In this interdisciplinary project, a consortium of labs with complementary expertise aims to develop novel methods to image the ultrastructure of chromatin on the level of individual genes and their regulatory regions in cells, using in situ fluorescent chemical labeling, 3D nanoscopy and sequencing-based methods to obtain distance constraints. First, they intend to establish a method to fluorescently label a specific genetic locus in the cell, via a combination of genetic engineering and chemical biology. Second, they will employ and further develop nanoscopic imaging techniques, allowing to determine the 3D conformation of the chromatin locus to an accuracy of individual nucleosomes. Third, they aim to develop methods to integrate 3D nanoscopic data and pairwise distance correlation data from sequencing experiments into a locus model of chromatin structure.
The co-applicants intend to apply these methods to resolve the conformation of regulatory chromatin contacts in an enhancer cluster which controls a critical regulatory gene (AXIN2) in B-cells. This genomic locus is of particular interest, as the Deplancke lab has recently found that this gene is involved in in the prevention and progression of chronic lymphocytic leukemia (CLL) and exhibits a dramatic, genotype-dependent activation profile, thus allowing to directly compare chromatin conformation in the ON vs. OFF state. In summary, the imaging methods developed in this proposal aim to significantly advance our ability to provide a detailed view of the 3D organization of the genome at a locus-specific level. These innovations will be of great interest to the community and allow to tackle new long-standing problems in the field of gene regulation and beyond.
