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. Overall design: 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.