Figure 1: Single-molecule footprinting makes it possible to deconvolute the multiple binding events that are co-occurring at promoters in vivo.
Figure 2: Single-molecule visualisation of the footprints created by the pre-initiation complex and RNA Pol II at the Fur1 promoter in Drosophila S2 cells (Krebs et al., Mol Cell 2017).
The Krebs group develops and employs innovative genomics strategies to understand the multiple regulatory layers that control gene expression.
Previous and current research
In higher eukaryotes, the establishment of correct gene expression patterns is crucial for organism development, and their perturbation is a key event in the manifestation of many diseases including cancer. Cis-regulatory elements are short DNA sequences located proximal (promoters) or distal (enhancers) to transcription start sites. The binding of transcription factors to these regions leads to the recruitment of multiple enzymatic activities that activate or repress transcription. In addition, their activity is regulated by the epigenetic modification of their chromatin environment. Despite our increasing knowledge of the genomic location of these regulatory sequences and the identity of the factors that bind them, our understanding of how they are mechanistically interpreted remains limited.
We are developing quantitative experimental strategies to dissect the fundamental principles of gene regulation. Our approach combines large-scale manipulation of the regulatory system using high–throughput genome engineering with the implementation of quantitative multi-dimensional readouts to determine the effects of the generated perturbations.
To test the contribution of genetic information in the establishment of epigenetic states, we developed an experimental strategy to insert several hundred DNA sequence variants in parallel into the same genomic locus and to determine their DNA methylation status. The comprehensive dataset generated by this approach revealed the importance of transcription factors in controlling the epigenetic state of regulatory regions (Krebs et al., eLife 2014).
Most methods currently used in genomics rely on averaging signal over large populations of cells. To overcome this limitation, we recently developed single molecule footprinting (SMF): a methodology to quantify protein-DNA contacts at the level of single DNA molecules in vivo (Figure 1). Applying this technique genome-wide enabled us to dissect the complex sequence of binding events that lead to transcription initiation (Krebs et al., Mol Cell 2017) (Figure 2).
Future projects and goals
The group will develop and apply novel experimental and computational strategies for genomics. We will attempt to improve the resolution of current genomics assays with the aim of measuring regulatory events at the level of single cells and single DNA molecules. We will devise methodologies to perform large-scale genetic manipulations to dissect the function of regulatory DNA. Additionally, we will implement strategies to promote the use of advanced genomics techniques to study complex multicellular systems through close collaborations with fellow groups at EMBL.
We will combine these advances to address several fundamental questions such as:
- How is genetic information translated into gene expression patterns?
- What is the functional impact of epigenetic modifications on the function of regulatory regions?