Vostok (CG11504): A New Looping Factor for the Organization of the Regulatory Genome in the Drosophila Brain

The Drosophila regulatory genome is organized in a hierarchical series of topological associating domains (TADs) and intra-TAD regulatory loops. High-resolution Micro-C maps identified ~645 such loops in the larval brain of Drosophila, spanning distances of 25 kb to 250 kb. Many of these loops are associated with genes encoding neural adhesion proteins implicated in the formation of specific synapses, including Dprs, Dips, Sides and Beats. They are formed by interactions of pairs of tethering elements that contain simple repeated sequence motifs, such as GAGAG. Approximately 10% (64/645) of the loops are lost or diminished in larval brains obtained from homozygous mutants lacking the GAF (GAGA-associated factor) POZ oligomerization domain. We also present evidence for a second looping factor, the MADF-containing Vostok protein, which recognizes a GCAACA motif that is over-represented in brain tethering elements. There is a loss of 47 of the loops (7%) in larval brains obtained from Vostok mutants. These losses are associated with diminished expression of associated genes, suggesting impaired enhancer-promoter interactions. Only 9 loops are disrupted in both Vostok and GAF mutants, raising the possibility of a combinatorial code for tether-tether interactions. Support for this possibility stems from the observation that two previously identified meta-loops spanning 6 Mb rely on both GAF and Vostok. We discuss the prospects of using different combinations of looping factors to produce unlimited 3D associations of genetic loci in animal genomes. Overall design: Micro-C and bulk RNA-seq on Vostok mutant to examine the gene function on chromatin organization and transcription

果蝇调控基因组以层级化的拓扑关联结构域（topological associating domains, TADs）与TAD内部调控环构成的串联体系进行组织。
高分辨率Micro-C图谱在果蝇幼虫脑中鉴定出约645个此类调控环，其跨度范围为25 kb至250 kb。
其中多数调控环与编码神经黏附蛋白的基因相关联，这类蛋白参与特定突触的形成，涵盖Dprs、Dips、Sides与Beats家族蛋白。
此类调控环由成对锚定元件的相互作用所形成，这些锚定元件包含诸如GAGAG的简单重复序列基序。
在缺失GAGA结合因子（GAGA-associated factor, GAF）POZ寡聚化结构域的纯合突变体幼虫脑中，约10%（64/645）的调控环出现丢失或信号减弱。
本研究还提供了第二种环形成因子的相关证据：含MADF结构域的Vostok蛋白，该蛋白可识别在脑锚定元件中过度富集的GCAACA序列基序。
在Vostok突变体的幼虫脑中，共有47个调控环（占比7%）发生丢失。此类环的丢失与靶基因的表达水平降低相关，提示增强子-启动子相互作用受损。
仅在Vostok与GAF双突变体中，有9个调控环被破坏，这提示锚定元件间的相互作用可能存在组合编码机制。
该推测的支持依据来自此前的观测结果：两个已被鉴定的、跨度达6 Mb的超级环（meta-loops）的形成同时依赖GAF与Vostok蛋白。
本研究还探讨了利用不同组合的环形成因子，在动物基因组中实现遗传位点无限三维关联的应用前景。
实验整体设计：对Vostok突变体开展Micro-C与批量RNA测序（bulk RNA-seq），以探究该基因在染色质组织与转录过程中的功能。