Genome wide views of chromatin occupancy in the control of Aquilegia development

描述（由申请人提供）：繁殖时间是植物发育的关键事件，由精细的遗传网络控制。为了调节开花时间，植物解释了多种信号，这些信号汇聚在茎尖上的多个遗传途径，在茎尖上实现了生殖过渡2,3。这些途径包括激素信号、白昼长度感知、发育阶段和资源可用性、环境温度、光质量和寒冷通道或春化2,3。这种转变的遗传基础已经在拟南芥和单叶草2-4中的几种草种中得到了很好的表征。作为研究开花时间遗传基础的一个系统，Aquilegia具有明显的优势，尤其是在其进化和生态学方面5-6。在开花植物中，拟南芥在系统发育上介于拟南芥和禾草之间，为深入的系统发育比较提供了关键的第三个数据点。与此同时，水仙属在最近才开始多样化，产生了几十个具有不同表型的物种，它们广泛分布在不同的环境中，物种之间的序列多样性非常低。因此，Aquilegia代表了一种模型适应辐射。低序列多样性加上大的表型变异，不仅极大地促进了适应性性状的遗传鉴定，而且为基因网络的功能研究提供了丰富的环境。该项目旨在通过利用我们对当前模型系统开花时间的了解，更好地理解转录因子网络的连通性。首先，它旨在对Aquilegia的生殖转变进行全基因组调查，以更好地了解控制这种适应性状的网络。通过染色质免疫沉淀结合深度测序（ChIP-seq）对控制生殖过渡的染色质状态变化进行全基因组鉴定，并通过转录组测序评估其结果。接下来，将通过病毒诱导基因沉默敲除、生化保护试验和原位杂交等功能分析，对拟南芥开花时间控制相关基因的遗传和生化功能、开花位点T （FT）、FD和LEAFY （LFY）的同源拟南芥同源基因进行鉴定。最后，该项目将利用关键整合子蛋白LFY的最新全基因组转录因子结合图谱，暗示数千个位点作为直接转录靶点。该项目将通过对Aquilegia属以及更远的亲缘物种进行全基因组LFY ChIP-seq分析，筛选出对LFY功能至关重要的保守的体内结合位点，并进行微观和宏观进化比较。综上所述，本研究的目的是利用现有的知识，利用广泛分类群的物种优势，对Aquilegia属适应性辐射模型中的进化重要性状进行功能研究，并解决中心转录因子遗传连通性的基本问题。！

DESCRIPTION (provided by applicant): The timing of reproduction is a critical event in plant development and is controlled by a refined genetic network. To regulate flowering time, plants interpret a variety of signals that converge from multiple genetic pathways at the shoot apex where the reproductive transition is effected2,3. These pathways include hormone signaling, perception of day length, developmental phase and resource availability, ambient temperature, light quality, and the passage of cold, or vernalization2,3. The genetic basis for this transition has been well characterized in the dicot Arabidopsis and several grass species in the monocots2-4. The basal dicot model genus Aquilegia has clear benefits as a system for studying the genetic basis of flowering time, most notably aspects of its evolution and ecology5-6. Within flowering plants, Aquilegia is phylogenetically intermediate between Arabidopsis and the grasses, providing a critical third data point for deep phylogenetic comparisons. At the same time, the genus Aquilegia has diversified very recently, leading to several dozen phenotypically distinct species that are broadly distributed in different environments, with very low sequence diversity between species7. Thus Aquilegia represents a model adaptive radiation. The low sequence diversity coupled with large phenotypic variation not only promises to greatly facilitate the genetic identification of adaptive traits, but also to provide a rich setting for the functional study of gene networks. This project aims to better understand the connectivity of transcription factor networks by leveraging our knowledge of flowering time in current model systems. First, it aims to perform a genome-wide interrogation of the reproductive transition in Aquilegia to better understand the network controlling this adaptive trait. Genome-wide identification of chromatin state changes implicated in governing the reproductive transition will be mapped with chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) and their output will be assessed with transcriptome sequencing. Next, the genetic and biochemical functions of genes implicated in flowering time control, the Aquilegia homologs of FLOWERING LOCUS T (FT), FD, and LEAFY (LFY) will be characterized by functional assays including knockdown by viral induced gene silencing and tests of biochemical conservation in Arabidopsis, and in situ hybridization. Finally, this project will leverage very recent genome- wide transcription factor binding maps of the key integrator protein LFY, implicating thousands of loci as direct transcriptional targets. This project will winnow out the conserved in vivo binding sites that are critical for LFY function by performing genome-wide LFY ChIP-seq assays in the Aquilegia genus in addition to more distantly related species for both micro- and macro-evolutionary comparisons. In summary, the aim is to perform functional studies of evolutionary important traits in the adaptive radiation model of the Aquilegia genus, and to address basic questions of genetic connectivity of central transcription factors by leveraging current knowledge of this network and taking advantage of species across a wide range of taxa. !