Specific positioning of chromatin at nuclear periphery in Arabidopsis thaliana

在所有生物体中，转录调控在协调基因活动中起着至关重要的作用。核空间不是一个同质的生化环境。大量研究发现，基因的转录活性与其在核空间中的位置有关。尽管付出了很多努力，但转录调控是如何发生在核膜上的基因上仍然不清楚，因为人工靶向染色质可能导致基因上调或抑制。对植物的了解就更少了。然而，近年来在鉴定植物核包膜的结构成分方面取得了实质性进展，有助于深入研究发生在核边界的各种生物事件，包括特定的染色质系缚。利用特异性定位于核孔复合体核质侧的拟南芥NUP1（也称为NUP136），我们发现位于染色体远端臂上的基因组片段和中心周围区域特异性定位于核外围。在这里，我们建议采用我们新开发的方法来详细研究核周围的特异性染色质栓系。该项目分为三个部分，第一部分将从各种组织中生成核外围染色质定位的高分辨率地图，并将分析它们与其他基因组和表观基因组景观的关联。在第二部分，遗传学和蛋白质组学方法将被用于研究染色质拴系的分子机制。将进行一系列的交叉，以评估核膜结构变化如何影响染色质系结；串联亲和纯化结合质谱法将用于鉴定与栓系染色质相关的蛋白质。在第三部分中，我们建议应用crispr - cas9衍生的方法人工将染色质系在核外周以检查其生物学影响。总之，我们的目标是解决核区隔现象的分子和功能特性，特别是在植物核周围系住染色质的背景下。

Transcriptional regulation plays critical roles in orchestrating gene activities in all living organisms. The nuclear space is not a homogenous biochemical environment. A large number of cases have been found that the transcriptional activities of a genes are linked to its positioning in the nuclear space. Despite much effort, it has still remained unclear how transcriptional regulation happens on genes tethered at nuclear envelope, as artificially targeting chromatin there could result in either gene upregulation or suppression. Even less is known in plants. However, substantial progress has been made recently in identifying structural components of the plant nuclear envelope, facilitating in-depth investigations on various biological events happened at nuclear boundary including specific chromatin tethering. By using the Arabidopsis thaliana NUP1 (also known as NUP136), which is specifically localized to nucleoplasmic side of nuclear pore complex, we found that pericentromeric regions and certain genomic fractions located on distal chromosome arm were specifically positioned at nuclear periphery. Here we propose to employ our newly developed methods to investigate specific chromatin tethering at nuclear periphery in detail. The proposed project has three parts, in the first part, high-resolution maps of chromatin positioning at nuclear periphery will be generated from various tissues, and will be analyzed to reveal their associations with other genomic and epigenomic landscapes. In the second part, both genetic and proteomic approaches will be used to study the molecular mechanism of chromatin tethering. A serial of crossing will be performed to assess how structural change on nuclear envelope will affect chromatin tethering; and tandem affinity purification coupled with mass spectrometry will be done to identify proteins associated with tethered chromatin. In the third part, we propose to apply a CRISPR-CAS9-derived method to artificially tether chromatin to nuclear periphery to examine its biological impact. Altogether, we aim to address the molecular and functional properties of the phenomenon of nuclear compartmentation in a context of specifically tethering chromatin at nuclear periphery in plants.