原生动物嗜热四膜虫染色质重塑过程中组蛋白分子伴侣的功能分析

In eukaryotic cells, nuclear DNA is packaged into chromatin. The basic building block of chromatin is the nucleosome core particle, which contains 147 base pairs of double-stranded DNA around the surface of an octamer of histone proteins. Appropriate nucleosome assembly plays a role in gene silencing and spatial organization of silenced chromatin. Therefore, histone chaperones, which regulate nucleosome assembly, are important in regulating chromatin assembly and dynamics. Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain nuclear stability. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. Tetrahymena contains spatially differentiated macronucleus and micronucleus within a single cytoplasm. The polyploid somatic macronucleus is involved in active transcription, while the transcriptionally inert diploid micronucleus stores the genetic information for sexual progeny. The current project will be working on: specific histone chaperones identification basing on the Tetrahymena Genome Database and Tetrahymena Functional Genomics Database; localization of the chaperones using immunofluorescence staining; functional analysis the genes by gene knockout /RNAi technique and gene overexpression; establishment of protein-protein interaction network by Co-Immunoprecipitation/Mass Spectrometry and pull down techniques. The main aims are: explore specific crosstalk between histone and their chaperones and between chaperones and chaperones, highlighting dynamic regulation mechanism of of two different nuclear structure and remoding in ciliate. In addition, our studies might be helpful for understanding structure and function of chromatin and epigenetics.

真核生物有关DNA的生命活动都在基因组与其所缠绕的组蛋白组装形成的染色质上进行，染色质结构的动态变化在基因转录沉默和激活过程中起重要作用，组蛋白修饰、组蛋白变体及其相关的分子伴侣的相互作用影响了特定的染色体结构域。嗜热四膜虫具有转录活跃进行无丝分裂的多倍体大核和转录沉默进行有丝分裂的二倍体小核，两种细胞核含不同的组蛋白变体和修饰类型。本研究基于四膜虫基因组数据库和功能基因组数据库，筛选特异的组蛋白分子伴侣基因；通过免疫荧光定位, 分析组蛋白分子伴侣的细胞定位；通过基因敲除/敲减技术和基因过表达技术分析基因缺失或过表达对染色质结构的影响；通过免疫共沉淀、蛋白质谱分析和Pull-down技术建立组蛋白和组蛋白分子伴侣之间以及不同组蛋白分子伴侣之间的相互作用网络。阐明组蛋白分子伴侣在纤毛虫不同细胞核结构形成和染色质动态重塑过程的功能，对深入了解真核生物染色质的结构以及表观遗传调控机制具有重要意义

在真核生物中， DNA缠绕组蛋白折叠形成染色质。核心组蛋白是构成真核生物核小体的主要成分，核小体装配和解离推动着染色质的动态变化。细胞以精准调控的方式改变组蛋白修饰来促进基因组转录或维持基因沉默，进而实现基因的选择性表达。组蛋白修饰和核小体装配主要通过酶和组蛋白分子伴侣来实现，组蛋白修饰酶在组蛋白修饰中的作用已进行了深入分析，但组蛋白分子伴侣在该过程中的分子机制并不清楚。嗜热四膜虫（Tetrahymena thermophila）是一种单细胞原生动物，具有核的二态性。转录激活的大核（macronucleus，MAC）和沉默的生殖小核（micronucleus，MIC）在染色质结构和分裂周期上并不相同。嗜热四膜虫具有复杂的细胞周期和独特的核发育事件，是揭示组蛋白分子伴侣功能的良好模式体系。本研究首次鉴定了四膜虫不同的组蛋白分子伴侣。促进染色质转录因子(Facilitate Chromatin Transcriptions，FACT)，染色质组装因子（chromatin assembly factor-1，CAF1），N1/N2相关蛋白（N1/N2 related protein，Nrp1），抗沉默因子（Anti-Silencing Factor 1，ASF1），核小体组装蛋白（Nucleosome Assembly Protein 1，NAP1）的功能，并对不同因子之间的相互作用及在维持染色质稳定性和核分裂中的功能进行了系统研究。促进染色质转录因子亚基Pob3参与调控嗜热四膜虫功能合子核和新大核的形成，对有性生殖的完成是必需的。Nrp1调控组蛋白H3的核输入，同时影响大核的无丝分裂和小核的有丝分裂。Nrp1调控H3K56的乙酰化水平，进而影响染色质的稳定性和基因组的转录。组蛋白分子伴侣的功能研究为进一步阐明高等真核生物组蛋白翻译后修饰和核输入的分子机制提供了新的数据。

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