Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5′ untranslated regions (5′UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.
细胞状态、功能等特性的形成是生物学的基本问题之一，而最终决定细胞类型的因素是基因表达，在细胞分化和对内外信号产生响应的过程中，基因表达的调控往往是最终的结果，基因表达调控机制本身也是生物学研究的核心问题之一。基因表达调控在线性DNA水平主要表现为调控元件的选择和对靶基因调控的参与，其中研究较多的调控元件为增强子。增强子的功能不依赖在基因组中和靶基因间的距离、方向和位置，这些特点很大程度上增加了增强子鉴定的难度。调控元件自转录测序方法（STARR-seq）通过同时测量千百万条DNA片段的自转录水平从而可以达到鉴定增强子的目的。我们使用该技术在国际上首次实现了对水稻基因组的分析实现定量确定全基因组范围内增强子活性的分布图谱，并揭示了多种水稻增强子特点。绝大多数增强子位于基因序列内，尤其是在5'端非翻译区和编码的外显子区，这和哺乳动物中增强子一般被认为分布在远离基因的位置不同。相当数量的增强子位于转座子富集区域内沉默和非转录基因的附近，一定程度上近年来转座序列可以演化为新的增强子的发现相吻合。我们对水稻增强子的表观遗传特征进行了多层次的分析，发现功能性增强子大多数并不和DNA酶超敏感点相重叠，同时和动物体系里增强子的组蛋白修饰H3K4me1重叠不多。通过聚类分析进一步发现仅有约40%增强子具有一种及一种以上的表观特征，有趣的是H3K4me3和H3K27ac的组合而不是H3K4me1和H3K27me3的组合更富集在功能性鉴定的增强子上。同时，H3K27me3和H3K4me3和H3K27ac可以同时富集，可能是细胞和不同染色体的差异性。我们还借鉴基于超敏感点位置预测增强子的方法预测增强子，并与STARR-seq鉴定的增强子相比较，我们的结果显示基于纯粹超敏感点位置的增强子预测结果和功能性鉴定获得的结果具有巨大的差异，几乎不重叠。鉴定增强子的方法有多种，各种方法的结果间可能可以互补，我们的工作不仅提供了一个新的增强子数据，更重要的是揭示了不同方法鉴定的增强子具有完全不同的特点，为进一步推动增强子鉴定的研究和对增强子功能和作用机制的研究提供了资源基础





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