Affiliations: 1) Stanford University, Stanford, CA; 2) Genentech, South San Francisco, CA
Keywords: a. chromatin structure; e. enhancers
In metazoans, cis-regulatory DNA sequences (enhancers) often lie kilobases away from the target genes’ proximal transcription start sites (promoters), where transcriptional machinery is assembled. Various assays of the physical arrangement of sequences in 3D space have not yet solved controversial debates about which mechanism, or combination of mechanisms mediate such long-range communication in Drosophila. In mammals, ATP-dependent motors on the cohesin complex walking in opposite directions along DNA while tethered to one another may bridge distally located enhancers and promoters. This mechanism requires a DNA-bound factor such as the CCCTC binding factor (CTCF) to stall cohesin at the right loci. Despite a non-conserved function for CTCF in Drosophila, perturbations of cohesin components and regulators quantitatively change gene expression and suggest a possible role for active loop extrusion in enhancer-promoter contact. Alternatively, point-to-point loops could be formed by protein tethers that interact when thermal fluctuations in the DNA polymer bring their binding sites together. Several Drosophila DNA-binding factors are found near the boundaries of topologically associating domains (TADs) and may mediate physical tethering of select DNA loci by interacting or phase separating with one another.
We investigate these proposed mechanisms for Drosophila genome folding with optical reconstruction of chromatin architecture (ORCA), a super-resolution imaging technique. We have fluorescently labeled 80 5-kilobase regions spanning TADs that harbor enhancer-promoter loops for the genes scyl and chrb in Drosophila tissues. From the imaging data, we have reconstructed individual polymers tracing the DNA path in single cells. ORCA has been performed on embryos depleted of loop-associated factors, including a candidate tether, GAGA binding factor (GAF), the cohesin component, Rad21, the cohesin loader, NipB, and the cohesin unloader, WAPL. We find that GAF depletion specifically disrupts enhancer-promoter loop formation. Positive and negative perturbations of cohesin function have opposing effects on overall TAD compaction without disrupting the GAF-dependent loops. I will discuss how these results, and the insights of the Drosophila model system, are promising to expand current models of genome organization.