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

项目概述：转录因子在调节基因表达中起着关键作用，但我们的能力， 通过鉴定转录因子结合位点或预测调节功能来解释调节DNA， 缺乏一个通常未被认识到的贡献因素是每个转录因子都有潜力， 与辅因子相互作用，有时在结构上不同的方式，从而增加了转录因子的能力， 复合物结合不同的DNA序列。也许最能说明这一想法的是霍克斯家族， 蛋白质，其在所有后生动物中形成前/后轴。当与辅因子结合时， (Exd)，Hox蛋白在DNA结合特异性方面表现出潜在的差异，这些差异是进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， 说明了DNA结合特异性和亲和力之间的基本权衡。在这份提案中，我将 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， Hox蛋白和它们的辅因子之间的联系Hox和Exd之间的物理相互作用，由一个 典型的基序存在于所有的Hox蛋白，已经特别好地研究。然而，一些Hox蛋白 包含额外的图案。这些基序可以促进交替的Hox-Exd-DNA构象，这是一种潜在的 旁系同源特异性活动的潜在机制。为了检验这一假设，Exd相互作用的组合 基序将在三个Hox基因的内源位点发生突变：超双胸（Ubx），腹部-A（abd-A） 和Abdominal-B（Abd-B）。ChIP-seq（染色质免疫沉淀）将用于评估 体内DNA结合谱，以鉴定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利用一种形式的SELEX-seq （通过指数富集的配体系统进化）修饰以捕获低亲和力DNA结合。 比较ChIP-seq和SELEX-seq数据集将生成仅结合的基序特异性位点列表 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的列表。的 本研究的第二个目的是评估Exd相互作用基序在调控转录中的作用， Hox蛋白具有激活和抑制基因表达的双重功能。为了解决这个 问题，RNA-seq，ATAC-seq和表观遗传标记特异性ChIP-seq将在 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 同型胸（Hth），另一种Hox辅因子，是Exd核定位所需的， Hox活动。研究Hox特异性和调节活性的机制是重要的 由于（1）Hox基因在高等生物中高度保守，（2）其他转录因子表现出 低亲和力DNA结合的相同现象和（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节位点是重要的。

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.