Identification And Functional Studies Of G-Quadruplex Structures In The HOXC10 Promoter

As a member of the homeobox family,HOXC10 plays an important role in cell differentiation and embryonic development in mammals.Increasing evidence also demonstrates a regulatory role of HOXC10 in the development and progression of cancers,but the regulation of HOXC10 gene expression is relatively understudied.In this study,we discovered multiple consecutive guanine tracts in the antisense strand of the HOXC10 promoter with high potential to form G-quadruplex structure predicted by a website-based algorithm.Oligonucleotides based on the region of high G-quadruplex potential in the HOXCIO promoter were synthesized in vitro.In the circular dichroism analysis,the signature molar ellipticity at 262 nm of G-quadruplex structure was determined suggesting the presence of G-quadruplex structure in the HOXC10 promoter.Furthermore,analysis by gel-shifting assay using native polyacrylamide gel electrophoresis revealed the formation of G-quadruplex structures in presence of potassium ion.The oligonucleotides were also transfected into HeLa cells and fluorescence signal was detected by immunofluorescence using an antibody,BG4 against G-quadruplex,indicating that the oligonucleotides could also form G-quadruplex structures in a cellular environment.Then,we amplified the HOXC10 promoter by PCR and generated a reporter construct with this promoter driving the expression of Gaussia luciferase(Gluc).We also generated constructs with the predicted G-quadruplex regions being mutated or deleted.These reporter constructs were transfected into HeLa cells,and Gluc expression was determined by detection of Gluc activity.The reporter assays showed that deletion or mutation of prediected G-quadruplex regions could differentially impact Gluc expression driven by the HOXC10 promoter.It has been reported that helicase can mediate the unwinding of G-quadruplex structure.In this study,we analyzed samples of 759 breast cancer patients and discovered a chromatin remodeling protein CHD7 with DNA helicase activity was positively correlated with the expression of the HOXC10 gene.Reporter assays and RNAi-mediated knockdown showed that CHD7 is a helicase that likely unwinds the G-quadruplex structure in the HOXC10 promoter to enhance its gene expression.In conclusion,we discovered that the G-rich regions in the HOXC10 promoter can form G-quadruplex structures that can be unwind by a helicase CHD7 to regulate HOXC10 gene expression.