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起关键作用，但我们的能力 通过鉴定转录因子结合位点或通过预测调节功能来解释调节性DNA 缺乏。一个通常没有批准的促成因素是每个转录因子都有潜力 与辅因子相互作用，有时在结构上不同，从而增加了转录因子的能力 复合物结合不同的DNA序列。也许这个想法的最好的例证是Hox家族 蛋白质，在所有后生动物中对前轴进行模拟。当结合到辅因子内外 （EXD），HOX蛋白在DNA结合特异性上表现出潜在的差异，以执行其独特 活动。此外，它们通过识别低亲和力DNA结合位点，表现出旁程特异性结合， 说明了DNA结合特异性与亲和力之间的基本权衡。在此提案中，我将扩展 这些观察通过研究介导相互作用的蛋白质蛋白相互作用（PPI）的作用 在HOX蛋白质及其辅因子之间。 HOX和EXD之间的物理相互作用，由A介导 所有HOX蛋白质中存在的规范基序都被特别研究了。但是，一些HOX蛋白 包含其他主题。这些基序可能会促进交替的HOX-EXD-DNA构象，一个潜力 寄生虫特异性活动的基础机制。为了检验这一假设，EXD相互作用的组合 基序将在三个HOX基因的内源基因座突变：Ultrabithorax（UBX），腹部A（ABD-A） 和腹部B（ABD-B）。 CHIP-SEQ（染色质免疫沉淀）将用于评估IN的差异 体内DNA结合曲线以识别基序冗余和基序特异性结合位点。接下来，DNA- 突变体HOX-EXD复合物之间的绑定亲和力将通过使用SELEX-SEQ版本来确定 （通过指数富集对配体的系统演变）修改以捕获低亲和力DNA结合。 比较chip-seq和selex-seq数据集将生成一个仅绑定的特定于特定位点的列表 通过HOX和EXD之间的相互作用，以及由其他机制引起的结合事件列表。这 这项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蛋白作为HOX蛋白的潜力既可以激活和抑制基因表达。解决这个问题 问题，RNA-seq，atac-seq和表观遗传标记的特定芯片seq将在组织上进行 具有突变EXD相互作用基序的表达HOX蛋白。这项研究的第三个目的是研究如何不同 同胸（HTH）的同工型，这是EXD核定位所需的另一个HOX辅助因子 HOX活动。研究指导HOX旁他特异性和调节活动的机制很重要 由于（1）较高生物体中HOX基因的深度保护，（2）其他转录因子表现出 低亲和力DNA结合的相同现象和（3）研究转录因子如何与DNA相互作用 解释与疾病相关的调节基因座数量增加很重要。