G4解旋酶G4R1通过解旋G4调控基因表达和在细胞增殖中的作用机制研究

G-quadruplex (G4) are four-stranded tetrad structures linked together by Hoogsteen hydrogen bonds formed between four guanines in G-rich nucleic acid sequence. G4 are widely present in the genome, and have important roles in the genome stability maintenance and gene expression regulation. Some helicases specifically bind and resolve G4-DNA, which is important for the genome metabolism. G4 resolvase 1 (G4R1)，which is responsible for the major G4-DNA resolving activity in HeLa cells, binds and unwinds G4-DNA and G4-RNA with high affinity in vitro. The Knockout of G4R1 affects the expression of many genes having G4-DNA in their promoter region. Our previous study has demonstrated that G4R1 enhances YY1 expression through resolving the G4-DNA in YY1 promoter. These studies suggested that G4R1 play important role in the regulation of gene expression based on its' G4 resolving activity. We also found that G4R1 is over-expressed in breast cancer, which suggests G4R1 may play a role in cell proliferation and tumorigenesis. However, the mechanism of G4R1 binding and resolving of G4-DNA in vivo, and its' role in gene expression regulation and cell proliferation are less studied. In this study, using ChIP-Seq, IP-MS, cell proliferation assay and other methods, we intend to work at the DNA, proteins and cells levels in order to identify the target genes of G4R1 through binding and resolving G4-DNA, elucidate the cofactor or post-modification associated with G4R1 resolving G4-DNA activity, and study the role of G4R1 in normal and pathological cell proliferation. Our study will demonstrate the mechanism of G4R1 resolving G4-DNA in vivo, and its' roles in gene expression regulation and cell proliferation, Thus providing evidences for the role of G4-DNA and G4R1 in cell physiological activity.

四联体（G4）是由富含鸟嘌呤的核苷酸链所形成的四链二级结构，广泛存在于基因组中，在维持基因组稳定和调控基因表达等方面具有重要作用。G4能够为特定解旋酶所解旋。在细胞中起主要G4解旋作用的解旋酶是G4R1。G4R1具有高度的G4-DNA和G4-RNA结合和解旋活性。敲除G4R1能够影响大量具有G4的基因表达，提示G4R1在调控基因表达中具有重要作用。然而对于G4R1在体内如何解旋G4和调控基因表达，及在细胞增殖中作用还尚不清楚。本研究拟从DNA，蛋白和细胞三个层面入手，通过ChIP-Seq，IP-MS和细胞学检测等技术手段，鉴别G4R1通过解旋G4所调控的靶基因；鉴定参与G4R1解旋G4的互作蛋白或修饰；检测G4R1在正常和病理细胞增殖中的作用，旨在证实G4R1通过解旋G4-DNA的基因表达调控机制和在细胞增殖中的作用,为揭示G4-DNA及G4R1在细胞中的作用机制奠定基础。

四联体（G4）是一种由富含鸟嘌呤的核酸链所形成的四链二级结构，参与调控与细胞生长相关基因的表达调控。G4R1是细胞中的主要G4解旋酶，能够特异解旋G4，从而调节基因的复制和转录，以及核酸动态稳定。然而G4R1在细胞内体内如何解旋G4和调控基因表达，及在细胞增殖中作用还尚不清楚。本研究拟鉴别G4R1通过解旋G4所调控的靶基因和鉴定G4R1解旋G4的互作蛋白来阐明G4R1的G4解旋机制；检测G4R1在乳腺癌细胞中的作用，揭示G4R1与肿瘤发生的相关性。通过本项目的研究，证实了G4R1在乳腺癌中异常过表达，提示G4R1可能是乳腺癌治疗的新靶标；细胞学检测发现干扰G4R1能够抑制乳腺癌细胞生长，表明G4R1在乳腺癌发生发展过程中其到类似癌基因样的作用；转录组芯片检测发现干扰G4R1能够抑制乳腺癌细胞中一系列基因的表达，证实G4R1参与基因的表达调控；证实G4配体TMPyP4，槲皮素和大豆黄素具有良好的抗肿瘤活性，是潜在的抗肿瘤药物。通过本项目的开展，揭示了G4和G4解旋酶G4R1在乳腺癌发生过程中具有重要作用，为乳腺癌治疗提供了一个新靶标。