Dissecting the molecular building principles of locally formed transcriptional hubs

Numerous DNA variants have already been identified that modulate inter-individual molecular traits – most prominently gene expression.

However, since finding mechanistic interpretations relating genotype to phenotype has proven challenging, the focus has shifted to higher-order regulatory features, i.e. chromatin accessibility, transcription factor (TF) binding and 3D chromatin interactions.

This revealed at least two enhancer types: “lead” enhancers in which the presence of genetic variants modulates the activity of entire chromatin domains, and “dependent” ones in which variants induce subtle changes, affecting DNA accessibility, but not transcription.

Although cell type-specific TFs are likely important, it remains unclear which sequence features are required to establish such enhancer hierarchies, and under which circumstances genetic variation results in altered enhancer-promoter contacts and differential gene expression.

Here, we propose to investigate the molecular mechanisms that link DNA variation to TF binding, chromatin topology, and gene expression response.

We will leverage data on enhancer hierarchy and sequence-specific TF binding to identify the sequence signatures that define “lead” enhancers.

The results will guide the design of a synthetic locus that serves as an in vivo platform to systematically vary the building blocks of local transcriptional units: i) DNA sequence – including variations in TF binding site affinity and syntax, ii) molecular interactions between TFs, and iii) chromatin conformation.

To validate our findings, we will perform optical reconstruction of chromatin architecture for a select number of DNA variants.

By simultaneously perturbing co-dependent features, this proposal will provide novel mechanistic insights into the formation of local transcriptional hubs.