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G-quadruplexes are promoter elements and chromatin organizers

ABSTRACT: Despite many efforts in the past it has been difficult to define universal sequence features associated to eukaryotic promoters. Using a thorough sequence analysis associated to experimental TSSs in mammalian cells, we shed light on G-rich motifs able to form G-quadruplex (G4) secondary structures of DNA at promoters. G4 forming sequences are located at the deepest point of nucleosome exclusion at promoters and correlate both with experimental G4s and maximum of promoter activity. A genome-wide analysis indicated that predicted G4s (PG4s) exclude nucleosomes both at promoters and intergenic areas and also display a strong nucleosome positioning potential. Mutating PG4s at integrated model promoters coupled to MS2 reporter resulted in chromatin closure and reduction of transcription activity at the single cell level associated to longer periods of transcriptional shutdown. Further functional investigation using a G4 ligand in vivo demonstrated a global effect on chromatin organization and proximal promoter pausing at PG4 locations. Altogether, our data introduce G4s as bona fide promoter elements with both nucleosome exclusion and positioning properties in mammalian cells.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE159276 | GEO | 2025/04/15

REPOSITORIES: GEO

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Project description:DNA secondary structures are important for fundamental genome functions such as transcription and replication1. The G-quadruplex (G4) structural motif has been linked to gene regulation2,3 and genome instability4,5 and may be important to cancer development and other diseases6-8. Recently, ~700,000 discrete G4s have been observed in naked human single-stranded genomic DNA using G4-seq, a high-throughput sequencing technique that detects structural features in vitro.9 It is of vital importance to investigate G4 structures within an endogenous chromatin context, which until now remained elusive10,11. Herein, we address this via the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We identified ~10,000 endogenous G4 structures and show that G4s are predominantly seen in regulatory, nucleosome-depleted, chromatin regions. G4s were enriched in the promoters and 5âUTR regions of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Reorganization of the chromatin landscape using a histone deacetylase inhibitor, resulted in de novo G4 formation in new and more prominent regulatory, nucleosome-depleted regions associated with increased transcriptional output. Our findings suggest a striking relationship between promoter nucleosome-depleted regions, G4 formation and elevated transcriptional activity. Comparison between normal human epidermal keratinocytes and their immortalized counterparts revealed a ï¾7-fold greater G4 abundance in immortalized cells, of which 80 % were found in regulatory, nucleosome-depleted regions common to both cell types. Consequently, cells exhibiting more G4s displayed significantly increased transcriptional output and were more sensitive to growth inhibition by a small molecule G4 ligand. Overall, our results provide new mechanistic insights into where and when DNA adopts G4 structure in vivo. Our findings show for the first time that regulatory, nucleosome-depleted chromatin and transcriptional states predominantly shape the endogenous G4 DNA landscape. Two cell lines, treated with entinostat or untreated, analyzed to detect gene expression differences, presence of G-Qudruplexes and chromatin state. Each combination of conditions replicated in duplicates or triplicates.

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G-quadruplexes are promoter elements and chromatin organizers

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