Small Molecule Metabolite Hemin Binds With Genome-wide G-quadruplexes And Promotes G-quadruplex Formation

G-quadruplex(G-4)is a unique atypical nucleic acid secondary structure that is widely found in living organisms and is involved in a variety of life activities,such as transcriptional translation of genes and DNA replication.G-quadruplex are thought to be associated with transcription factors and chromatin remodeling occurrence,regulating cell-specific transcription and chromatin landscape.In a previous study,we established a novel G4-CUT&Tag method for detecting G-quadruplex on the whole genome.Using this method,we found that G-quadruplex are mostly present in transcriptional regulatory regions such as promoters and enhancers of genes and that they inhibit gene transcription initiation when the classical G-quadruplex ligand TMPy P4 is used to induce stable formation of G-quadruplex structures.The natural metabolite heme(Hemin)has a porphyrin ring structure similar to that of the G-quadruplex ligand TMPy P4,and in vitro studies have shown that Hemin can bind directly to the G-quadruplex.Therefore,we expect to investigate whether this naturally occurring porphyrin metabolite has a function similar to that of TMPy P4.whether Hemin can bind G-quadruplexes intracellularly and promote their formation,through a genome-wide study.First,to detect the genome-wide distribution of Hemin,we synthesized a biotinlabeled Hemin,Biotin-PEG4-Hemin,which specifically binds the G-quadruplex and does not interact with single and double-stranded DNA.We have combined BiotinPEG4-Hemin with CUT&Tag technology to develop in situ capture sequencing of heme binding sites.The assay showed that a large number of Biotin-PEG4-Hemin binding signal peaks overlapped with G4-CUT&Tag signal peaks on the genome of HEK293 T cells,and 5,031 of them were sensitive to the treatment of TMPy P4,indicating that BiotinPEG4-Hemin can bind to G-quadruplex at the chromatin level.It has been shown that the G-quadruplex-Hemin complex has peroxidase activity and can autobiotinize the G-quadruplex in the presence of hydrogen peroxide and biotin tyramine.Therefore,we performed G-quadruplex autobiotinylation reaction in Hemin-treated HEK293 T cells,and then combined with CUT&Tag method to detect the autobiotinylated G-quadruplex sites,i.e.Hemin-bound G-quadruplex sites.In this assay,we identified 8,195 Hemin binding sites sensitive to TMPy P4 treatment,of which about 80% were present in the gene promoter region and about 72% overlapped with G4-CUT&Tag signaling peaks.These results further illustrate the binding of Hemin to chromatin G-quadruplexs and the possible involvement of Hemin in G tetrasomal regulation.Next,to investigate the effect of Hemin on G-quadruplexs,we treated Hela cells with 80 u M Hemin and then stained the intracellular G-quadruplexs using the Gquadruplex-specific antibody BG4-EGFP and counted the average fluorescence intensity in the nuclei.We found that the mean fluorescence staining intensity of cells was enhanced after Hemin treatment,indicating that at the cellular level Hemin promotes the formation of G-quadruplexs.Then we further combined G4-CUT&Tag to detect genome-wide G-quadruplex formation and found that Hemin treatment rapidly promoted G-quadruplex formation in promoter and enhancer regions.All these results demonstrate the relationship of Hemin in the regulation of transcription by Gquadruplex,which may be involved in the transcriptional regulation of genes through G-quadruplex.In summary,we examined the genome-wide binding sites of the natural porphyrin metabolite Hemin by two high-throughput sequencing means,demonstrated the binding of Hemin to G-quadruplexs at the chromatin level and at the cellular level,and found that Hemin treatment could rapidly promote the formation of Gquadruplexs genome-wide,suggesting that Hemin may have a function similar to that of the G tetrasomal ligand TMPy P4,i.e.,to participate in gene transcriptional regulation by binding to G-quadruplexs and promoting their formation.Available reports suggest that intracellular metabolic small molecules can act as cofactors of epimodifying enzymes or agonist/antagonist molecules of transcription factors,affecting the activity of epimodifying enzymes or transcription factors and thus regulating epigenetic modifications and gene transcription.his study reveals a possible new mechanism for the involvement of small molecule metabolites in transcriptional regulation,namely,Hemin may act as a ligand for chromatin secondary structure Gquadruplexs and regulate gene transcription by altering G-quadruplex formation.