Elucidating the Role of Hox Transcription Factors in the Chromatin Context

许多转录因子在多种细胞类型中表达，但它们在不同的细胞环境中以最高的精度控制着不同的转录程序。这种广泛表达但具有特定作用的转录因子的一个最突出的例子是Hox蛋白。有趣的是，尽管Hox转录因子在许多细胞类型中都很活跃，但它可以重新编程细胞的区域身份，细胞的区域身份受到动物前后轴强烈的表观遗传控制。这一行为表明，Hox蛋白可以诱导染色质开放，使它们能够调整特定谱系的基因表达程序，以适应地区要求。然而，HOX蛋白在体内对局部染色质状态和构型的调节作用及其潜在的机制仍然知之甚少。基于我们最近的发现，即Hox Tf Ubx以一种细胞谱系受限的方式与梵天染色质重塑复合体的组件相互作用，以及Hox Tf与开放和封闭的染色质结合以在区域和谱系命运调控中发挥双重作用的事实，我们现在将探索Hox TFS开创染色质的可能性。我们假设HOX染色质的开创性依赖于与核小体上的低亲和力DNA基序的结合，并且通用细胞谱系计划通过与谱系受限的(先驱者)TF相互作用来调整以满足区域特定的要求。为了严格检验我们的假设，我们将解决HOX结合的方式、序列要求以及与其他(先驱)TF的功能相互作用。为此，我们将使用最先进的基因组技术，这将使我们能够同时探测TF染色质结合和体内局部染色质环境，而不需要结合部位预测，以及补充生化和遗传方法。我们将使用UBX及其谱系特异性相互作用伙伴TIN和GRH以及通用相互作用伙伴EXD作为模型，EXD已被证明可以增加HOX与封闭染色质的结合，并将使用体外、细胞和体内方法研究它们在染色质开放中的作用。总之，这项工作将有助于解决发育生物学中的一个主要难题，即Hox转录因子如何在高度特异地分配区域身份的同时履行细胞谱系稳定的一般功能。

Many transcription factors (TFs) are expressed in multiple cell types, yet they control distinct transcriptional programs with highest precision in different cellular contexts. One of the most prominent examples of such broadly expressed but specifically acting TFs are the Hox proteins. Intriguingly, Hox TFs, despite being active in many cell types, can “reprogram” the regional identity of cells, which is under strong epigenetic control along the anterior-posterior axis of animals. This behavior suggests that Hox proteins can induce chromatin opening, allowing them to adapt lineage-specific gene expression programs to the regional requirements. However, the role of Hox proteins in the regulation of local chromatin state and configuration in vivo and the underlying mechanisms are still poorly understood. Building on our recent findings that the Hox TF Ubx interacts in a cell lineage restricted fashion with components of the Brahma chromatin remodelling complex and the fact that Hox TFs bind to open and closed chromatin to carry out dual roles in regional and lineage fate regulation, we will now explore the possibility that Hox TFs pioneer chromatin. We hypothesize that Hox chromatin pioneering is dependent on binding to low-affinity DNA motifs on nucleosomes and that generic cell lineage programs are tuned to region-specific requirements by interacting with lineage-restricted (pioneer) TFs. In order to rigorously test our hypothesis, we will resolve the modalities of Hox binding, the sequence requirements and the functional interaction with other (pioneer) TFs. To this end, we will employ state of the art genomic techniques, which will allow us to simultaneously probe TF chromatin binding and the local chromatin environment in vivo, without the need of binding site predictions, as well as complementing biochemical and genetic approaches. We will use Ubx and its lineage-specific interaction partners Tin and Grh as well as the generic interaction partner Exd, which has been shown to increase Hox binding to closed chromatin, as models, and will study their role in chromatin opening using in vitro, in cellulo and in vivo approaches. In sum, this work will be instrumental in resolving one of the major puzzles in Developmental Biology, which is how Hox TFs can function highly specifically in assigning regional identity while fulfilling at the same time generic functions in cell lineage stabilization.