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

基本信息

批准号：
10396632
负责人：
Charles G Sagerstrom
金额：
$ 36.4万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10396632
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

项目摘要

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.

TALE 家族转录因子 (TF) 普遍表达，并作为几类转录因子的辅助因子胚胎发生和细胞稳态中必需的转录因子（最突出的是 Hox 蛋白），但它们的确切例如，两个家族成员——Prep 和 Meis——结合了相同的序列。基序在体外并具有与 Pbx 蛋白二聚化的能力，表明它们可以互换功能。然而，几个系统的功能丧失分析表明它们在体内的作用是不同的。第三个 TALE 家族成员 (TGIF) 与 Prep 和 Meis 具有相同的 DNA 结合基序偏好，但尚不清楚是否这三个 TF 在体内相互竞争或补偿，因为大多数已知的 TF 属于较大的 TF。共享 DNA 和蛋白质的家族：蛋白质相互作用特性，关于共享和蛋白质的类似问题不同的功能困扰着我们对大多数 TF 家族的理解。此外，由于体内 TF 功能受到影响。由于在体外没有遇到限制，因此有必要评估它们在本地系统中的功能特性。事实上，关于体内 TF 活性的许多基本问题仍有待解决。重要的是，具有相似体外结合选择性的 TF 在体内是否会选择不同的基序？其机制是什么？不同的体内结合选择性的基础以及它如何影响功能？将对我们对胚胎发生和疾病的理解产生深远的影响，但这一领域的进展具体来说，访问多个全基因组数据集受到了重大障碍的阻碍。相关的转录因子受到限制——特别是在胚胎发生过程中，此时细胞数量通常很小。通过为多个 TF 家族的 TALE 家族成员生成 ChIP-seq 和 RNA-seq 数据解决了这个问题斑马鱼发育阶段 - 建立可用的最全面的数据集之一对于单个 TF 家族，尽管包含几乎相同的同源域并且与无法区分的结合。在体外，我们发现 Prep 和 Meis TF 在体内表现出不同的结合偏好。 Meis，在体内占据了一个新的非 Hox 相关基因组元件。这些初步观察结果强调了这一点。在原生环境中探索 TF 功能的重要性，并强调强大的技术和能力根据我们的初步研究结果，我们的研究小组进行这些进一步分析的概念立场。我们发现体内出现的限制限制了 TALE TF 基序的选择性，并且动态 TALE 成员之间的交换控制转录结果。为了检验这一假设，我们首先将在斑马鱼中表达野生型和结构域交换的 TF 构建体，以定义 TALE TF 的机制基础其次，我们将使用功能获得和丧失的方法来操纵平衡。的 TALE TF，以便定义占用相同内容的不同 TALE TF 的功能后果最后，我们将使用功能丧失的动物和 CRISPR 介导的缺失来识别体内基因组结合位点。检查新的 TALE 占据基序的体内作用，因为 TALE TF 与多种癌症有关，我们对 TALE 家族成员之间动态相互作用的体内描述将直接影响我们的从概念上讲，我们的发现也适用于胚胎发生和肿瘤发生。其他同源域 TF（构成第二大类 TF）和其他多成员 TF 家族。