拟南芥组蛋白H3 Lysine 9甲基化酶识别机制的研究

It has been intriguing for biologists to understand how protein recognize chromatin and how epigenetic marker is established and maintained. Our previous studies show that Histone H3 Lycine 9 (H3K9) methylation offers a great opportunity to solve these puzzles. In Arabidopsis,SUVH4 controls the majority of H3K9me genome-wide, whereas SUVH5 and SUVH6 act preferentially at specific loci. The mechanism underling this plant unique phenominon remains unknown. Here we propose to survay the SUVH5 and SUVH6 prefered targets genome wide with next generation sequencing using unique transgenic and mutant strains from our previous studies.The prefered targets of SUVH5 and SUVH6 will be further compared with known epigenomic pattern to determined the local epigenetic indicator of SUVH5 and SUVH6 respectively. Mutants from known gene scilencing pathways will be investigated to confirm the crosstalks between SUVH5/SUVH6 and other epigenetics pathways. Taken together, this project will provide detailed information of SUVH5 and SUVH6 targets in Arabidopsis genome as well as the genetic/epigenetic requirements to determine the locus specificity of SUVH5 and SUVH6.A new solution to track the chromatin modification enzyme will be tested. The knowledge produced from this project will not only deepen our understanding on how to establish and maintain the silencing marker H3K9me, but also review the mechanism of protein and chromatin recognition among high eukaryotic species.

蛋白与染色体的识别、表观遗传标记的起源和维持的机制一直是生物学关心的关键问题。我们已有的研究表明拟南芥组蛋白H3 Lysine 9的甲基化(H3K9me)是很好的切入点：H3K9me由SUVH4、SUVH5和SUVH6三个同源的甲基化酶催化，其中SUVH4在基因组中起主导作用，SUVH5和SUVH6则作用于基因组的不同位点。这种植物中特有现象的具体机制尚不清楚。本申请在前期工作基础上，利用已获得的独特的转基因和突变体材料，通过高通量测序来比较SUVH5和SUVH6的作用位点异同，结合生化和遗传手段，探索这两个组蛋白修饰酶的作用方式。本研究在深入分析H3K9甲基化的分子机制，以及不同表观遗传途径间的相互作用的同时，将为在基因组水平追踪染色体修饰酶提供新的解决方案，更有助于解决高等真核生物蛋白与染色体识别的一般性方式。

蛋白与染色体的识别、表观遗传标记的起源和维持的机制一直是生物学关心的关键问题。我们已有的研究表明拟南芥组蛋白H3 Lysine 9 的甲基化(H3K9me)是很好的切入点：H3K9me 由SUVH4、SUVH5 和SUVH6 三个同源的甲基化酶催化，其中SUVH4 在基因组中起主导作用，SUVH5 和SUVH6 则作用于基因组的不同位点。本研究利用甲基化来追踪SUVH5和SUVH6在基因组中的作用位点，通过检测和比较野生型、suvh4、suvh4suvh5、suvh4suvh6和suvh4suvh5suvh6全基因组甲基化，发现SUVH5 和SUVH6 的作用位点，并通过生物信息学分析，总结SUVH5 和SUVH6 各自偏好的靶位点的规律。多数情况下SUVH5对甲基化的贡献大于且多于SUVH6。根据贡献不同，可以将转座元件分为四类。其中SUVH4 > SUVH5 > SUVH6类型的位点最多。在异染色质中CHH甲基化由SUVH5和SUVH6共同控制。在很多位点中出现了如下现象：46双突变体甲基化高于4单突变体，说明存在某种负反馈机制，维持DNA甲基化。