Collaborative Research: The Epigenetic Regulatory Role of the Maize Ufo1 Gene

智力价值：允许形式和功能多样化的机制之一是表观遗传调控。表观遗传变化是指影响基因表达的染色质DNA或蛋白质成分的变化。表观遗传修饰在植物界中广泛存在，但很少被分离或鉴定，因为相关的表型不容易看到，而且这些修饰在一代又一代中不稳定。利用玉米果皮颜色1(P1)基因及其红色着色作为标记，p1基因座上的等位基因变异被证明是基因结构和组织的函数，并进一步受到表观遗传机制的调节。为了了解基因表达的表观遗传调控所调控的等位基因多样性的基础，我们对一种名为Ufo1(橙色不稳定因子)的显性突变进行了分子和遗传特征分析。含有一种名为P1-WR的特殊p1等位基因的植物，会产生果皮无色、果穗呈红色的果穗，这种沉默的果皮表型是稳定的；然而，在Ufo1存在的情况下，P1-WR等位基因在几个组织中表现出不同程度的增色表型。表达研究表明，色素沉着的增加与P1转录本的稳定水平增加有关。在P1-WR；Ufo1植株中P1的表达增强与P1-WR等位基因启动子和编码序列上DNA甲基化的减少有关。在本项目中，将进行ufo1基因的图位克隆。利用转座子诱变将产生ufo1的新等位基因。将使用高通量测序方法对Ufo1突变植物和野生型植物的染色质、转录本(mRNA和小RNA)和DNA甲基化进行全球图谱分析，以分析Ufo1介导的表观遗传调控效应的证据。将开发一个数据库，以可视化和分析CHIP-SEQ、RNA-SEQ和亚硫酸氢盐测序产生的数据。这些结果将确定Ufo1突变(和野生型ufo1基因)在影响转座子、DNA甲基化、组蛋白修饰和RNA图谱的全球变化中的作用。由于Ufo1在玉米中的作用似乎与其他已知的表观遗传调控因子不同，该项目将扩大对重叠的表观遗传途径如何调控这种重要作物基因表达的理解。广泛影响：该项目将提供植物表观遗传学的培训机会和植物基因的等位基因变异研究。宾夕法尼亚州立大学和特拉华大学之间的合作将加强植物生物学和生物信息学领域的专业互动。具体地说，两名博士后研究员和两名研究生将接受计算生物学方面和表观基因调控方面的交叉培训。本科生，包括女性和少数族裔学生，将学习经典和尖端的植物生物学技术，了解并剖析组织特异性基因表达调控的分子基础。此外，高中学生和老师将参加一个夏季生物技术研讨会，学习玉米的基因表达技术。该项目由分子和细胞生物科学部的遗传机制计划和综合组织系统部的植物基因组研究计划共同资助。

Intellectual Merit: One of the mechanisms that allow diversification of form and function is epigenetic regulation. Epigenetic changes are alterations in the DNA or protein components of chromatin that affect gene expression. Epigenetic modifications that are of transient nature are widespread in the plant kingdom, but are rarely isolated or identified because the associated phenotypes are not easy to see and the modifications are not stable from generation to generation. Using the pericarp color1 (p1) gene and its red pigmentation as a marker in maize, allelic variation at the p1 locus has been shown to be a function of gene structure and organization, which is further regulated by epigenetic mechanisms. To understand the basis of allelic diversity modulated through epigenetic regulation of gene expression, molecular and genetic characterization of a dominant mutation called Ufo1 (Unstable factor for orange1) has been performed. Plants with a particular p1 allele, called P1-wr, produce ears with colorless kernel pericarps and red cob glumes, and this silent pericarp phenotype is stable; however, in the presence of Ufo1 the P1-wr allele confers variably enhanced pigmentation phenotypes in several tissues. Expression studies demonstrated that the gain of pigmentation is associated with increased steady state levels of P1 transcript. Enhanced P1 expression in P1-wr;Ufo1 plants was shown to correlate with a decrease in DNA methylation at the promoter and coding sequence of the P1-wr allele. In the current project, map based cloning of ufo1 gene will be carried out. New alleles of ufo1 will be generated using transposon mutagenesis. Evidence for Ufo1-mediated effects on epigenetic regulation will be analyzed using high throughput sequencing methods for global profiling of chromatin, transcript (mRNA and small RNA), and DNA methylation of Ufo1 mutant and wild type plants. A database will be developed to visualize and analyze data generated from ChIP-seq, RNA-seq and bisulfite sequencing. The results will establish the role of the Ufo1 mutation (and wild type ufo1 gene) in effecting global changes in transposons, DNA methylation, histone modifications and RNA profiles. Because Ufo1 does not appear to act in the same way as other known epigenetic regulators in maize, this project will expand understanding of how overlapping epigenetic pathways regulate gene expression in this important crop plant.Broader Impacts: This project will provide training opportunities in plant epigenetics and study of allelic variation of plant genes. The collaboration between Pennsylvania State University and the University Delaware will enhance professional interactions in the area of plant biology and bioinformatics. Specifically, two postdoctoral fellows and two graduate students will be cross-trained in computational biological aspects and epigenetic gene regulation. Undergraduate students, including women and minority students, will learn classical and cutting-edge plant biology techniques that are used understand and dissect the molecular basis of regulation of tissue-specific gene expression. In addition, high school students and teachers will participate in a summer biotechnology workshop to learn gene expression techniques in maize.This project is co-funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences and by the Plant Genome Research Program in the Division of Integrative Organismal Systems.