Functional Interplay of Chromatin Remodellers and Histone Modifiers in Plants

在包括植物在内的高等生物中，DNA高度折叠成称为染色质的DNA-蛋白质（主要是组蛋白）复合物。染色质水平的基因表达调控，通常被称为表观遗传调控，是由两种酶复合物的联合活性介导的，所述酶复合物催化共价组蛋白修饰并改变染色质结构。Polycomb组（PcG）和Trithorax组（TrxG）蛋白是分别介导表观遗传基因抑制和激活的进化上保守的蛋白。PcG蛋白形成多聚体蛋白复合物，包括多梳抑制复合物2（PRC 2），其催化组蛋白3（H3 K27 me 3）的赖氨酸27的抑制性三甲基化。TrxG蛋白在生物化学上是异质的，包括模式植物拟南芥中的染色质重塑剂BRM和H3 K27 me 3脱甲基酶（擦除器）REF 6，但都具有拮抗Polycomb活性的功能。破译这两组蛋白质如何在全基因组范围内拮抗，以及它们如何靶向特定的基因组位点是非常有趣的。使用尖端的基因组学技术，如染色质免疫沉淀，然后进行高通量测序（ChIP-seq），我们最近研究了BRM，REF 6和PRC 2之间的全基因组功能相互作用。我们首先研究了BRM和PRC 2在调节H3 K27 me 3水平中的相互作用，并确定了这两种蛋白在控制开花时间中的关键作用。然后我们研究了BRM和REF 6之间的相互作用，发现REF 6通过识别特定的DNA基序（CTCTGYTY，Y代表C或T）直接靶向基因组位点，然后促进BRM的募集。这项工作为植物表观遗传学研究提供了一个新的范例。在这里，我们建议继续研究BRM/REF 6和PcG蛋白之间的功能相互作用。首先，我们将通过研究BRM的同源物SYD在调节PRC 2功能中的作用来扩展我们以前关于BRM-PRC 2相互作用的工作。我们还将分析SYD的全基因组占有率，并确定BRM和SYD的独特和共同靶点。其次，我们建议进一步研究REF 6的基因组靶向机制。我们将进行一系列的实验，以确定染色质的背景和/或其他因素，决定了整个拟南芥基因组的REF 6结合基序的可及性。提出的工作是至关重要的了解关键的表观遗传调节，影响参与重要的发育途径和生物和非生物胁迫反应的基因的表达之间的功能相互作用。我们相信这些知识可以应用于加拿大作物的遗传改良。这些项目将对学生和博士后非常有益，使他们能够获得多个学科的培训，包括遗传学，表观遗传学，表观基因组学，生物信息学，分子生物学等。

In higher organisms including plants, DNAs are highly folded into DNA-protein (mainly histones) complexes called chromatins. Gene expression regulation at chromatin level, often referred to as epigenetic regulation, is mediated by the joint activities of two enzymatic complexes that catalyze covalent histone modifications and alter chromatin structure. The Polycomb group (PcG) and Trithorax group (TrxG) proteins are evolutionarily conserved proteins that mediate epigenetic gene repression and activation, respectively. PcG proteins form multimeric protein complexes including the Polycomb Repressive Complex 2 (PRC2) that catalyzes the repressive trimethylation of lysine 27 of histone 3 (H3K27me3). TrxG proteins are biochemically heterogeneous, including the chromatin-remodeller BRM and the H3K27me3 demethylase (eraser) REF6 in the model plant Arabidopsis, but all function to antagonize Polycomb activities. It is of great interest to decipher how these two groups of proteins antagonize genome-wide, and how they are targeted to specific genomic sites. Using cutting edge genomics techniques such as Chromatin Immuno-Precipitation followed by high throughput sequencing (ChIP-seq), we have recently investigated the genome-wide functional interplay between BRM, REF6 and PRC2. We first examined the interaction between BRM and PRC2 in regulating H3K27me3 levels and identified a key role of the two proteins in controlling flowering time. We then examined the interaction between BRM and REF6 and found that REF6 targets directly to genomic loci by recognizing a specific DNA motif (CTCTGYTY, Y representing C or T) and then facilitate the recruitment of BRM. This work provides a new paradigm in the field of plant epigenetics. Here, we propose to continue our work on the functional interplay between BRM/REF6 and PcG proteins. First, we will expand our previous work on BRM-PRC2 interaction by examining the role of BRM's close homolog SYD in regulating PRC2 function. We will also profile the genome-wide occupancy of SYD and identify the unique and shared targets of BRM and SYD. Second, we propose to further investigate the genomic targeting mechanism of REF6. We will conduct a series of experiments to identify the chromatin context and/or other factors that determine the accessibility of the REF6 binding motif across the Arabidopsis genome. The work proposed is critical for understanding the functional interplay between the key epigenetic regulators that affect the expression of genes involved in important developmental pathways and biotic and abiotic stress responses. We believe that this knowledge can be applied to genetic improvement of Canadian crops. The projects will be very beneficial to students and postdocs, allowing them to obtain training in several disciplines, including genetics, epigenetics, epigenomics, bioinformatics, molecular biology, and others.