Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmid-based reporter assays of gene expression. Currently, histone marks are used heavily to identify and define enhancers but both methods do not consider the endogenous role of an enhancer in the context of native chromatin. We employed a combination of genomic editing, single cell analyses, and sequencing approaches to investigate a Nanog -associated cis -regulatory element (CRE) which has been reported by others to be either an alternative promoter or a super-enhancer (SE). We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We also demonstrate that the dominant SE modulates Nanog globally and operates by recruiting and/or initiating RNA Polymerase II. Our studies have important implications to how transcriptional enhancers are defined and how they regulate gene expression.

中文翻译：

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使用以染色质为重点的方法在Nanog附近定义关键的增强子可确定表达所需的RNA Pol II募集

转录增强子已经定义为它们在基因表达的基于质粒的报告基因分析中独立于距离和方向的作用能力。当前，组蛋白标记被大量用于鉴定和定义增强子，但是两种方法都没有考虑在天然染色质的背景下增强子的内源性作用。我们结合了基因组编辑，单细胞分析和测序方法来研究与Nanog相关的顺式调控元件（CRE），其他人已报道其为替代启动子或超增强子（SE）。我们首先展示了天然染色质的距离和方向独立性，消除了基于质粒的方法带来的问题。我们还证明了占优势的SE在全球范围内调节Nanog，并通过募集和/或启动RNA聚合酶II来运作。我们的研究对如何定义转录增强子以及它们如何调控基因表达具有重要意义。