Building synthetic gene regulatory domains from scratch

Dr. Daniel Ibrahim

 

Precise, cell-type specific gene expression is crucial for cell identity and differentiation. The genome encodes this information in short cis-regulatory elements, such as promoters and enhancers. Enhancers are distributed in regulatory domains surrounding their target gene, which in mammals typically span hundreds of kilobases. Usually multiple enhancers act together to control when, where and how strong a gene is expressed. While the active regulatory DNA elements active in a given cell type can be readily identified, their mechanism of action and their interdependencies are currently not well understood.

How does linear distance to a target gene influence activity of an enhancer? How does an enhancer identify the “correct” promoter? How do multiple enhancers synergize with each other? How do CTCF sites and other features affect enhancer activity?

The answers in the scientific literature are confusing and sometimes contradictory, because most of our insights are derived from dissection of individual loci, each with its own peculiarity. The reason for our lack of understanding lies in the technical challenge to systematically manipulate genomic sequences in the size of regulatory domains. This hinders the design of tailored experiments that assess enhancers and promoters in “neutral” regulatory domains changing only parameters, such as enhancer-promoter distance, enhancer order or presence/absence of CTCF sites.

This proposed project entails a collaboration between the Ibrahim lab at the Berlin Institute of Health (BIH)/MPIMG and the Buecker lab at Max Perutz labs in Vienna. We will combine our recently established methods in synthetic genomics and mammalian genome engineering to build synthetic gene regulatory domains from the bottom up.

In this project the student will have the chance to perform synthetic DNA assembly and various types of genome engineering in mESCs. For analysis we will use a combination of functional genomics approaches including ChIP-/ATAC-seq, Cut & Tag, as well as expression profiling using RNA-seq and smRNA-FISH, where the student will learn to apply standard bioinformatic analyses.

We will employ a synthetic genomics-based approach to create ~100kb large DNA fragments that carry a neutral sequence with randomized DNA (e.g. without repeats, transposons or putative enhancers). In addition, the neutral locus will carry so-called landing pads that allow the systematic knock-in of individual enhancers, promoters and reporter genes at various positions. The synthetic regulatory domain will then be introduced into mESCs, where it can serve as a novel model locus for efficient genome engineering to systematically alter parameters such as enhancer type, order, and distance to the target gene. Thereby we will dissect the importance of individual parameters in these synthetic regulatory domains to test how they affect enhancer synergy and thereby precise, cell-type specific gene expression.

For more information please visit the websites of the Ibrahim group at the MPIMG and Berlin Institute of Health.