拟南芥HAM互作蛋白HIP介导的组蛋白H4乙酰化在叶绿体发育中的机制研究

Histone acetylation is one of the most important epigenetic modifications in eukaryotic organisms and involves in the processes of gene transcription regulation, DNA replication, recombination and repair, etc. Previous functional studies mainly focus on the histone H3 acetylation and the enzymes catalyzing this specific post-translational modification in Arabidopsis. However, the functional consequence of HAM1, HMA2 and HAMs-derived H4 acetylation is largely unknown due to the embryonic lethality caused by ham1:ham2 double mutant. Here, we plan to study the functions of HAM in H4 acetylation through HAM-interacting proteins (HIPs). We have identified HIP1 and HIP2, two homologous proteins using yeast two-hybrid screening. Comparing to WT, the hip1:hip2 double mutant decreased in histone H4 acetylation level with the disrupted chloroplast structures observed under transmission electronic microscope. These data suggested that HIP1 and HIP2 play important roles in regulating chloroplast development through HAM-mediated H4 acetylation. In this project, transcriptome deep-sequencing and ChIP-sequencing will be performed to analyze the function of HIPs in regulating mRNA transcription and the H4 acetylation status of the Arabidopsis genome, so as to elucidate the association between H4 acetylation and gene transcription as well as the directly affected genes in the plant chloroplast development，and provide clues for the understanding of H4 acetylation in the plant growth and development.

真核生物组蛋白乙酰化是一类重要的表观遗传修饰，调控基因转录、DNA复制、重组和修复等基本生命过程。对植物组蛋白乙酰化的研究多集中在拟南芥H3乙酰化及其催化酶的生物功能上，而其它组蛋白的乙酰化对植物生长发育也很重要；组蛋白H4功能冗余的乙酰化酶HAM1，2的纯合双突变胚胎致死，导致H4乙酰化的功能研究难以深入进行。本项目利用HAM的互作蛋白来研究其介导的H4乙酰化在植物生长发育中的作用，筛选到HAM的互作同源蛋白HIP1和HIP2。hip1:hip2双突变体内组蛋白H4乙酰化严重下调，叶绿体发育异常，提示HIP1和HIP2介导的H4乙酰化调控叶绿体发育。为明确其功能和作用机理，将通过深度测序确定HIP1和HIP2对mRNA转录及基因组水平H4乙酰化的作用，明确植物中H4乙酰化与基因转录的关系以及HIP1和HIP2在叶绿体发育过程中直接调控的靶标基因，阐述H4乙酰化修饰在植物生长发育中的功能。

叶绿体是植物进行光合作用的重要细胞器，叶绿体发育依赖于一系列的核基因组与叶绿体基因组编码光合成相关基因的协调控制，在这个过程中组蛋白H4乙酰化是否及如何作用不为人知。由于拟南芥中组蛋白H4乙酰化酶HAM1/2的双突变体胚胎致死，我们通过对HAM互作蛋白EPL1和EPL2的研究来解析组蛋白H4乙酰化的生物学功能。我们利用免疫共沉淀和双分子荧光互补确认了AtHAM1/2与AtEPL1/2在植物体内的相互作用，并且发现两者相互作用可促进体内H4乙酰化水平的增加；我们对atepl1:atepl2突变体进行了叶绿体发育的相关生理及分子表型分析，发现atepl1:atepl2双突体的叶片黄化，叶绿素含量降低，叶绿体结构异常，光合作用中光系统I与光系统II内关键基因的转录和蛋白含量降低；通过VIGS沉默烟草中的HAM基因发现HAM敲低的烟草植株也具有组蛋白H4乙酰化水平下降，叶片黄化，叶绿体异常及显著的PSI和PSII基因的下调表达；通过深度测序系统解析了AtEPL1/2的突变对叶绿体发育过程中关键基因转录的影响，并综合NbHAM基因沉默的烟草相关RNA-Seq和ChIP-seq等深度测序结果，明确EPL作为HAM-EPL组蛋白乙酰化酶复合物的核心组分，通过调控叶绿体发育过程中的重要基因的启动子及编码区的组蛋白H4K5乙酰化水平来影响其转录。我们的研究阐述了EPL介导的组蛋白H4乙酰化修饰调控植物叶绿体发育的分子机制。