Modification of 3D genome architecture and gene expression at the Fgf8 locus by transposable elements and structural variations

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The genome of vertebrates consists mainly of non-coding sequence of which more than half is of repetitive in nature. In previous studies we were able to show that structural variants (SVs) can result in gene misexpression and disease by altering the 3D conformation of chromosomes. I here propose that repetitive elements can interfere with 3D genome folding thereby inducing ectopic contacts over TAD-boundaries with subsequent misexpression and disease and that this mechanism can produce similar phenotypes as genomic rearrangements by SVs. We will investigate this hypothesis by studying the pathology of SVs at the Fgf8 locus that are causal for split-hand-foot-malformation (SHFM) in humans. A SHFM phenotype is also caused by retrotransposon (MusD) insertions at the same locus in the mouse mutant dactylaplasia (Dac). We will use CRISPR/Cas9 genome editing to re-engineer the human SVs in mice to study their effect on gene regulation and limb development. The pathology of the MusD insertion in the Dac mutant will be studied with a detailed expression analysis in dac limb buds using expression profiling and single cell RNA sequencing. In addtition, we will investigate histone modification and CTCF binding and perform capture HiC from Dac/Dac embryos as well as the SHFM rearrangements to investigate their effect on chromatin modification and configuration. In a next step we will manipulate the Dac genome in ES cell generated from Dac/Dac embryos in order to rescue the Dac phenotype. We will study the effect of MusD transposable elements (TEs) genome wide by 4C in suceptible (129) vs. non suceptible (C57B6) strains to identify regions in which active TEs interfer with neighboring regions thereby changing 3D genome architecture. At the same time we aim at identifying the molecular pathology of duplications at the human FGF8 locus and unravel, why the mouse and the human phenotypes are so similar. This study will not only provide insight into the regulatory effect TEs might have and how they interfere with gene regulation, it will also tell us how similar phenotypes, in this case SHFM, can arise from different pathologies. Lessons learned from the Dac mutation and the human SHFM locus can be transferred to other human diseases advancing other studies into the causes of malformation in genetic disease with unknown cause and/or unusual inheritance.