Koenecke Nina, Johnston Jeff, Gaertner Bjoern, Natarajan Malini, Zeitlinger Julia
Stowers Institute for Medical Research, Kansas City, MO, 64110, USA.
Present address: Department of Pediatrics, University of California, San Diego, La Jolla, CA, 92093, USA.
Genome Biol. 2016 Sep 27;17(1):196. doi: 10.1186/s13059-016-1057-2.
Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to date, and illustrates the fundamental role of enhancers in controlling patterning, cell fate specification, and morphogenesis during development. Histone acetylation such as H3K27ac is an excellent marker for active enhancers, but it is challenging to obtain precise locations for enhancers as the highest levels of this modification flank the enhancer regions. How to best identify tissue-specific enhancers in a developmental system de novo with a minimal set of data is still unclear.
Using DV patterning as a test system, we develop a simple and effective method to identify tissue-specific enhancers de novo. We sample a broad set of candidate enhancer regions using data on CREB-binding protein co-factor binding or ATAC-seq chromatin accessibility, and then identify those regions with significant differences in histone acetylation between tissues. This method identifies hundreds of novel DV enhancers and outperforms ChIP-seq data of relevant transcription factors when benchmarked with mRNA expression data and transgenic reporter assays. These DV enhancers allow the de novo discovery of the relevant transcription factor motifs involved in DV patterning and contain additional motifs that are evolutionarily conserved and for which the corresponding transcription factors are expressed in a DV-biased fashion. Finally, we identify novel target genes of the regulatory network, implicating morphogenesis genes as early targets of DV patterning.
Taken together, our approach has expanded our knowledge of the DV patterning network even further and is a general method to identify enhancers in any developmental system, including mammalian development.
果蝇背腹轴（DV）模式形成是迄今为止理解最透彻的调控网络之一，它阐明了增强子在发育过程中控制模式形成、细胞命运特化和形态发生的基本作用。组蛋白乙酰化，如H3K27ac，是活性增强子的优良标记，但由于这种修饰的最高水平位于增强子区域两侧，因此获得增强子的精确位置具有挑战性。如何利用最少的数据从头识别发育系统中的组织特异性增强子仍不清楚。
以DV模式形成为测试系统，我们开发了一种简单有效的方法来从头识别组织特异性增强子。我们利用CREB结合蛋白辅因子结合数据或ATAC-seq染色质可及性数据对广泛的候选增强子区域进行采样，然后识别那些在不同组织间组蛋白乙酰化存在显著差异的区域。当与mRNA表达数据和转基因报告基因检测进行基准比较时，该方法识别出数百个新的DV增强子，并且优于相关转录因子的ChIP-seq数据。这些DV增强子使得能够从头发现参与DV模式形成的相关转录因子基序，并包含其他在进化上保守且相应转录因子以DV偏向方式表达的基序。最后，我们识别出调控网络的新靶基因，表明形态发生基因是DV模式形成的早期靶标。
综上所述，我们的方法进一步扩展了我们对DV模式形成网络的认识，并且是在任何发育系统中识别增强子的通用方法，包括哺乳动物发育。
