De-repression of tumor suppressor genes by DNA-binding polyamides at hypermethylated CpG promoters

项目摘要 DNA甲基化、组蛋白修饰和表观遗传调控机制是维持细胞内DNA甲基化的关键。 正常的细胞内稳态癌症和其他疾病的一个共同特征是表观遗传标记的失调， 富含CpG的启动子区。启动子CpG岛的异常甲基化和由此产生的基因沉默是一个基因突变。 建立了肿瘤抑制基因失活的机制。甲基化的DNA被一组蛋白质识别 称为甲基-CpG-结合结构域（MBD）蛋白，其募集修饰蛋白用于染色质组织， 表观基因组维持，导致DNA结构的改变和不适当的抑制性异染色质 阵研究表明，MBD蛋白的占据与肿瘤的高甲基化状态之间存在联系， 抑制基因，这有助于肿瘤的发生。靶向MBD蛋白的敲除研究已经证明了治疗 靶向潜力;然而，MBD蛋白的小分子抑制剂目前不存在。 该提案的总体目标是开发小分子吡咯-咪唑（Py-Im）聚酰胺， 人癌细胞系中肿瘤抑制基因的高甲基化CpG启动子区域，以破坏MBD蛋白- DNA结合。通过干扰MBD蛋白-DNA结合，我们的目标是恢复沉默的肿瘤抑制基因的表达， 这是由于表观遗传标记的错误调节和随后的染色质结构。以前的工作已经证明了 Py-Im聚酰胺通过阻止阻遏物-DNA相互作用来使基因表达去沉默。在这项工作中， 靶向富含CpG的DNA序列的序列特异性Py-Im聚酰胺将被设计、合成并测试其 抑制MBD蛋白-DNA结合的能力。这种抑制作用在细胞培养中的作用将在基因- 特异性和全基因组mRNA表达，以及细胞增殖和活力，以评估 聚酰胺介导的基因调控。 该提案描述了一种新的非诱变方法来对抗表观遗传驱动的癌症。消除沉默 通过在蛋白质-DNA界面抑制MBD蛋白来抑制肿瘤抑制基因代表了一种治疗癌症的新方法 治疗通过检测MBD蛋白的活性、作用方式、特异性和靶向结果，本研究将提供 更好地理解肿瘤发生中MBD蛋白依赖的基因沉默;我们相信这些努力将代表一个新的方向。 为癌症治疗的基因靶向系统治疗的发展做出了宝贵贡献。直接针对 DNA结合，而不会对DNA造成损害，代表了一种强大的和高度特异性的抗癌方法， 如果成功，将直接有益于公众健康。

PROJECT SUMMARY DNA methylation, histone modification, and the machinery involved in epigenetic regulation are key to maintaining proper cellular homeostasis. A common feature among cancer and other diseases is the misregulation of epigenetic marks in CpG-rich promoter regions. Aberrant hypermethylation of promoter CpG islands and the resulting gene silencing is an established mechanism for the inactivation of tumor suppressor genes. The methylated DNA is recognized by a set of proteins called methyl-CpG-binding domain (MBD) proteins, which recruit modification proteins for chromatin organization and epigenomic maintenance, leading to alterations of the DNA architecture and inappropriate repressive heterochromatin formation. Studies have shown a link between the occupancy of MBD proteins and the hypermethylation status of tumor suppressor genes, which contributes to tumorigenesis. Knockout studies targeting MBD proteins have demonstrated therapeutic targeting potential; however, small molecule inhibitors of MBD proteins currently do not exist. The overall objective of this proposal is to develop small molecule pyrrole-imidazole (Py-Im) polyamides targeted to hypermethylated CpG promoter regions of tumor suppressor genes in human cancer cell lines, to disrupt the MBD protein- DNA binding. By interfering with MBD protein-DNA binding, we aim to regain expression of silenced tumor suppressor genes due to misregulation of epigenetic marks and subsequent chromatin architecture. Previous work has demonstrated the ability of Py-Im polyamides to de-silence gene expression by preventing repressor-DNA interactions. In this work, a library of sequence-specific Py-Im polyamides targeted to CpG-rich DNA sequences will be designed, synthesized and tested for their ability to inhibit MBD protein-DNA binding. Effects of this inhibition in cell cultures will be examined in the context of gene- specific and genome-wide mRNA expression, as well as cell proliferation and viability, to assess the genome-wide specificity of polyamide-mediated gene regulation. This proposal describes a novel, non-mutagenic approach to combat epigenetic-driven cancers. The de-silencing of tumor suppressor genes by inhibition of MBD proteins at the protein-DNA interface represents a new approach to cancer treatment. By examining the activity, mode of action, specificity and outcome of targeting MBD proteins, this study will provide a better understanding of MBD protein-dependent gene silencing in tumorigenesis; we believe these efforts will represent a valuable contribution to the development of gene-targeted systemic treatments for cancer therapies. The direct targeting of DNA-binding, without causing damage to the DNA, represents a powerful and highly specific approach to fighting cancer, and if successful will directly benefit public health.