Interrogation of Androgen Receptor:Forkhead Interaction Using Py-Im Polyamides

雄激素受体的询问：使用 Py-Im 聚酰胺的叉头相互作用

基本信息

批准号：
8701250
负责人：
Thomas Farid Martinez
金额：
$ 3.89万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8701250
关键词：
AR gene Affinity Androgen Receptor Androgen Response Element Androgens Antineoplastic Agents Apoptosis Base Pairing Behavior Binding Cell Culture Techniques Cell physiology Cells ChIP-seq Complex DNA DNA Binding DNA Minor Groove Binding DNase-I Footprinting Data Development Diagnosis Electrophoretic Mobility Shift Assay Enhancers Family Family member Gene Expression Genes Genomics Growth Human Imidazole In Vitro Incubated Individual LNCaP Length Malignant Neoplasms Malignant neoplasm of prostate Metabolism Nylons PC3 cell line Pattern Promoter Regions Proteins Purines Pyrroles Recombinants Relative (related person)Response Elements Role Site Testing Therapeutic Titrations Transcriptional Regulation Work biophysical properties design drug development forkhead protein genome-wide human disease in vivo mRNA Expression men monomer novel prevent programs promoter prostate cancer model protein protein interaction purine receptor binding research study small molecule therapeutic target transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Many human diseases are caused by dysregulated gene expression. The oversupply or over activity of one or more transcription factors (TF) may be required for the survival, growth, and metastatic behavior of all human cancers, and therefore provide an attractive therapeutic target. The NCI estimates that in the US alone 217,730 men will be diagnosed with and 32,050 men will die of prostate cancer in 2010 (6). Androgen receptor (AR) is the aberrant TF responsible for expression changes leading to prostate cancer. Preliminary ChIP-Seq data on androgen treated LNCaP cells, a prostate cancer cell line, revealed a novel motif sequence, an androgen response element (ARE) plus a forkhead (Fkhd) response element (FHRE) separated by 4 base pairs (bps), enriched relative to background genomic data. This novel IS plus FHRE motif is suggestive of co-occupancy of DNA by AR and a forkhead TF to regulate specific genes in an unprecedented regime. Forkhead TFs are integral to a number of cancer related cellular functions, such as metabolism, development, proliferation, and apoptosis, and therefore may prove important to understanding prostate cancer (17). To demonstrate that co-occupancy of AR and a Fkhd is possible, an electrophoretic mobility shift assay (EMSA) will be employed. Recombinant human AR and human FoxA1, a candidate forkhead family member believed to interact with AR, will be incubated with a 30 bp DNA fragment containing the ARE plus FHRE motif (18). The cooperatively of the AR:Fkhd interaction will be quantitated using DNase I footprint titrations, and then repeated with truncated forms of the TFs in order to determine which domains are responsible for the protein-protein or allosteric interactions. The biophysical importance of the 4 bp spacer to the interaction will be assessed by repeating the EMSA experiments with DNA fragments containing spacers ranging from 0 to 8 bps. Pyrrole- imidazole (Py-Im) polyamides, sequence specific DNA minor-groove binding molecules, will be designed to disrupt the Fkhd:DNA interface to probe the in vivo role of the AR:Fkhd interaction. The effects of disrupting the Fkhd:DNA or AR:DNA interactions in genes containing the ARE plus FHRE motif in an enhancer or promoter region will be tested using qRT-PCR, ChIP-Seq, and RNA-Seq on LNCaP cells treated with an androgen, the designed Fkhd polyamides, and existing AR:DNA disrupting polyamides. qRT-PCR experiments will be used to test the ability of the polyamides to effectively disrupt the Fkhd:DNA and ARE:DNA interactions, as well as test the hypothesis that disruption of either AR or the Fkhd will restore mRNA expression to basal levels in genes containing the ARE plus FHRE motif. In order to understand the genome wide roles of the AR:Fkhd:DNA interaction on transcriptional programs, ChIP-Seq and RNA-Seq experiments will be performed on LNCaP cells treated with polyamides to disrupt each individual interaction. Biophysical characterization of this novel transcription factor complex combined with genome-wide characterization of genes regulated by AR:Fkhd using polyamides can reveal a new target for prostate cancer drug development.

描述（由申请人提供）：许多人类疾病是由基因表达失调引起的。一种或多种转录因子（TF）的过量供应或过度活性可能是所有人类癌症的存活、生长和转移行为所必需的，因此提供了有吸引力的治疗靶点。 NCI 估计，2010 年仅美国就有 217,730 名男性被诊断患有前列腺癌，32,050 名男性将死于前列腺癌 (6)。雄激素受体 (AR) 是一种异常的 TF，其表达变化会导致前列腺癌。雄激素处理的 LNCaP 细胞（一种前列腺癌细胞系）的初步 ChIP-Seq 数据揭示了一种新的基序序列，即雄激素反应元件 (ARE) 加上由 4 个碱基对 (bps) 分隔的叉头 (Fkhd) 反应元件 (FHRE)，相对于背景基因组数据有所丰富。这种新颖的 IS 加 FHRE 基序表明 AR 和叉头 TF 共同占据 DNA，以前所未有的方式调节特定基因。叉头 TF 是许多癌症相关细胞功能（例如代谢、发育、增殖和凋亡）不可或缺的一部分，因此可能对了解前列腺癌很重要 (17)。为了证明 AR 和 Fkhd 共存是可能的，将采用电泳迁移率变动测定 (EMSA)。重组人 AR 和人 FoxA1（据信与 AR 相互作用的候选叉头家族成员）将与包含 ARE 加 FHRE 基序的 30 bp DNA 片段一起孵育 (18)。 AR:Fkhd 相互作用的协同作用将使用 DNase I 足迹滴定进行定量，然后用截短形式的 TF 重复，以确定哪些结构域负责蛋白质-蛋白质或变构相互作用。 4 bp 间隔区对相互作用的生物物理重要性将通过使用含有 0 至 8 bp 间隔区的 DNA 片段重复 EMSA 实验来评估。吡咯-咪唑 (Py-Im) 聚酰胺是序列特异性 DNA 小沟结合分子，将设计用于破坏 Fkhd:DNA 界面，以探测 AR:Fkhd 相互作用的体内作用。将使用 qRT-PCR、ChIP-Seq 和 RNA-Seq 在用雄激素处理的 LNCaP 细胞上测试破坏增强子或启动子区域中包含 ARE 加 FHRE 基序的基因中的 Fkhd:DNA 或 AR:DNA 相互作用的影响、设计的 Fkhd 聚酰胺和现有的 AR:DNA 破坏聚酰胺。 qRT-PCR 实验将用于测试聚酰胺有效破坏 Fkhd:DNA 和 ARE:DNA 相互作用的能力，并测试破坏 AR 或 Fkhd 会将 mRNA 表达恢复到基因基础水平的假设包含 ARE 加 FHRE 基序。为了了解 AR:Fkhd:DNA 相互作用对转录程序的全基因组作用，将在用聚酰胺处理的 LNCaP 细胞上进行 ChIP-Seq 和 RNA-Seq 实验，以破坏每个单独的相互作用。这种新型转录因子复合物的生物物理表征与使用聚酰胺的 AR:Fkhd 调节基因的全基因组表征相结合，可以揭示前列腺癌药物开发的新靶点。