植物分生组织中染色质环调控WUSCHEL基因表达的分子机制解析

Plant meristems harbor small populations of stem cells that are responsible for the generation of all plant tissues and organs. In model plant Arabidopsis, WUSCHEL (WUS) plays a central role in the establishment and maintenance of the shoot apical meristems. In eukaryotic cells, gene activity is regulated by the interaction between DNA regulatory regions such as promoters and enhancers and trans-acting factors. This interaction spans thousands base pairs nucleotides resulting in the formation of intrachromosomal or interchromosomal DNA looping. Our previous research results revealed that AGAMOUS (AG), a key floral meristem terminator, and TERMINAL FLOWER2 (TFL2), a component of PcG complex, repressed WUS expression by specifically binding to the promoter and downstream regions of WUS gene that is important for the spatio-temporal expression of WUS. However, the molecular mechanisms underlying the regulation event are still largely unknown. In this work, we will investigate the existence of DNA looping at WUS locus and its function in regulating WUS expression by chromosome conformation capture (3C). Using CRISPR/Cas9 and transgenic technology, we will study the role of the downstream region of WUS in the formation and function of DNA looping. Through inducible system, we will uncover the function of AG and TFL2 in the formation of DNA looping during flower development. Meanwhile, we will find more trans-acting factors involved in the formation of DNA looping by yeast one-hybrid screening and forward genetic screening. Eventually, our work will reveal the novel regulatory mechanism in which DNA looping is involved and shed light on the mechanisms of plant meristem maintenance.

植物体来源于植物分生组织中的干细胞。拟南芥中，WUSCHEL(WUS)基因表达对植物干细胞产生及维持具有决定作用。真核细胞中，基因活性调节是DNA调控元件如启动子及增强子与反式作用因子相互作用的结果。这种相互作用往往跨越长片段基因组而形成染色质环。我们前期研究表明，干细胞终止关键因子AGAMOUS(AG)及PcG蛋白复合体中TERMINAL FLOWER2 (TFL2)可共同结合WUS基因上下游特定区域并抑制其表达，且WUS下游调控区域对其表达的时空性具重要作用。但是调控的分子机制还不清楚。本研究将通过染色质捕获技术研究WUS基因位点染色质环形成及调控基因表达的机制；利用CRISPR/Cas9及转基因技术研究WUS下游调控序列在染色质环形成及功能中的作用；利用诱导表达系统研究花发育中AG及TFL2介导染色质环形成的作用；利用酵母单杂交系系统筛选介导染色质环形成的关键组分及其生物学功能。

植物体来源于植物分生组织，而其维持及分化取决于其中植物干细胞池的平衡。模式植物拟南芥中，WUSCHEL（WUS）基因的表达对植物干细胞的维持具有决定作用。真核细胞中，基因活性的调节是DNA调控元件如启动子，增强子及抑制子与反式作用因子相互作用的结果。这种相互作用往往需要跨越长片端基因组以染色质环的形式实现。在本研究中我们通过染色质捕获技术研究WUS基因位点染色质环的形成及对基因表达的调控机制；利用CRISPR/Cas9及转基因技术研究WUS下游调控序列在染色质环形成及功能中的作用；利用基因诱导表达系统研究花发育过程中AG及TFL2介导染色质环形成中的作用；最终解析染色质环调控WUS表达的分子机制。我们的结果加深了染色质构象变化调控基因表达的认识。