Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy.
Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy.
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DOI：
10.1038/s41467-020-20714-y
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发表时间：
2021-01-19
影响因子：
16.6
通讯作者：
Spitz F
中科院分区：
文献类型：
--
作者：
Hörnblad A;Bastide S;Langenfeld K;Langa F;Spitz F
发育基因往往受多个具有重叠活性的元件的调控。然而，在大多数情况下，这些元件的相对功能及其对内源基因表达的贡献仍然没有得到很好的描述。这种现象的一个例子是，已经提出了不同组的增强剂来指导肢体顶端外胚层脊和中脑-后脑边界的Fgf8。利用体内CRISPR/Cas9基因组工程，我们从功能上剖析了这个复杂的调控集合，并展示了两种不同的调控逻辑。在顶端外胚脊中，Fgf8的表达调控似乎分布在不同的促进剂之间。相反，我们发现在中脑-后脑边界，三个活性增强剂中的一个是必不可少的，而另外两个是可有可无的。我们进一步剖析了基本的中脑-后脑边界增强器，揭示了它也是由基本模块和可有可无的模块组成的。对这种增强子的跨物种转基因分析表明，它的组成可能在脊椎动物谱系中发生了变化。发育基因通常受多种因素的调控，但它们对基因表达的相对贡献仍然知之甚少。在这里，作者应用体内CRISPR/Cas9基因组工程，在肢体顶端外胚层脊和中脑-后脑边界找到两种不同的调控逻辑来指导Fgf8。
Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage. Developmental genes are often regulated by multiple elements, yet their relative contribution to gene expression remains poorly understood. Here the authors apply in vivo CRISPR/Cas9 genome engineering to find two distinct regulatory logics directing Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary.
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