哺乳动物基因组中，70-80%的CpG是甲基化的。然而富含GC的CpG岛区域却很少被甲基化。根据已有注释，70%的基因都含有CpG岛启动子，不仅包括大多数管家基因，还包括一些组织特异性表达的基因，CpG岛很可能参与了对这些基因的转录调控。然而，非甲基化CpG岛调控基因表达的机制尚不清楚。目前已知一些可以依赖ZF-CXXC结构域来特异性结合非甲基化CpG的蛋白。利用体外pull down实验，我们发现BEND3可以特异性结合非甲基化的CpG。但是，BEND3并没有ZF-CXXC结构域，说明存在一种新的机制可以解读非甲基化的CpG。本申请试图阐明BEND3结合非甲基化CpG的分子机制；利用免疫染色和ChIP-seq技术研究BEND3在染色质上的定位；通过建立敲除细胞系，结合高通量测序技术研究BEND3对于转录调节的功能。研究结果将有助于理解非甲基化CpG信号的解读及其对转录调控的影响。

It’s estimated that approximately 70% to 80% of CpG dinucleotides are methylated in mammalian genomes. However, CpG islands (CGIs) which are characterized by increased CpG content and GC percentage remain free of DNA methylation. Approximately 70% of annotated gene promoters are associated with CGIs, including most housekeeping genes and many tissue-specific genes, which suggests a function of CGIs in regulating gene expression. However, the precise mechanisms by which CpG islands contribute to regulatory element function remain poorly understood. It has been demonstrated that un-methylated CpG signals in CGIs can be interpreted by some proteins, which contain a specific un-methylated CpG binding motif, ZF-CXXC motif. With DNA pull down assay in vitro, we found that BEND3 preferentially bound to un-methylated CpG fragments. Moreover, BEND3 doesn’t possess the ZF-CXXC motif, which may suggest a novel binding mechanism. In this application, we prepare to investigate the molecular mechanism by which BEND3 specifically recognizes un-methylated CpG; we also intend to explore the chromatin localization of BEND3 using immunostaining and ChIP-seq technology. In addition, we will use Bend3 knock-out cell lines and high-throughput sequencing technology to unravel the functions of BEND3. Our results may give more clues about how signals of un-methylated CpG are interpreted and involved in regulation of gene expression.