Identification of novel cis-regulatory elements of Eya1 in Xenopus laevis using BAC recombineering

Santosh Kumar Maharana,Nicolas Pollet,Gerhard Schlosser

doi:10.1038/s41598-017-15153-7

Abstract

The multifunctional Eya1 protein plays important roles during the development of cranial sensory organs and ganglia, kidneys, hypaxial muscles and several other organs in vertebrates. Eya1 is encoded by a complex locus with candidate cis-regulatory elements distributed over a 329 kbp wide genomic region in Xenopus. Consequently, very little is currently known about how expression of Eya1 is controlled by upstream regulators. Here we use a library of Xenopus tropicalis genomic sequences in bacterial artificial chromosomes (BAC) to analyze the genomic region surrounding the Eya1 locus for enhancer activity. We used BAC recombineering to first create GFP reporter constructs, which were analysed for enhancer activity by injection into Xenopus laevis embryos. We then used a second round of BAC recombineering to create deletion constructs of these BAC reporters to localize enhancer activity more precisely. This double recombineering approach allowed us to probe a large genomic region for enhancer activity without assumptions on sequence conservation. Using this approach we were able to identify two novel cis-regulatory regions, which direct Eya1 expression to the somites, pharyngeal pouches, the preplacodal ectoderm (the common precursor region of many cranial sensory organs and ganglia), and other ectodermal domains.

Highlights

Eya1 is a multifunctional protein that acts as a transcriptional coactivator of the transcription factor Six1 and has additional functions in the cytoplasm, where it affects cell polarity and promotes asymmetric cell divisions, partly mediated by its phosphatase activity1,2
The X. tropicalis bacterial artificial chromosomes (BAC) library contains several BACs covering the region between the Eya1 locus and its neighboring genes MSCI and XKR9
Our findings illustrate how a double BAC recombineering strategy with a reporter gene cassette and various deletion cassettes can identify cis-regulatory elements in large genomic regions. Applying this strategy to the Eya1 locus in X. tropicalis where candidate enhancers are distributed over a 329 kbp wide genomic region, we identified two novel genomic regions with enhancer activity, one mapping to the Eya1 locus itself and containing several of its introns (BAC15P2D2-P2D3), the other one mapping far upstream (135 kbp) of the first Eya1 exon (BAC16P2D3-P2D4)

Summary

Introduction

Eya is a multifunctional protein that acts as a transcriptional coactivator of the transcription factor Six and has additional functions in the cytoplasm, where it affects cell polarity and promotes asymmetric cell divisions, partly mediated by its phosphatase activity. Eya and Six play multiple important roles during the development of sensory organs and ganglia derived from cranial placodes in vertebrates such as the inner ear and the lateral line system They have been implicated in the development of kidneys, hypaxial muscles and several other organs. Eya is first expressed at the beginning of gastrulation and becomes upregulated in a horseshoe shaped region of the ectoderm surrounding the anterior neural plate at the end of gastrulation, where it is coexpressed with Six112. Eya is first expressed at the beginning of gastrulation and becomes upregulated in a horseshoe shaped region of the ectoderm surrounding the anterior neural plate at the end of gastrulation, where it is coexpressed with Six112–17 This expression domain, the preplacodal region (PPR) delineates a panplacodal primordium that will subsequently give rise to all cranial placodes. Ectoderm is competent to express Eya and Six in response to inducing signals and showed that Dlx3/5, GATA2/3, FoxI, AP2, and Msx1/2 are among the transcription factors required for non-neural competence

Methods

Results

Conclusion