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

DNA 结合聚酰胺在高甲基化 CpG 启动子处消除肿瘤抑制基因的抑制

基本信息

批准号：
9393114
负责人：
Paul B Finn
金额：
$ 5.67万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2018-07-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9393114
关键词：
Affect Affinity Animals Architecture Base Pair Mismatch Behavior Binding Binding Proteins Biological Assay Breast Carcinoma Burkitt Lymphoma Cancer cell line Cell Culture Techniques Cell Line Cell Proliferation Cell Survival Cells ChIP-seq Characteristics Chromatin CpG Islands CpG dinucleotide DNA DNA Binding DNA Methylation DNA Sequence DNA Sequence Alteration DNA methyltransferase inhibition DNA-Binding Proteins DNA-Protein Interaction Deoxycytidine Development Disease Dose-Limiting Electrophoretic Mobility Shift Assay Epigenetic Process Event FGF19 gene Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Genes Genetic Transcription Hela Cells Heterochromatin Homeostasis Human Hypermethylation Imidazole In Vitro Intercalating Agents Knock-out LTBP2 gene Libraries Link MCF7 cell Maintenance Malignant Neoplasms Malignant neoplasm of cervix uteri Measures Mediating Messenger RNA Methyl-CpG-Binding Protein 2 Minor Groove Nylons Outcome Pathway interactions Permeability Post-Translational Protein Processing Promoter Regions Protein Binding Domain Proteins Public Health Pyrroles Quantitative Reverse Transcriptase PCR Reader Recruitment Activity Repression S Phase Secondary to Set protein Small Interfering RNA Solid Specificity Structure System Techniques Testing Time Tumor Cell Line Tumor Suppressor Genes Tumor-Suppressor Gene Inactivation Work base cancer cell cancer therapy chromatin immunoprecipitation chromatin protein combat design ds-DNA epigenetic regulation epigenomics fighting gene repression genome-wide histone modification inhibitor/antagonist knock-down mRNA Expression methylation pattern novel novel strategies prevent promoter response small molecule small molecule inhibitor therapeutic target tool transcriptome transcriptome sequencing tumorigenesis

项目摘要

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.

项目摘要 DNA甲基化，组蛋白修饰和表观遗传调节所涉及的机械是维持的关键适当的蜂窝稳态。癌症和其他疾病中的一个共同特征是表观遗传标记的不利富含CPG的启动子区域。启动子CpG岛的异常高甲基化和由此产生的基因沉默是一种建立的肿瘤抑制基因失活的机制。甲基化的DNA通过一组蛋白质识别称为甲基-CPG结合结构域（MBD）蛋白，该蛋白募集染色质组织和表观基因组维护，导致DNA架构的改变和不当抑制异染色质形成。研究表明，MBD蛋白的占用与肿瘤的高甲基化状态之间有联系抑制基因，有助于肿瘤发生。靶向MBD蛋白的敲除研究已证明治疗靶向潜力；但是，目前不存在MBD蛋白的小分子抑制剂。该提案的总体目的是开发针对的小分子吡咯 - 咪唑（PY-IM）聚酰胺。人类癌细胞系中肿瘤抑制基因的高甲基化的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，我们旨在恢复沉默的肿瘤基因的表达由于表观遗传标记和随后的染色质结构的不正调。以前的工作证明了能力 PY-IM聚酰胺以防止抑制剂-DNA相互作用而脱光基因表达。在这项工作中，一个库将设计，合成和测试其针对富含CpG的DNA序列的序列特异性PY-IM聚酰胺将抑制MBD蛋白-DNA结合的能力。这种抑制在细胞培养物中的影响将在基因的背景下进行检查特异性和全基因组mRNA表达以及细胞增殖和活力，以评估全基因组特异性聚酰胺介导的基因调节。该提案描述了一种新型的非毒素方法来对抗表观遗传驱动的癌症。解放通过在蛋白质-DNA界面抑制MBD蛋白来抑制肿瘤抑制基因，代表了一种癌症的新方法治疗。通过检查靶向MBD蛋白的活动，作用方式，特异性和结果，本研究将提供更好地理解肿瘤发生中MBD蛋白依赖性基因沉默；我们相信这些努力将代表对癌症疗法的基因靶向系统治疗的发展有价值的贡献。直接定位 DNA结合而不会对DNA造成损害，代表了一种对抗癌症的强大而高度特定的方法，并且如果成功将直接受益于公共卫生。