A Role for CAF1 in the Incorporation of H3.3 in Arabidopsis [ChIP-seq]

Eukaryotic organisms organize their genetic material in chromatin rather than unprotected DNA. Efficient chromatin assembly is essential for all DNA-templated processes, including replication, transcription, and DNA repair. Our research focused on two major histone H3 deposition complexes: Chromatin Assembly Factor (CAF1), which deposits canonical H3 on naked DNA immediately after replication during the S-phase in yeast and metazoans, and the Histone Regulator A (HIRA) complex, which is thought to incorporate the H3.3 variant in a transcription-dependent manner. By combining biochemical, genetic, genomic, and proteomic data, we surprisingly discovered that CAF1 incorporates the histone variant H3.3 into Arabidopsis chromatin. Moreover, CAF1's role inH3.3 deposition is particularly pronounced in the transcribed regions of the genome. ChIP-seq of FAS2 in fas2-4 and hira-1,fas2-4 backgrounds and ChIP-seq of histone H3.1 and histone H3.3 in Columbia-0 (WT), as well as in fas2-4 and hira-1 mutant backgrounds.The experimental tissues were collected form 12 day after germination (12DAG) from seedlings cultured on GM medium (1x MS salts, 1% sucrose, pH=5.7).

真核生物的遗传物质组装为染色质（chromatin），而非裸露的DNA分子。高效的染色质组装对于所有以DNA为模板的生物学过程至关重要，包括DNA复制、转录与DNA损伤修复。本研究聚焦于两类主要的组蛋白H3沉积复合物：染色质组装因子1（Chromatin Assembly Factor 1, CAF1），该复合物可在酵母与后生动物的S期复制完成后，将经典组蛋白H3沉积至裸露DNA之上；以及组蛋白调控因子A（Histone Regulator A, HIRA）复合物，其被认为以转录依赖的方式整合组蛋白H3变体H3.3。通过整合生物化学、遗传学、基因组学与蛋白质组学数据，我们意外发现CAF1可将组蛋白变体H3.3沉积至拟南芥（Arabidopsis）的染色质中。此外，CAF1在H3.3沉积中的功能作用在基因组的转录区域尤为突出。我们分别在fas2-4单突变体、fas2-4与hira-1双突变体背景下开展FAS2的染色质免疫共沉淀测序（ChIP-seq）；同时在哥伦比亚生态型（Columbia-0, WT）、fas2-4及hira-1突变体背景下，对组蛋白H3.1与组蛋白H3.3进行ChIP-seq实验。实验所用组织采集自萌发后12天（12DAG）的幼苗，该幼苗培养于GM培养基（含1×MS盐、1%蔗糖，pH=5.7）中。