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 已经根据这些数据进行了推断，从而得出了 TF 作为主要监管者的模型 或接近监管层级的顶部，而不是谱系或条件特定的转录因子，这些转录因子是 这些监管机构的下游。然而，这些方法都没有直接识别哪些 因子参与难以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，可被大多数 TF 结合 细胞，通过与核小体 DNA 稳定结合（“先锋结合”），从而增加 DNA 染色质的酶促可及性（“先锋活性”），允许顺序招募 其他转录因子位于非活性染色质上。通过启动顺式调控元件进行后续 转录调控活性，先锋因子充当细胞的“看门人” 差异化。 尽管先驱因素很重要，但人们对它们知之甚少，而且只有少数几个因素被研究过。 特点。表征先锋因素的一个主要障碍是缺乏稳健的、高 通量功能测定。在这个项目中，我们将开发一项新技术，称为 Pioneer Interactions On Nucleosomal Engineered Arrays (PIONEAR)，用于高通量 先锋结合的表征。我们将使用 PIONEAR 检测来调查先驱者 数十个人类 TF 的相互作用。 PIONEAR 检测鉴定出的 TF 具有先锋地位 将在体内评估结合，以在更广泛的范围内检查染色质解压缩。 鉴定表现出先锋活性的转录因子可能会导致定向细胞方面的突破 分化和重编程以及针对癌症的改进疗法的开发 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.