Abstract
Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.
Highlights
Enhancers are short, cis-acting regulatory elements that modulate transcription of target genes, relatively independently of their orientation or distance with respect to the promoter
Animal development relies on the early delimitation of specific embryonic territories that will later participate in the formation of tissues and organs
This process is governed by sets of so-called developmental genes
Summary
Cis-acting regulatory elements that modulate transcription of target genes, relatively independently of their orientation or distance with respect to the promoter. They act as platforms to recruit multiple transcription factors [1] that interact with the transcription machinery at the promoter via cofactors [2]. The functions of enhancers have been mainly investigated through analysis of transgenic constructs carrying a reporter gene driven by a minimal promoter and linked to the enhancer [8]. The advent of easy and efficient genome editing techniques, in particular those based on the CRISPR/Cas system, have facilitated mutation of putative enhancers in their natural genomic context [9,10], enabling the direct dissection of enhancer function in various species, including vertebrates [11]
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