Jin Meng, Aibar Sara, Ge Zhongqi, Chen Rui, Aerts Stein, Mardon Graeme
Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA.
Laboratory of Computational Biology, KU Leuven Center for Human Genetics, Leuven, Belgium.
Dev Biol. 2016 Jul 1;415(1):157-167. doi: 10.1016/j.ydbio.2016.05.007. Epub 2016 May 10.
Drosophila eye development is a complex process that involves many transcription factors (TFs) and interactions with their cofactors and targets. The TF Sine oculis (So) and its cofactor Eyes absent (Eya) are highly conserved and are both necessary and sufficient for eye development. Despite their many important roles during development, the direct targets of So are still largely unknown. Therefore the So-dependent regulatory network governing eye determination and differentiation is poorly understood. In this study, we intersected gene expression profiles of so or eya mutant eye tissue prepared from three different developmental stages and identified 1731 differentially expressed genes across the Drosophila genome. A combination of co-expression analyses and motif discovery identified a set of twelve putative direct So targets, including three known and nine novel targets. We also used our previous So ChIP-seq data to assess motif predictions for So and identified a canonical So binding motif. Finally, we performed in vivo enhancer reporter assays to test predicted enhancers from six candidate target genes and find that at least one enhancer from each gene is expressed in the developing eye disc and that their expression patterns overlap with that of So. We furthermore confirmed that the expression level of predicted direct So targets, for which antibodies are available, are reduced in so or eya post-mitotic knockout eye discs. In summary, we expand the set of putative So targets and show for the first time that the combined use of expression profiling of so with its cofactor eya is an effective method to identify novel So targets. Moreover, since So is highly conserved throughout the metazoa, our results provide the basis for future functional studies in a wide variety of organisms.
果蝇眼睛发育是一个复杂的过程，涉及许多转录因子（TFs）以及它们与辅因子和靶标的相互作用。转录因子无眼（So）及其辅因子眼缺失（Eya）高度保守，对眼睛发育而言既是必需的也是充分的。尽管它们在发育过程中发挥了许多重要作用，但So的直接靶标仍大多未知。因此，对调控眼睛决定和分化的So依赖性调控网络了解甚少。在本研究中，我们将从三个不同发育阶段制备的so或eya突变体眼组织的基因表达谱进行交叉分析，并在果蝇基因组中鉴定出1731个差异表达基因。通过共表达分析和基序发现相结合的方法，确定了一组十二个假定的So直接靶标，包括三个已知靶标和九个新靶标。我们还利用之前的So染色质免疫沉淀测序（ChIP-seq）数据评估So的基序预测，并鉴定出一个典型的So结合基序。最后，我们进行了体内增强子报告基因分析，以测试来自六个候选靶基因的预测增强子，发现每个基因至少有一个增强子在发育中的眼盘中表达，并且它们的表达模式与So的表达模式重叠。我们进一步证实，对于有抗体可用的预测直接So靶标，其在so或eya有丝分裂后敲除的眼盘中表达水平降低。总之，我们扩展了假定的So靶标集，并首次表明将so与其辅因子eya的表达谱分析结合使用是鉴定新的So靶标的有效方法。此外，由于So在整个后生动物中高度保守，我们的结果为未来在多种生物体中的功能研究提供了基础。
