G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.

Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.

G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific single-chain antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. This technology does not require expression of the G4 antibody in situ, enabling broad applicability to theoretically all chromatin sources. Beginning with chromatin isolation and antibody preparation, the entire protocol can be completed in <1 week, including basic computational analysis.

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C‐rich DNA has the capacity to form a tetra‐stranded structure known as an i‐motif. The i‐motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i‐motif structures form in complex environment of living cells is not currently known. Herein, using state‐of‐the‐art in‐cell NMR spectroscopy, we evaluate the stabilities of i‐motif structures in the complex cellular environment. We show that i‐motifs formed from naturally occurring C‐rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i‐motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i‐motifs in vitro with their stability in vivo and provide essential information for the design and development of i‐motif‐based DNA biosensors for intracellular applications.

BackgroundThe binding of transcription factors (TF) to genomic targets is critical in the regulation of gene expression. Short, double-stranded DNA sequence motifs are routinely implicated in TF recruitment, but many questions remain on how binding site specificity is governed.ResultsHerein, we reveal a previously unappreciated role for DNA secondary structures as key features for TF recruitment. In a systematic, genome-wide study, we discover that endogenous G-quadruplex secondary structures (G4s) are prevalent TF binding sites in human chromatin. Certain TFs bind G4s with affinities comparable to double-stranded DNA targets. We demonstrate that, in a chromatin context, this binding interaction is competed out with a small molecule. Notably, endogenous G4s are prominent binding sites for a large number of TFs, particularly at promoters of highly expressed genes.ConclusionsOur results reveal a novel non-canonical mechanism for TF binding whereby G4s operate as common binding hubs for many different TFs to promote increased transcription.

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