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

脊椎动物的基因组主要由非编码序列组成，其中一半以上是重复序列。在以前的研究中，我们能够证明结构变异（SV）可以通过改变染色体的3D构象导致基因错误表达和疾病。我在这里提出，重复的元素可以干扰3D基因组折叠，从而诱导异位接触超过TAD边界与随后的错误表达和疾病，这种机制可以产生类似的表型基因组重排SV。我们将通过研究Fgf 8基因座SV的病理学来研究这一假设，这些SV是人类分裂手足畸形（SHFM）的原因。SHFM表型也是由小鼠突变型指状体发育不全（Dac）中相同基因座处的反转录转座子（MusD）插入引起的。我们将使用CRISPR/Cas9基因组编辑来重新设计小鼠中的人类SV，以研究它们对基因调控和肢体发育的影响。将使用表达谱和单细胞RNA测序，通过在无肢芽中的详细表达分析来研究Dac突变体中的MusD插入的病理学。此外，我们将研究组蛋白修饰和CTCF结合，并从Dac/Dac胚胎中捕获HiC以及SHFM重排，以研究它们对染色质修饰和构型的影响。在下一步中，我们将操纵从Dac/Dac胚胎产生的ES细胞中的Dac基因组以拯救Dac表型。我们将研究MusD转座因子（TE）在易感（129）与非易感（C57 B6）菌株中的4C全基因组效应，以确定活性TE干扰邻近区域的区域，从而改变3D基因组结构。与此同时，我们的目标是确定在人类FGF 8基因座重复的分子病理学和解开，为什么小鼠和人类的表型是如此相似。这项研究不仅将深入了解TE可能具有的调节作用以及它们如何干扰基因调节，还将告诉我们如何从不同的病理中产生相似的表型，在这种情况下，SHFM。从Dac突变和人类SHFM基因座中吸取的教训可以转移到其他人类疾病中，从而推进对未知原因和/或异常遗传的遗传疾病中畸形原因的其他研究。

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.