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

花同源异型转录因子的器官特异性 DNA 结合位点选择和转录调控

• 批准号: 408478570
• 负责人: Dr. Cezary Smaczniak
• 金额: --
• 依托单位: Lehrstuhl für Pflanzliche Zell- und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/408478570?language=en
• 关键词: Organ; specific; DNA; binding; site

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.

揭示转录因子如何与特定的DNA序列相互作用对于理解它们如何控制基因表达以及最终如何发挥其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，充当所谓的同源异型调节因子。它们的作用是指定四种不同类型的花器官-萼片，花瓣，雄蕊和心皮-起源于分生组织干细胞。同源异型调节剂相互作用，从而形成器官特异性二聚体和高阶蛋白质复合物。每种蛋白质复合物可能具有不同的DNA结合特异性，这是由个体二聚体的DNA结合偏好、结合位点的优选间隔、与非MADS辅因子的相互作用以及DNA结构引起的。虽然我们以前发现使用SELEX-seq，个别MADS二聚体的DNA结合特异性有助于花同源异型复合物的DNA结合特异性，DNA结合特异性的其他主要决定因素仍然知之甚少。然而，这是至关重要的了解顺式调控代码的花器官specification.In这个项目中，我建议功能特性复杂的特异性DNA结合位点的同源异型调节。我们将使用DAP-seq分析结合位点间距对高阶复合物的DNA结合特异性的影响，并确定参与确定DNA结合特异性的蛋白质结构域。我们还将研究与非MADS辅因子的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，我们将研究辅阻遏物复合物如何被招募到MADS转录因子结合的基因组区域的子集，以及不同的同源异型复合物如何使用（辅因子）ChIP-seq和遗传方法的组合影响这种招募。在我们的分析中，我们将整合新的结果与可用的组织特异性结合和染色质可及性数据。最终，我们将功能性地表征DNA调控元件在拟南芥基因组负责花器官规格直接下游的同源异型调节。为此，我们将采用CRISPR-Cas9方法对MADS结构域转录因子结合位点进行靶向诱变，并研究突变植物的花表型。此外，我们将进行详细的表达分析的新的目标基因，预测花器官规格的关键目标。综上所述，本项目将联合收割机的几个国家的最先进的技术来了解植物花器官的顺式调控密码子的特化，并确定新的同源异型调节的关键器官特异性靶基因。因此，该项目提供了一个概念基础，确定植物干细胞发育编程的基因组基础，使用花发育作为模型系统。