To loop or not to loop: what is the role of TADs in enhancer function and gene regulation?

The past decade has seen a huge jump in the resolution and scale at which we can interrogate the three-dimensional properties of the genome. This revealed different types of chromatin structures including topologically associating domains, partitioning genes and their enhancers into interacting domains. While the visualisation of these topologies and their dynamics has dramatically improved, our understanding of their underlying mechanisms and functional roles in gene expression has lagged behind. A suite of recent studies have addressed this using genetic manipulations to perturb topological features and loops at different scales. Here we assess the new biological insights gained on the functional relationship between genome topology and gene expression, with a particular focus on enhancer function.

Predictive features of gene expression variation reveal mechanistic link with differential expression.

For most biological processes, organisms must respond to extrinsic cues, while maintaining essential gene expression programmes. Although studied extensively in single cells, it is still unclear how variation is controlled in multicellular organisms. Here, we used a machine-learning approach to identify genomic features that are predictive of genes with high versus low variation in their expression across individuals, using bulk data to remove stochastic cell-to-cell variation. Using embryonic gene expression across 75 Drosophila isogenic lines, we identify features predictive of expression variation (controlling for expression level), many of which are promoter-related. Genes with low variation fall into two classes reflecting different mechanisms to maintain robust expression, while genes with high variation seem to lack both types of stabilizing mechanisms. Applying this framework to humans revealed similar predictive features, indicating that promoter architecture is an ancient mechanism to control expression variation. Remarkably, expression variation features could also partially predict differential expression after diverse perturbations in both Drosophila and humans. Differential gene expression signatures may therefore be partially explained by genetically encoded gene-specific features, unrelated to the studied treatment.

Chromosome topology guides the Drosophila Dosage Compensation Complex for target gene activation.

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity.

Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.