Organ-specific DNA-binding site selection and transcriptional regulation by floral homeotic transcription factors

了解转录因子如何与特定的DNA序列相互作用，对于了解它们如何控制基因表达，以及最终它们如何发挥生物学功能至关重要。在植物中，MADS结构域转录因子是花发育的主要调节因子，起着所谓的同源异型调节作用。它们的作用是确定四种不同类型的花器官--萼片、花瓣、雄蕊和心皮--来自分生组织干细胞。同源异型调节因子相互作用，从而形成器官特异的二聚体和高阶蛋白质复合体。每个蛋白质复合体可能具有不同的DNA结合特异性，这是由单个二聚体的DNA结合偏好、结合位点的优选间距、与非MADS辅助因子的相互作用以及DNA结构造成的。虽然我们以前使用SELEX-SEQ发现单个MADS二聚体的DNA结合特异性有助于花的同源异型复合体的DNA结合特异性，但其他决定DNA结合特异性的因素仍然知之甚少。然而，这对于理解植物花器官规范背后的顺式调控代码是至关重要的。在这个项目中，我建议从功能上表征同源异型调控子的复杂特异性DNA结合位点。我们将使用DAP-SEQ分析结合位点间距对高阶复合体DNA结合特异性的影响，并确定与决定DNA结合特异性有关的蛋白质结构域。我们还将研究与非MADS辅助因子相互作用对结合位点选择和调控特异性(靶基因的激活或抑制)的影响。在这里，我们将研究共抑制物复合体是如何招募到MADS转录因子结合的基因组区域的子集，以及不同的同源异源复合体如何使用(辅因子)芯片序列和遗传方法相结合的方式影响这种招募。在我们的分析中，我们将把新的结果与可用的组织特异性结合和染色质可及性数据结合起来。最终，我们将从功能上表征拟南芥基因组中负责花器官规范的DNA调控元件，这些元件直接位于同源异型调节器的下游。为此，我们将使用CRISPR-Cas9方法对MADS结构域转录因子结合位点进行定向突变，并研究突变植株的花型。此外，我们还将对新的目标基因进行详细的表达分析，这些基因被预测为花器官规范的关键目标。综上所述，该项目将结合几种最先进的技术来了解植物花器官规范所依据的顺式调控代码，并确定新的关键器官特异的同源异型调控靶基因。因此，本项目以花发育为模型系统，为确定植物干细胞发育规划的基因组基础提供了概念基础。

Unraveling how transcription factors interact with specific DNA sequences is crucial to understand how they control gene expression and ultimately how they exert their biological functions. In plants, MADS-domain transcription factors are prominent master regulators of flower development, acting as so-called homeotic regulators. Their role is to specify four different types of floral organs - sepals, petals, stamens and carpels - originating from meristematic stem cells. Homeotic regulators interact with each other, thereby forming organ-specific dimeric and higher-order protein complexes. Each protein complex potentially possesses a different DNA-binding specificity, resulting from DNA-binding preferences of individual dimers, preferred spacing of binding sites, interactions with non-MADS cofactors as well as DNA structure. While we previously found using SELEX-seq that DNA-binding specificities of individual MADS dimers contribute to DNA-binding specificity of floral homeotic complexes, other major determinants of DNA-binding specificity are still poorly understood. This is however crucial to understand the cis-regulatory code underlying floral organ specification in plants.In this project, I propose to functionally characterize complex-specific DNA binding sites of homeotic regulators. We will analyze the impact of binding site spacing on DNA-binding specificity of higher-order complexes using DAP-seq, and identify protein domains involved in determining DNA-binding specificity. We will also investigate the impact of interactions with NonMADS co-factors on binding site selection and regulatory specificity (activation or repression of target genes). Here, we will study how co-repressor complexes are recruited to a subset of MADS transcription factor-bound genomic regions, and how different homeotic complexes affect this recruitment using a combination of (cofactor) ChIP-seq and genetic approaches. In our analysis, we will integrate novel results with available tissue-specific binding and chromatin accessibility data. Ultimately, we will functionally characterize DNA regulatory elements in the Arabidopsis genome responsible for floral organ specification directly downstream of the homeotic regulators. For this, we will employ the CRISPR-Cas9 methodology for targeted mutagenesis of MADS-domain transcription factor binding sites and investigate flower phenotypes of the mutant plants. In addition, we will perform detailed expression analyses of novel target genes that are predicted key targets for floral organ specification. In summary, this project will combine several state-of-the art technologies to understand the cis-regulatory code underlying specification of floral organs in plants, and to determine novel key organ-specific target genes of homeotic regulators. Therefore, this project offers a conceptual basis for determining the genomic basis of developmental programming of plant stem cells, using flower development as a model system.