Identification And Functional Studies Of G-Quadruplex Structures In The Human ARID1A Promoter

As a member of the SWI/SNF family,ARID1A plays an essential role in modulating chromatin structure and gene expression.The tumor suppressive function of ARID1A has been well-defined and its downregulation in cancers is attributed to genomic deletion,DNA methylation and microRNA-mediated inhibition.Whether restoring the activity of ARID1A can be used as an anticancer approach remains to be explored.G-quadruplex structures are widely present in the human genome,and play an important role in regulating chromosome DNA replication,RNA editing and RNA stability.However,targeting G-quadruplex structures in promoter regions to treat diseases,including cancers,has not been extensively studies and this research field remains fertile to be explored.In this study,we used a variety of experimental methods to demonstrate the formation of the G-quadruplex structures in the region of the ARID1A promoter,and found that the G-quadruplex structures could enhance the transcription activity of the ARID1A promoter,which provided helpful insights into cancer treatments.The specific research results are as follows:1.The website-based QGRS Mapper algorithm was used to predict the potential G-quadruplex structures in the human ARID1A promoter region.Oligonucleotides were synthesized according to the sequence of the G-quadruplex-forming region of the ARID1A promoter.The G-quadruplex formation of these oligonucleotides was first evaluated by circular dichroism and thermal stability assay.The structural differences of G-quadruplexes formed by these oligonucleotides under different conditions were tested by native polyacrylamide gel electrophoresis.Chromatin immunoprecipitation was used to determine the presence of G-quadruplex structures in the ARID1A promoter.The specific G-quadruplex-forming sequence in the ARID1A promoter was delineated by the dimethyl sulfate(DMS)footprinting approach.Immunofluorescence assay was used to visualize the G-quadruplex formation in cells by the oligonucleotides derived from the ARID1A promoter.2.Reporter assays showed that G-quadruplexes in the ARID1A promoter could promote its transcriptional activity.The DNA replication blockage experiment showed that the G-quadruplex structures in the ARID1A promoter could impede DNA synthesis.3.We discovered relatively strong positive correlation between ARID1A expression and two helicases CHD2 and CHD8.However,knockdown of two DNA helicases,CHD2 and CHD8,could promote the transcription activity of the ARID1A promoter in reporter assays and increase the expression of the endogenous ARID1A gene.Thus,CHD2 and CHD8 unlikely mediate the unwinding of the G-quadruplex structures in the ARID1A promoter,but may be involved in stabilizing the chromatin in this region and consequently repress ARID1A gene expression.4.We tested the effects of TMPyP4,small molecules with ability of stabilizing G-quadruplex structures,on the ARID1A promoter.We observed that TMPyP4 could inhibit the ARID1A promoter-mediated transcription in both reporter assays and RT-qPCR for the endogenous ARID1A expression in HeLa cells.These results suggested that the binding of these small molecules to the G-quadruplex structures may interfere with the transcription activity of the ARID1A promoter,although these molecules could stabilize the structures.Indeed,using native polyacrylamide gel electrophoresis,the oligonucleotides derived from the ARID1A promoter showed smeared bands when treated by TMPyP4,suggesting the formation of various intermolecular G-quadruplex structures.Despite the function of TMPyP4 in stabilizing G-quadruplex structures,we observed its role in reducing the transcription mediated by the ARID1A promoter.It is possible that the integration of TMPyP4 molecule into the G-quadruplexes of the ARID1A promoter could abolish the binding of transcription factors and consequently reduce its transcription activity.Furthermore,we discovered the binding motif of SP1,a transcription factor.However,the knockdown and overexpression of SP1 did not alter the transcription mediated by the ARID1A promoter.Thus,it is likely that the ARID1A promoter did not contain the structure needed for SP1 binding and the presence of SP1 binding motif is insufficient to cause its binding to the ARID1A promoter.5.When the oligonucleotides with the full-length,partial and mutated sequences of the G-quadruplex region in the ARID1A promoter were transiently transfected into HeLa and MDA-MB-231 cells,only the full-length oligonucleotide could enhance the transcription of the endogenous ARID1A and reduce the proliferation of the tumor cells;the oligonucleotides with partial or mutated sequences did not show these activities.In conclusion,this study demonstrated that the promoter region of human ARID1A gene forms G-quadruplex structures,and these structures can positively regulate the transcription mediated by the ARID1A promoter.Additionally,the transcriptional activity of the ARID1A promoter is regulated by CHD2 and CHD8.The small molecules TMPyP4 can also alter ARID1A promoter-mediated transcription through binding to the G-quadruplex structures.Importantly,the oligonucleotide based on the sequence of G-quadruplex region in the ARID1A promoter can promote ARID1A gene expression and inhibit tumor cell proliferation.