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.

中文翻译：

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通过全面鉴定视黄酸调节的表观遗传标记，发现身体轴和肢体形成所需的基因。


介导转录因子下游所需功能的靶基因的鉴定受到大量基因的阻碍，当转录因子从特定组织中去除时，这些基因的表达会发生变化，并且基因组中存在该因子的众多结合位点。视黄酸 (RA) 通过与 RA 反应元件 (RARE) 结合的 RA 受体调节转录，脊椎动物基因组中有数千个 RARE 反应元件。在这里，我们结合了表观遗传标记的染色质免疫沉淀测序 (ChIP-seq) 和对野生型和缺乏 RA 合成（表现出体轴和前肢缺陷）的 Aldh1a2-/- 胚胎的躯干组织进行 RNA 测序。我们发现，当 RA 缺失时，相对少数基因的表达发生改变，这些基因附近也存在与 RA 调节的组蛋白 H3 K27 乙酰化 (H3K27ac)（基因激活标记）或组蛋白 H3 K27 三甲基化 (H3K27me3)（基因抑制标记）相关的沉积。具有保守的 RARE，表明这些基因在 RA 下游发挥作用。在已知身体轴和肢体形成所需的 RA 靶基因附近发现了 RA 调节的表观遗传标记，从而验证了我们的方法；此外，还发现了许多其他候选 RA 靶基因。通过我们的方法鉴定了核受体2f1（Nr2f1）和核受体2f2（Nr2f2）以及Meis同源盒1（Meis1）和Meis同源盒2（Meis2）基因家族成员，并且每个家族的双敲除表明了以前未知的身体要求轴和/或肢体形成。类似的表观遗传学方法可用于确定任何可进行敲除的转录调节因子的靶基因。