| As a molecular chaperone protein,hypoxia up-regulated protein 1(HYOU1)is involved in maintaining and restoring cellular homeostasis.In cancers,the HYOU1 gene can be activated via endoplasmic reticulum stress response and other pathways,promoting cancer cell proliferation,invasion,metastasis,and anti-apoptotic effects.G-quadruplexes(G4s)are secondary structures formed by folding of guanine-rich regions in DNA and RNA,with regulatory functions in gene expression,RNA splicing,and so on.A previous study has indicated a strong positive correlation between the expression of transcription factor Yin Yang 1(YY1)gene and the HYOU1 gene.Analysis of the HYOU1 gene promoter region revealed a guanine-rich region in the vicinity of its transcription start site(TSS).In the current study,we predicted that the HYOU1 gene promoter region could form G4 structures,and YY1 could regulate HYOU1 gene expression by binding to the G4 s in its promoter.In this study,using liver cancer cells as a model,we identified the G4 structure formation in the HYOU1 gene promoter and investigated the mechanism of G4-regulated HYOU1 gene expression.The results are shown as follows:1.Analysis of the HYOU1 gene promoter revealed a guanine-rich region near the TSS,and two regions with G4 forming potential located in-33 to-55 and-81 to-100 upstream of the TSS were predicted by bioinformatics methods.2.Oligonucleotides containing the two guanine-rich regions in the HYOU1 promoter were synthesized.Circular dichroism spectroscopy,DMS footprinting and gel-shift assays were used to demonstrate that the regions could form G4 structures.Luciferase reporter assay was also employed to determine that the G4 structures in the HYOU1 promoter could inhibit gene expression.3.Analyses of the data from the ENCODE database showed that YY1 and specificity protein1(SP1)were highly expressed in liver cancer and showed significantly positive correlations with HYOU1.Ch IP-seq analyses revealed that YY1 and SP1 were highly enriched in the HYOU1 gene promoter.Furthermore,RT-q PCR analyses indicated that individual knockdown of endogenous YY1 and SP1 could significantly reduce endogenous HYOU1 gene expression,suggesting a positive regulation of HYOU1 expression by YY1 and SP1.4.Due to the presence of multiple SP1 binding sites in the G4 sequences of the HYOU1 promoter,oligonucleotides were synthesized with mutations at the SP1 binding motifs in combination with the mutations of G4 sequences.Circular dichroism spectroscopy,DMS footprinting and gel shift assays showed that the oligonucleotides with mutated SP1 binding motifs could form G4 structures.Gel-shift assays also demonstrated that both YY1 and SP1 could bind to the G4 structures in the HYOU1 promoter.5.Luciferase reporter assay revealed that YY1 could strongly activate HYOU1 gene expression by binding to its G4 sequences.With the mutation of the G4 sequences,SP1 could show regulatory activity in HYOU1 expression through its binding sites.In summary,this study identified two G4 sequences in the HYOU1 gene promoter that inhibit HYOU1 expression.YY1 and SP1 regulate HYOU1 gene expression,and YY1 has strong activity in promoting HYOU1 gene expression by binding to its G4 structures.The regulatory effect of SP1 on HYOU1 gene expression can only become detectable through its interaction with YY1 when G4 structures are disrupted.The study provides insights into the regulatory mechanisms of HYOU1 gene expression and suggests potential therapeutic targets for cancer therapies. |
