Vostok: 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 (CG11504), 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. ChIP-Seq analyses were performed on the brains of wandering larvae (4-6h before puparium formation). All experiments were performed using wild type or Vostok null mutant larvae.

果蝇调控基因组以层级化的拓扑关联结构域（topological associating domains, TADs）及TAD内部调控环的形式进行组织。高分辨率Micro-C图谱在果蝇幼虫脑组织中鉴定出约645个此类调控环，其跨度介于25 kb至250 kb之间。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，包括Dprs、Dips、Sides及Beats家族蛋白。此类调控环由成对的锚定元件相互作用形成，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组织中，约10%（64/645）的调控环出现丢失或信号减弱。本数据集还提供了第二种环化因子的相关证据：含MADF结构域的Vostok蛋白（CG11504）可识别在脑锚定元件中过度富集的GCAACA基序。在Vostok突变体的幼虫脑组织中，共有47个调控环（占总环数的7%）出现丢失。此类环的丢失与对应基因的表达量下降相关，提示增强子-启动子相互作用受到损伤。在Vostok与GAF双突变体中，仅有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。该推测的佐证来自一项观测结果：此前鉴定出的两个跨度达6 Mb的元环（meta-loops）同时依赖GAF与Vostok蛋白发挥功能。本文还探讨了利用不同环化因子组合，在动物基因组中实现遗传位点无限三维关联的应用前景。染色质免疫共沉淀测序（ChIP-Seq）分析的样本为游走幼虫（化蛹前4~6小时）的脑组织。所有实验均使用野生型或Vostok纯合缺失突变体幼虫完成。