Discovery of genes required for body axis and limb formation by global identification of retinoic acid-regulated epigenetic marks.

Jun Yin,Gregg Duester,Karolin F. Meyer,Yi-Hsien Su,Marie Berenguer

doi:10.1371/journal.pbio.3000719

Jun Yin, Gregg Duester + Show 3 more

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https://doi.org/10.1371/journal.pbio.3000719

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Abstract

Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined chromatin immunoprecipitation sequencing (ChIP-seq) for epigenetic marks and RNA-seq on trunk tissue from wild-type and Aldh1a2-/- embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of histone H3 K27 acetylation (H3K27ac) (gene activation mark) or histone H3 K27 trimethylation (H3K27me3) (gene repression mark) associated with conserved RAREs, suggesting these genes function downstream of RA. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach; plus, many other candidate RA target genes were found. Nuclear receptor 2f1 (Nr2f1) and nuclear receptor 2f2 (Nr2f2) in addition to Meis homeobox 1 (Meis1) and Meis homeobox 2 (Meis2) gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. A similar epigenetic approach can be used to determine the target genes for any transcriptional regulator for which a knockout is available.

Highlights

Retinoic acid (RA) is generated from retinol by the sequential activities of retinol dehydrogenase 10 (RDH10) [1] and aldehyde dehydrogenase 1A2 (ALDH1A2) [2,3]
Gene discovery guided by RA-regulated epigenetic marks additional genes that our findings indicate are candidate RA target genes for trunk, including Nuclear receptor 2f1 (Nr2f1), nuclear receptor 2f2 (Nr2f2), Meis homeobox 1 (Meis1), Meis homeobox 2 (Meis2), and Spry4 that were further examined here (Table 1); differential expression of these genes in E8.5 wild-type versus Aldh1a2-/- trunk was validated by quantitative reverse transcription-polymerase chain reaction (S2 Fig)
Our epigenetic ChIP-seq studies combined with RNA-seq on wild-type versus Aldh1a2-/- RAdeficient trunk tissue provides a means for identifying new candidate RA target genes that may be required for development

Summary

Introduction

Retinoic acid (RA) is generated from retinol by the sequential activities of retinol dehydrogenase 10 (RDH10) [1] and aldehyde dehydrogenase 1A2 (ALDH1A2) [2,3]. 100%/ $2,145,500 of the total project costs were financed with Federal funding. 0%/$0 of the total costs were financed with non-Federal funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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