Characterization and testing of looping and non-looping enhancers
The spatial organization of the genome has been identified as a critical factor in controlling gene expression. How enhancers and promoters interact within the three-dimensional organization of chromatin is just beginning to be understood. Genome-wide assays revealed that mammalian genomes fold into distinct units of ~1Mb called topologically associated domains (TADs), in which enhancers and their target genes are located. However, to what degree 3D chromatin looping between enhancers and promoters within TADs is required for gene regulation is less clear. While some studies show a direct connection between enhancer-promoter looping and gene activity, other studies find less evidence. This discrepancies in part stem from methodological differences in analyzing 3D chromatin structure, in particular differences between sequencing-based (chromosome conformation capture – 4C-seq, HiC) or imaging-based (DNA-FISH) methods. Furthermore, the local influence of other regulatory elements within the TAD can impact the interpretation of enhancer-promoter looping. Despite these specific challenges, in analyzing high-resolution epigenomic and 3D-chromatin folding data of mouse and chicken embryonic hearts we found that indeed distinct enhancer classes exist: looping and non-looping enhancers. This poses the fundamental question how important 3D-chromatin looping is for enhancer-driven gene activity and if some enhancers rely more on 3D looping than others. However, current functional reporter assays are not well suited to systematically test the influence of 3D looping for enhancer function, since they typically position enhancer and reporter gene within a few kilobases at best. In this proposal, we aim to overcome these limitations and elucidate the role of long-range enhancer-promoter looping using a combination of bioinformatic analysis, genome-engineering, and microscopy. Starting from our cardiac enhancer dataset, we will first identify the genomic signatures that distinguish looping from non-looping enhancers. By adapting and expanding genome-engineering techniques, we will develop a novel long-range enhancer-reporter assay that is tailored to investigate long-range enhancer activity and can be combined with sequencing- and imaging-based analyses. We will combine the long-range enhancer assay with 4C-seq, DNA-FISH, and expression analysis, which will deliver complimentary readouts linking 3D-chromatin looping to gene activation. This setup will allow us to characterize the genetic features driving enhancer-promoter looping, establish a novel experimental platform for enhancer analysis, and directly compare the how chromatin looping relates to gene expression.
Dr. Daniel Murad Ibrahim,Berlin
Max-Planck-Institut für molekulare Genetik (MPIMG)