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CRISPR -Cas9 Targeting of G-Quadruplex DNA in ADH1 promoter Highlights its role in Transcriptome and Metabolome Regulation

ABSTRACT: G-quadruplex (G4) structures are crucial regulators of gene expression, yet their roles within native chromatin remain underexplored. Using CRISPR/Cas9, we introduced guanine-to-thymine mutations at a G4-forming motif in the adh1+ (alcohol dehydrogenase 1) promoter of Schizosaccharomyces pombe, generating two mutant strains: one with G4-only mutations (ADH1_G4_MUT) )and another with both G4 and TATA-box mutations (ADH1_G4-TATA_MUT). Bulk-RNAseq analyses revealed that these mutations significantly reduce adh1 transcript levels, with G4 TATA-box mutant causing the strongest transcriptional suppression. This indicates a positive regulatory role for the Adh1 G4 structure in adh1+ gene expression. Furthermore, both mutants displayed altered transcriptomic profiles, particularly impacting the oxidoreductase pathway. Global transcriptomic shifts further underscored the regulatory influence of the Adh1 G4 structure, with downstream effects on pathways critical for cellular metabolism. These findings demonstrate the pivotal role of a single G4 structure in modulating transcriptomic profiles and offer insights into its potential as a therapeutic target for reprogramming gene expression and metabolic pathways.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE285129 | GEO | 2025/09/24

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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CRISPR -Cas9 Targeting of G-Quadruplex DNA in ADH1 promoter Highlights its role in Transcriptome and Metabolome Regulation

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