In vivo motif selectivity and functionality of TALE family TFs

TALE 家族 TF 的体内基序选择性和功能

• 批准号: 10583395
• 负责人: Charles G Sagerstrom
• 金额: $ 6.85万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10583395
• 关键词: Address; Affect; Animals; Area; Binding; Binding Proteins; Binding Sites; Biological Process; Cell Count; ChIP-seq; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Abnormality; DNA; DNA Binding; Data; Data Set; Disease; Elements; Embryo; Embryonic Development; Environment; Equilibrium; Family; Family member; Gene Expression; Gene Structure; Genes; Genetic Transcription; Genomics; HOX protein; Homeostasis; In Vitro; Malignant Neoplasms; Mediating; Mutation; Oogenesis; Outcome; Output; Phenotype; Play; Positioning Attribute; Preparation; Property; Proteins; Reporting; Repression; Research; Role; Site; System; Testing; Transcriptional Regulation; Work; Zebrafish; base; cancer type; cofactor; dimer; experimental study; functional outcomes; genome-wide; homeodomain; human disease; in vivo; insight; loss of function; member; novel; preference; programs; protein protein interaction; transcription factor; transcriptome sequencing; tumor progression; tumorigenesis; zebrafish development

TALE family transcription factors (TFs) are ubiquitously expressed and act as cofactors to several classes of essential TFs – most prominently Hox proteins – in embryogenesis and cellular homeostasis, but their exact functions remain enigmatic. For instance, two family members – Prep and Meis – bind identical sequence motifs in vitro and share the ability to dimerize with Pbx proteins, suggesting that they function interchangeably. However, loss-of-function analyses in several systems suggest that their roles diverge in vivo. Furthermore, a third TALE family member (TGIF) shares DNA binding motif preference with Prep and Meis, but it is unclear if these three TFs compete or compensate for each other in vivo. Because most known TFs belong to larger families that share DNA and protein:protein interaction properties, similar questions about shared and divergent functions vex our understanding of most TF families. Further, since TF function in vivo is subject to constraints not encountered in vitro, it is essential to evaluate their functional properties in native systems. Indeed, numerous fundamental questions about TF activity remain to be addressed in vivo. Perhaps most importantly, do TFs with similar in vitro binding selectivity select distinct motifs in vivo? What is the mechanistic basis of divergent in vivo binding selectivity and how does it impact function? The answers to these questions will have profound implications for our understanding of embryogenesis and disease, but progress in this area has been hampered by major barriers. Specifically, access to multiple genome-wide data sets for closely related TFs has been limited – particularly during embryogenesis, when cell numbers are often small. We have addressed this by generating ChIP-seq and RNA-seq data for members of the TALE family of TFs at multiple stages of zebrafish development – thereby establishing one of the most comprehensive sets of data available for a single TF family. In spite of containing near-identical homeodomains and binding to indistinguishable motifs in vitro, we find that Prep and Meis TFs display divergent binding preferences in vivo. Also, Prep, but not Meis, occupies a novel non-Hox related genomic element in vivo. These initial observations underscore the importance of exploring TF function in their native environment and highlight the strong technical and conceptual position of our research group to pursue these analyses further. Based on our preliminary findings, we hypothesize that emergent in vivo constraints restrict TALE TF motif selectivity and that dynamic exchange among TALE members controls transcriptional outcome. To test this hypothesis, we will first express wild-type and domain-swapped TF constructs in zebrafish to define the mechanistic basis of TALE TF binding selectivity in vivo. Second, we will use gain- and loss-of-function approaches to manipulate the balance of TALE TFs in order to define the functional consequences of different TALE TFs occupying the same genomic binding sites in vivo. Lastly, we will use loss-of-function animals and CRISPR-mediated deletions to examine the in vivo role for a novel TALE-occupied motif. Since TALE TFs are implicated in several cancers, our in vivo delineation of the dynamic interplay between TALE family members will directly impact our understanding of both embryogenesis and oncogenesis. Conceptually, our findings will also be applicable to other homeodomain TFs (constituting the 2nd largest class of TFs) and other multi-member TF families.

故事家庭转录因子（TFS）普遍表达，并充当几类的辅助因子 必需的TF（最突出的HOX蛋白）在胚胎发生和细胞稳态中，但它们的确切 功能仍然神秘。例如，两个家庭成员 - 准备和梅斯 - 绑定相同的顺序 在体外基序，并具有与PBX蛋白二聚体的能力，表明它们可以互换起作用。 但是，在几种系统中的功能丧失分析表明，它们的角色在体内差异。此外， 第三个故事家庭成员（TGIF）与Prep和Meis分享了DNA绑定基序的偏好，但目前尚不清楚是否存在 这三个TF在体内竞争或相互补偿。因为大多数已知的TF属于较大的TF 共享DNA和蛋白质的家庭：蛋白质相互作用的特性，关于共享和 发散的功能使我们对大多数TF家庭的理解感到厌烦。此外，由于体内的TF功能受到 在体外没有遇到的约束，评估其在天然系统中的功能特性至关重要。 确实，关于TF活动的许多基本问题仍有待在体内待解决。也许最大 重要的是，具有相似体外结合选择性的TF是否在体内选择不同的基序？什么是机械 在体内结合选择性的基础上，它如何影响函数？这些问题的答案 对我们对胚胎发生和疾病的理解将具有深远的影响，但是在这一领域的进展 受到重大障碍的阻碍。具体而言，访问多个全基因组数据集以密切 相关的TF受到限制 - 尤其是在胚胎发生期间，当细胞数通常很小时。我们有 通过为TFS的故事家族的成员生成chip-seq和RNA-seq数据来解决此问题。 斑马鱼开发的阶段 - 从而建立了最全面的数据集之一 对于一个单一的TF家庭。尽管含有几乎相同的同源固定酶，并且与无法区分的结合 在体外的基序，我们发现PrEP和MEIS TFS在体内显示出不同的结合偏好。另外，准备，但没有 Meis在体内占据了一种新型的非Hox相关基因组元素。这些最初的观察结果强调了 在其本地环境中探索TF功能的重要性，并强调强大的技术和 我们研究小组的概念立场进一步追求这些分析。根据我们的初步发现 我们假设在体内限制中出现的出现限制了TF TF基序的选择性和动态 故事成员之间的交流控制转录结果。为了检验这一假设，我们将首先 在斑马鱼中表达野生型和域折叠的TF构建体，以定义故事TF的机械基础 在体内结合选择性。其次，我们将使用功能丧失方法来操纵平衡 故事tfs的故事，以定义占据相同的不同故事的功能后果 体内基因组结合位点。最后，我们将使用功能丧失的动物和CRISPR介导的删除 检查一个新的故事占领的基序的体内作用。由于在几种癌症中实现了故事TFS，因此 我们的体内描述故事家庭成员之间动态相互作用将直接影响我们的 对胚胎发生和肿瘤发生的理解。从概念上讲，我们的发现也适用于 其他同源域TF（构成第二大的TFS级）和其他多人TF家庭。