Administrative Supplement: Surveying transcription factor pioneer interactions with nucleosomal DNA

许多研究都集中在对基因组“部分列表”进行编目，包括转录本， 转录因子（TF）结合位点和染色质状态。转录调控网络 基于这些数据进行了推断，从而得出了TF是主调节器的模型， 或接近监管层级的顶部，而谱系或条件特异性TF， 这些监管机构的下游。然而，这些方法都没有直接识别出 这些因子参与不可接近的染色质以启动转录调控级联。 先锋因子是染色质可及性的关键，用于与大多数转录因子结合， 细胞，通过稳定地结合到核小体DNA（“先锋结合”），从而增加DNA 染色质的酶促可及性（“先锋活性”），允许顺序募集 其他转录因子在非活性染色质上的表达。通过启动顺式调节元件， 转录调节活性，先锋因子作为细胞的“看门人”， 分化 尽管它们很重要，但人们对先驱因素知之甚少，只有少数几个 表征了对先锋因素进行定性的一个主要障碍是缺乏一个强有力的、高- 通量功能测定。在这个项目中，我们将开发一种新技术，称为先锋 用于高通量的核小体工程化阵列（PIONRAYS）上的相互作用 先锋绑定的特性。我们将使用PIONARY分析来调查先驱者 数十种人类TF的相互作用。通过PIONELINE试验鉴定的TF表现出先锋 将在体内评价结合以在更广泛的范围内检查染色质解压缩。 鉴定具有先锋活性的TF可能会导致定向细胞凋亡的突破。 分化和重编程以及开发针对癌症的改进疗法 干细胞

Numerous studies have focused on cataloging the genome `parts list', including transcripts, transcription factor (TF) binding sites, and chromatin states. Transcriptional regulatory networks have been inferred based on these data, leading to models of what TFs are master regulators at or near the top of the regulatory hierarchy versus lineage- or condition-specific TFs, which are downstream of those regulators. However, none of these approaches directly identifies which factors engage inaccessible chromatin to initiate the transcriptional regulatory cascades. Pioneer factors serve as keys to chromatin accessibility for binding by the majority of TFs in a cell, by binding stably to nucleosomal DNA (`pioneer binding') and thus increase the DNA enzymatic accessibility of the chromatin (`pioneer activity'), allowing the sequential recruitment of other TFs on inactive chromatin. By priming cis regulatory elements for subsequent transcriptional regulatory activity, pioneer factors serve as `gatekeepers' to cellular differentiation. Despite their importance, little is known about pioneer factors, and only a handful have been characterized. A major hurdle in characterization of pioneer factors is the lack of a robust, high- throughput functional assay. In this project, we will develop a new technology, termed Pioneer Interactions On Nucleosomal Engineered ARrays (PIONEAR), for high-throughput characterization of pioneer binding. We will use PIONEAR assays to survey the pioneer interactions of dozens of human TFs. TFs identified by PIONEAR assays to exhibit pioneer binding will be evaluated in vivo to examine chromatin decompaction on a broader scale. Identification of TFs that exhibit pioneer activity may lead to breakthroughs in directed cellular differentiation and reprogramming and the development of improved therapies to target cancer stem cells.