Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy

A Hörnblad,S Bastide,F Spitz,F Langa,K Langenfeld

doi:10.1038/s41467-020-20714-y

Abstract

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.

Highlights

Developmental genes are often regulated by multiple elements with overlapping activity
Using in vivo CRISPR/Cas[9] genome editing, we demonstrate extensive redundancy between enhancers in the limb, while in the midbrain-hindbrain boundary (MHB), one distant primary enhancer is essential for Fgf[8] expression
A previous study identified a set of putative limb and MHB enhancers in the Fgf[8] locus with the potential to drive gene expression in these tissues[6] (Fig. 1a)

Summary

Introduction

Developmental genes are often regulated by multiple elements with overlapping activity. 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 Fgf[8] in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Next-generation sequencing approaches, such as chromosome conformation capture, ChIP-seq, DNAse-seq and ATAC-seq allowed for more comprehensive identification of candidate regulatory regions[1,2,4,8] These studies have demonstrated that the regulatory architecture of developmental genes is complex: it frequently includes multiple regulatory elements, dispersed over large genomic regions that often display overlapping and/or redundant activity[9]. We demonstrate that albeit sequence conservation may predict similar enhancer activity in fish and mouse, the functional subunits of the enhancer appear to have diverged and reorganized their regulatory logic

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