Re-constructing gene regulatory landscapes using synthetic biology
Dr. Daniel Ibrahim
The vast majority of our genomes consists of non-coding sequences that are believed to contribute to the regulation of gene expression. However, the rules of how the non-coding genome controls gene regulation are not well understood, which poses a major challenge to interpret non-coding genetic variants detected in patients with a suspected genetic disease or which can be observed in evolutionary comparisons. Most genes require complex regulatory control to ensure precise gene expression. For this, gene regulatory elements, such as enhancers, are scattered over hundreds of kilobases surrounding their target genes in so-called gene regulatory landscapes. Enhancers are thought to act in a modular fashion, whereby each contains the information for a cell-type-specific fraction of the overall gene expression. However, how these enhancers interact with one another and how they activate their target gene is unknown.
This is mainly due to current technical limitations in genome engineering. Our ability to study how several enhancers in a gene regulatory landscape interact and influence each other remains limited because it is difficult to manipulate large genomic regions. In this project we will overcome these limitations by combining advances in the field of synthetic biology with genome engineering in mouse embryonic stem cells.
We will employ a synthetic genomics-based approach using a DNA assembly process in yeast to create large DNA fragments that carry a re-organised regulatory landscape (e.g. without repeats or with rearranged enhancers). We will then replace an endogenous regulatory landscape with the engineered synthetic regulatory landscape in mouse embryonic stem cells and study how this affects gene regulation during embryonic development.
As a model locus, we will manipulate a known and well characterized regulatory landscape that controls the expression of Indian hedgehog (Ihh), a gene essential for skeletal development and growth. We have previously shown that Ihh is controlled by at least 9 enhancers with overlapping specificity (Will et al. Nat Genet. 2017), located in an intron of another gene, surrounded by sequences of unknown fucntion including repetitive elemnets.
In this project, the student will have the chance to experimentally perform synthetic DNA assembly, genome engineering in mESCs and generation of transgenic mouse embryos. For analysis we will use a combination of imaging and functional genomics approaches including ChIP-/ATAC-seq, Hi-C and scRNA-seq, where the student will be able to apply standard bioinformatics analysis steps.
The methods developed in this project will allow us to address long unsolved questions in genome function and gene regulation such as: What happens if enhancers are moved around and taken out of their natural context? How do transposon and repetitive elements influence gene expression? Is it possible to create entire regulatory landscapes from scratch?
For more information please visit the website of the research group Development & Disease or check out some of our papers.
Our thoughts on gene regulation:
Ibrahim, DM and Mundlos S (2020). The role of 3D chromatin domains in gene regulation: a multi-facetted view on genome organization. Curr Opin Genet Dev, 61, 1-8. doi:10.1016/j.gde.2020.02.015
Recent experimental work to understand the link between 3D chromatin structure and gene regulation:
Despang A, Schopflin R, Franke M, Ali S, Jerkovic I, Paliou C, Chan WL, Timmermann B, Wittler L, Vingron M, Mundlos S and Ibrahim DM (2019). Functional dissection of the Sox9-Kcnj2 locus identifies nonessential and instructive roles of TAD architecture. Nat Genet 51 (8): 1263-1271