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

• 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
• 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

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。所有人类癌症的生存，生长和转移行为可能需要一个或多个转录因子（TF）的过度供应或过度活性，因此提供了一个有吸引力的治疗靶标。 NCI估计，仅在美国，将被诊断出217,730名男性，32,050名男性将在2010年死于前列腺癌（6）。雄激素受体（AR）是导致前列腺癌的表达变化的异常TF。关于雄激素处理的LNCAP细胞（前列腺癌细胞系）的初步CHIP-SEQ数据显示了一个新的基序序列，雄激素响应元件（AS）加上叉子（FKHD）响应元件（FHRE），由4个碱基对（BPS）分离，相对于背景基因组数据富集。这部小说是Plus FHRE基序提示AR和叉子TF的DNA共占领，以调节前所未有的政权中的特定基因。叉状TFs是许多与癌症相关的细胞功能不可或缺的一部分，例如代谢，发育，增殖和凋亡，因此对于理解前列腺癌可能很重要（17）。为了证明AR和FKHD的同居率是可以使用电泳迁移率转移测定（EMSA）。重组人AR和人类FOXA1（据信与AR相互作用的候选叉子家族成员）将与30 bp的DNA片段孵育，其中包含res fhre基序（18）。 AR：FKHD相互作用的合作性将使用DNASE I足迹滴定进行定量，然后以TF的截断形式重复，以确定哪些域是负责蛋白质蛋白质或变构相互作用的原因。 4 bp间隔物对相互作用的生物物理重要性将通过重复EMSA实验，其中包含含有0至8 bps的隔离剂的DNA片段的EMSA实验。吡咯-咪唑（PY-IM）聚酰胺，序列特异性DNA次要结合分子将被设计为破坏FKHD：DNA界面，以探测AR：FKHD相互作用的体内作用。破坏FKHD：DNA或AR的效果：包含该基因中的DNA相互作用在增强子或启动子区域中的DNA相互作用将使用QRT-PCR，ChIP-Seq和RNA-Seq在LNCAP细胞上使用Androgen处理，设计的FKHD Polodamides和现有的polyamides进行测试。 QRT-PCR实验将用于测试聚酰胺有效破坏FKHD：DNA的能力：DNA和：DNA相互作用，并检验了一个假说，即AR或FKHD的破坏将将mRNA表达恢复到包含基因的基础水平，其中包含含有的基础水平。为了理解AR：FKHD：DNA相互作用在转录程序上的宽泛作用，将对用聚酰胺处理的LNCAP细胞进行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结合了使用聚酰胺调节的AR：FKHD的基因的全基因组表征，可以揭示前列腺癌药物发育的新靶标。