The role of protein-protein interaction motifs in coordinating the DNA binding and regulatory specificity of Hox proteins

蛋白质-蛋白质相互作用基序在协调 Hox 蛋白 DNA 结合和调节特异性中的作用

• 批准号: 9788759
• 负责人: William Joseph Glassford
• 金额: $ 6.37万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788759
• 关键词: ATAC-seq; Abdomen; Address; Affect; Affinity; Animals; Anterior; Binding; Binding Proteins; Binding Sites; Biochemistry; Biological Assay; CRISPR/Cas technology; Cell Nucleus; Cells; ChIP-seq; Chromatin; Complex; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; Data Set; Development; Disease; Embryo; Enzymes; Epigenetic Process; Event; Evolution; Exhibits; Gene Expression; Genes; Genetic Transcription; Goals; HOX protein; Homeobox Genes; In Vitro; Knowledge; Ligands; Mediating; Minor Groove; Modeling; Modification; Molecular Conformation; Morphology; Mutate; N-terminal; Nuclear; Observational Study; Organism; Pattern; Play; Process; Protein Family; Protein Isoforms; Proteins; Role; Site; Specificity; Structure; Techniques; Testing; Tissue-Specific Gene Expression; Tissues; Transcriptional Regulation; Variant; Vertebrates; Work; arm; chromatin immunoprecipitation; chromatin remodeling; cofactor; experimental study; genome sequencing; genome-wide; homeodomain; in vivo; insight; monomer; mutant; preference; protein protein interaction; transcription factor; transcriptome sequencing

Project Summary: Transcription factors perform a key role in regulating gene expression, but our ability to interpret regulatory DNA by identifying transcription factor binding sites or by predicting regulatory function is lacking. One generally unappreciated contributing factor is that each transcription factor has the potential to interact with cofactors, sometimes in structurally distinct ways, thus adding to the ability of transcription factor complexes to bind distinct DNA sequences. Perhaps the best illustration of this idea is the Hox family of proteins, which pattern the anterior/posterior axis in all metazoans. When bound to the cofactor Extradenticle (Exd), Hox proteins exhibit latent differences in DNA-binding specificity necessary to carry out their unique activities. In addition, they exhibit paralogue specific binding by recognizing low-affinity DNA-binding sites, illustrating a fundamental tradeoff between DNA binding specificity and affinity. In this proposal, I will extend these observations by studying the role of protein-protein interaction (PPI) motifs that mediate the interaction between Hox proteins and their cofactors. The physical interaction between Hox and Exd, mediated by a canonical motif present in all Hox proteins, has been particularly-well studied. However, some Hox proteins contain additional motifs. These motifs may promote alternate Hox-Exd-DNA conformations, a potential mechanism underlying paralogue-specific activity. To test this hypothesis, combinations of Exd interaction motifs will be mutated in the endogenous loci of three Hox genes: Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B). ChIP-seq (Chromatin ImmunoPrecipitation) will be used to assess differences in in vivo DNA-binding profiles to identify motif-redundant and motif-specific binding sites. Next, differences in DNA- binding affinity between mutant Hox-Exd complexes will be determined by utilizing a version of SELEX-seq (Systematic Evolution of Ligands by Exponential Enrichment) modified to capture low affinity DNA-binding. Comparing the ChIP-seq and SELEX-seq datasets will generate a list of motif-specific sites driven to bind only by the interaction between Hox and Exd, and a list of binding events caused by other mechanisms. The second aim of this study is to evaluate the role of Exd interaction motifs in regulating the transcriptional potential of Hox proteins as Hox proteins can both activate and repress genes expression. To address this question, RNA-seq, ATAC-seq and epigenetic mark specific ChIP-seq will be performed on tissues that express Hox proteins with mutant Exd interaction motifs. The third aim of this study is to study how different isoforms of Homothorax (Hth), another Hox cofactor that is required for the nuclear localization of Exd, regulate Hox activity. Studying the mechanisms guiding Hox paralogue specificity and regulatory activity is important because of (1) the deep conservation of Hox genes in higher organisms, (2) other transcription factors exhibit the same phenomenon of low affinity DNA binding and (3) studying how transcription factors interact with DNA is important to interpreting the increasing number of regulatory loci associated with disease.

项目摘要：转录因子在调节基因表达中发挥着关键作用，但我们的能力 通过识别转录因子结合位点或预测调控功能来解释调控 DNA 缺乏。一个普遍不被重视的因素是每个转录因子都有可能 与辅因子相互作用，有时以结构不同的方式，从而增加转录因子的能力 复合物结合不同的DNA序列。也许这个想法的最好例证是 Hox 家族 蛋白质，在所有后生动物中形成前/后轴。当与辅助因子外齿结合时 (Exd)，Hox 蛋白在 DNA 结合特异性方面表现出潜在的差异，这是执行其独特功能所必需的 活动。此外，它们通过识别低亲和力 DNA 结合位点表现出旁系同源物特异性结合， 说明 DNA 结合特异性和亲和力之间的基本权衡。在这个提案中，我将扩展 通过研究介导相互作用的蛋白质-蛋白质相互作用（PPI）基序的作用来观察这些结果 Hox 蛋白及其辅助因子之间的关系。 Hox 和 Exd 之间的物理相互作用，由 所有 Hox 蛋白中存在的规范基序已得到特别深入的研究。然而，一些 Hox 蛋白 包含额外的主题。这些基序可能会促进替代的 Hox-Exd-DNA 构象，这是一种潜在的 旁系同源物特异性活动的机制。为了检验这个假设，Exd 相互作用的组合 基序将在三个 Hox 基因的内源位点发生突变：Ultrabithorax (Ubx)、abdominal-A (abd-A) 和腹部-B (Abd-B)。 ChIP-seq（染色质免疫沉淀）将用于评估 体内 DNA 结合图谱可识别基序冗余和基序特异性结合位点。接下来，DNA的差异—— 突变体 Hox-Exd 复合物之间的结合亲和力将通过使用 SELEX-seq 版本来确定 （指数富集配体的系统进化）经过修改以捕获低亲和力 DNA 结合。 比较 ChIP-seq 和 SELEX-seq 数据集将生成仅驱动结合的特定基序位点列表 由 Hox 和 Exd 之间的相互作用，以及由其他机制引起的一系列绑定事件。这 本研究的第二个目的是评估 Exd 相互作用基序在调节转录中的作用 Hox 蛋白的潜力 因为 Hox 蛋白既可以激活也可以抑制基因表达。为了解决这个问题 问题，RNA-seq、ATAC-seq 和表观遗传标记特异性 ChIP-seq 将在以下组织上进行 表达具有突变 Exd 相互作用基序的 Hox 蛋白。本研究的第三个目的是研究不同的 Homothorax (Hth) 的亚型是 Exd 核定位所需的另一种 Hox 辅因子，调节 霍克斯活动。研究指导 Hox 旁系同源物特异性和调节活性的机制很重要 由于 (1) Hox 基因在高等生物中的深度保守，(2) 其他转录因子表现出 低亲和力 DNA 结合的相同现象以及 (3) 研究转录因子如何与 DNA 相互作用 对于解释与疾病相关的越来越多的调控位点很重要。