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

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）的过度供应或过度活性可能是所有人类癌症的生存、生长和转移行为所必需的，因此提供了一个有吸引力的治疗靶点。NCI估计，2010年仅在美国就有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）是导致前列腺癌的异常TF。对雄激素处理的前列腺癌细胞LNCaP细胞的ChIP-Seq初步数据显示了一个新的基序序列，雄激素反应元件（ARE）和叉头反应元件（Fkhd）相隔4个碱基对（bps），相对于背景基因组数据丰富。这种新颖的IS + FHRE基序提示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的组成部分，如代谢、发育、增殖和凋亡，因此可能对了解前列腺癌很重要（17）。为了证明AR和Fkhd的共存是可能的，将采用电泳迁移率转移试验（EMSA）。重组人AR和被认为与AR相互作用的候选叉头家族成员FoxA1将与含有ARE + FHRE基序的30 bp DNA片段一起孵育（18）。AR:Fkhd相互作用的合作将使用DNase I足迹滴定法进行定量，然后用截断形式的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间隔对相互作用的生物物理重要性将通过使用含有0至8 bps间隔的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小槽结合分子，将被设计用于破坏Fkhd:DNA界面，以探索AR:Fkhd相互作用的体内作用。在使用雄激素、设计的Fkhd聚酰胺和现有的AR:DNA破坏聚酰胺处理的LNCaP细胞上，将使用qRT-PCR、ChIP-Seq和RNA-Seq来测试在增强子或启动子区域中含有ARE + FHRE基序的基因中破坏Fkhd:DNA或AR:DNA相互作用的影响。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，以及测试AR或Fkhd的破坏将使含有ARE + FHRE基序的基因的mRNA表达恢复到基础水平的假设。为了了解AR:Fkhd:DNA相互作用在转录程序中的全基因组作用，ChIP-Seq和RNA-Seq实验将在聚酰胺处理的LNCaP细胞上进行，以破坏每个个体的相互作用。利用聚酰胺对这种新型转录因子复合物进行生物物理表征，并结合AR:Fkhd调控基因的全基因组表征，可以揭示前列腺癌药物开发的新靶点。

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.