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

通过转座元件和结构变异修改 Fgf8 基因座的 3D 基因组结构和基因表达

• 批准号: 422857683
• 负责人: Professor Dr. Stefan Mundlos
• 金额: --
• 依托单位: Institut für Medizinische Genetik und Humangenetik (CVK)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/422857683?language=en
• 关键词: Modification; 3D; genome; architecture; gene

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.

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（SVs）可以通过改变染色体的三维构象导致基因错误表达和疾病。我在这里提出，重复元件可以干扰三维基因组折叠，从而诱导tad边界上的异位接触，从而导致错误表达和疾病，并且这种机制可以产生与sv基因组重排相似的表型。我们将通过研究Fgf8位点SVs的病理学来研究这一假设，Fgf8位点SVs是导致人类手足裂形（SHFM）的原因。SHFM表型也是由小鼠突变型dactylaplasia （Dac）的反转录转座子（MusD）插入同一位点引起的。我们将利用CRISPR/Cas9基因组编辑技术对小鼠体内的人类SVs进行重组，研究其对基因调控和肢体发育的影响。MusD插入Dac突变体的病理将通过使用表达谱和单细胞RNA测序在Dac肢体芽中进行详细的表达分析来研究。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配置的影响。下一步，我们将在Dac/Dac胚胎产生的ES细胞中操纵Dac基因组，以挽救Dac表型。我们将研究易感菌株（129）和非易感菌株（C57B6）中4C对MusD转座因子（TEs）基因组宽度的影响，以确定活性te干扰邻近区域从而改变3D基因组结构的区域。同时，我们旨在确定人类FGF8位点重复的分子病理学，并解开小鼠和人类表型如此相似的原因。这项研究不仅将提供te可能具有的调节作用以及它们如何干扰基因调节的见解，还将告诉我们相似的表型（在本例中为SHFM）如何从不同的病理中产生。从Dac突变和人类SHFM基因座中获得的经验教训可以转移到其他人类疾病中，推动其他研究，以查明原因不明和/或不寻常遗传的遗传性疾病的畸形原因。