Determine The Formation Of G-quadruplexes In The BAP1 Promoter And Their Regulatory Role In Gene Expression

Accumulating evidence suggests a key role of BAP 1 in cancer development and progression,but mechanisms regulating BAP1 gene expression remain unexplored.G-quadruplexes(G4)are stable secondary structures of nucleic acids formed by four-chain DNA or RNA rich in guanines and stacked as three or more planar quartets stabilized by Hoogsteen hydrogen bonding.A myriad of studies indicate that G4 structures play an important role in regulating various important genes during oncogenesis,and thus targeting G4 structures and related helicases represents a new exploring area in cancer therapies.In this report,we revealed that the BAP1 promoter contains multiple G-tracts and repetitive sequences in its negative strand with high potential of forming G-quadruplex(G4)structures.In circular dichroism studies,synthesized oligonucleotides within the G-rich region of the negative strand upstream the BAP1 transcription start site showed molar ellipticity at specific wavelengths characteristic of G4 structures.Analyses of these oligonucleotides by native polyacrylamide gel electrophoresis also revealed the formation of multiple types of G-quadruplexes.In reporter assays,mutations or deletion of the predicted G4 structure forming sequences in the BAP1 promoter generally reduced expression of a reporter gene Gaussia luciferase,suggesting that G-quadruplex structures in the BAP1 promoter activates its gene expression.Consistently,we discovered that CHD2 and CHD7,chromatin remodeling proteins with DNA helicase activity,could bind to the BAP1 promoter and resolve the G4 structures to inhibit its gene expression.In summary,the BAP1 promoter contains sequences in its negative strand with high potential of G-quadruplex formation.These G4 structures promote BAP1 expression and DNA helicases CHD2 and CHD7 reduce BAP1 gene expression likely through unwinding its G4 structures.