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Fig. 2 | Genome Biology

Fig. 2

From: G-quadruplexes sense natural porphyrin metabolites for regulation of gene transcription and chromatin landscapes

Fig. 2

Genome-wide profiling of hemin binding sites using biotin-PEG4-hemin and G4 self-biotinylation reaction. A, B Synthesis and characterization of biotin-PEG4-hemin. With the coupling reagent dicyclohexyl carbodiimide (DCC), biotin-PEG4-hydrazide reacts with hemin to produce biotin-PEG4-hemin (A). TIC of biotin-PEG4-hemin was created with the precursor of m/z 1103.45 (A) and the products m/z were 616.25 and 666.22 (B). C Schematic diagram of profiling of hemin binding sites in the genome using CUT&Tag and biotin-PEG4-hemin. Isolated nuclei were conjugated to concanavalin A-coated magnetic beads, biotin-PEG4-hemin was used to bind with chromatin in situ. Anti-biotin antibody and secondary antibody were added sequentially to tether protein A-Tn5 transposase. After activation with magnesium, Tn5 cut the chromatin close to biotin-PEG4-hemin binding sites and simultaneously integrated to adapters. Then Tn5-tagmented genomic DNA was extracted and amplified for library preparation and second-generation sequencing. For negative controls, IgG was used to replace anti-biotin antibody. D UCSC genome browser snapshots of in situ biotin-PEG4-hemin binding signals and G4-CUT&Tag signals in HEK293T cells. Twenty micromolars TMPyP4 was used as a competitor for biotin-PEG4-hemin. E Heatmap and metaplot analysis of biotin-PEG4-hemin binding signals and G4-CUT&Tag signals. A total of 5013 biotin-PEG4-hemin binding peaks were identified to be sensitive to TMPyP4 treatment and colocalized with G4. F Profiling of hemin binding sites in cells using G4 self-biotinylation reaction and CUT&Tag. UCSC genome browser snapshots of G4 self-biotinylation signals in HEK293T cells in the presence or absence of 80 μM TMPyP4 or PDS competitors. G Heatmaps of G4 self-biotinylation in the HEK293T after treatment of vehicle or hemin. With the TMPyP4 competitor, we characterized 8195 G4 self-biotinylation peaks that were sensitive to TMPyP4 treatment. H Genome-wide annotation of the 8195 G4 self-biotinylation peaks in HEK293T cells. The Venn diagram shows the overlap of G4-CUT&Tag peaks with the 8195 hemin binding peaks. I Pearson’s correlation of in vivo G4 self-biotinylation signals and in situ biotin-PEG4-hemin binding signals at the 8195 G4 self-biotinylation peaks

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