arise from the self-stacking of two or more guanine quartets (G-quartets), in which the four guanine molecules form a square planar arrangement in a cyclic hydrogen-bonding pattern (Bochman et al. 2012; Varshney et al. 2020). G4s are formed in guanine-rich nucleic acids and further stabilized in the presence of monovalent cations (H?nsel-Hertsch et al. 2017; Spiegel et al. 2020). They are evolutionarily conserved and stable under physiologic conditions (Chen et al. 2018; Marsico et al. 2019). In mammals, the genomic distribution of G4s is not random but rather peculiar to specific genomic regions, such as telomeres, gene promoters, transcription factor binding sites, and sites with DNA double-strand breaks (Biffi et al. 2013; Varshney et al. 2020; Zheng et al. 2020). Although the chemistry of G4s has been under investigation for decades, the important biological functions of G4 have just begun to emerge recently. Based on their distribution in the genome, G4s have been implicated in several essential cellular processes, such as gene transcription, DNA replication, genomic instability, and telomere elongation and maintenance (Varshney et al. 2020). Gene transcription is Tenofovir (Viread) usually a driving pressure of chromatin relaxation and single-stranded DNA (ssDNA) exposure, which is a prerequisite for G4 formation. Chromatin immunoprecipitation with an designed G4 Tenofovir (Viread) structure-specific antibody BG4 followed by high-throughput sequencing (G4 ChIP-seq) has detected and mapped endogenous G4s in mammalian cells (Biffi et al. 2013; H?nsel-Hertsch et al. 2018). Using this method, 10,000 G4 structures on chromatin have been identified in human cells, the majority of which mainly reside upstream of the transcription start sites (TSSs) of actively transcribed genes (H?nsel-Hertsch et al. 2016; Tenofovir (Viread) Zheng et al. 2020), suggesting the potential interplay between G4s and transcriptional regulation. Dysfunctions of G4s have been seen Tenofovir (Viread) in neurodegenerative diseases and breast malignancy (H?nsel-Hertsch et al. 2020; Wang et al. 2021a), and G4s were suggested to serve as potential therapeutic targets for DNA-targeted therapies, particularly in anticancer drug design (Neidle 2016; Zyner et al. 2019; Carvalho et al. 2020). The application of multiple G4-stabilizing compounds (G4 ligands), such as TMPyP4, pyridostatin (PDS), and PhenDC3, as potential anticancer drugs is currently being evaluated. These compounds were initially developed to interfere with telomere functions and alter transcription of oncogenes (De Cian et al. 2007; Rodriguez et al. 2012; Carvalho et al. 2020). Additionally, G4 formation was elevated in immortalized cells compared Rabbit Polyclonal to 5-HT-1F to their normal counterparts (H?nsel-Hertsch et al. 2016), and the differentially enriched G4 regions can function as genomic markers of regions that drive breast malignancy and serve as predictors of drug response to G4 ligands (H?nsel-Hertsch et al. 2020). Furthermore, genetic interaction studies of G4s uncovered many genetic vulnerabilities to G4 ligands, raising new therapeutic possibilities for G4 ligands in anticancer treatment (Zimmer et al. 2016; Zyner et al. 2019). After decades of development, some G4 ligands have reached advanced phase I and phase II trials as candidate therapeutic agents against several types of tumors (Drygin et al. 2009; Xu et al. 2017; Carvalho et al. 2020). However, elucidating how G4s are regulated by G4 ligands, especially at promoters, and the specific mechanisms underlying the biological functions of G4s and G4 ligands are still challenging. Plenty of studies have linked G4 formation with transcriptional regulation, and different models have been proposed for G4 involved in transcription at promoters and gene bodies (Spiegel et al. 2020; Varshney et al. 2020). G4 has been reported to act as a direct or indirect roadblock for RNA polymerase II (Pol II) elongation (Varshney et al. 2020), promoting or inhibiting the recruitment of specific transcription factors Tenofovir (Viread) and cofactors (Raiber et al. 2012; Gao et al. 2015; Li et al. 2017; Makowski et al. 2018). However, this evidence is largely based on computationally predicted G4 motifs, correlations between G4 and gene expression, or manipulation of G4 structures on individual genes in plasmid constructs (Spiegel et al. 2020; Varshney et al. 2020). The genome-scale interplay between native G4 and transcription remains unknown. More explicit evidence of native G4 involvement in transcription and scrutiny of the potential interference of indirect or network effects are imperative and would promote a better characterization of the direct functions of G4 in genome-scale transcriptional regulation. In this study,.