Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmidbased reporter assays of gene expression. At present, histone marks are used to identify and define enhancers but 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, which has been reported by others to be either an alternative promoter or a super-enhancer. We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We next demonstrate that the dominant super-enhancer 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. This article contains supporting information. ‡ Equal contribution.

Acute myeloid leukemia (AML) affects tens of thousands of patients a year, yet survival rates are as low as 25% in certain populations. This poor survival rate is partially due to the vast genetic diversity of the disease. Rarely do 2 patients with AML have the same mutational profile, which makes the development of targeted therapies particularly challenging. However, a set of recurrent mutations in chromatin modifiers have been identified in many patients, including mutations in the cohesin complex, which have been identified in up to 20% of cases. Interestingly, the canonical function of the cohesin complex in establishing sister chromatid cohesin during mitosis is unlikely to be the affected role in leukemogenesis. Instead, the cohesin complex's role in DNA looping and gene regulation likely facilitates disease. The epigenetic mechanisms by which cohesin complex mutations promote leukemia are not completely elucidated, but alterations of enhancer-promoter interactions and differential histone modifications have been shown to drive oncogenic gene expression changes. Such changes commonly include HoxA upregulation, which may represent a common pathway that could be therapeutically targeted. As cohesin mutations rarely occur alone, examining the impact of common co-occurring mutations, including those in NPM1, the core-binding factor complex, FLT3, and ASXL1, will yield additional insight. While further study of these mutational interactions is required, current research suggests that the use of combinatorial genetics could be the key to uncovering new targets, allowing for the treatment of AML patients based on their individual genetic profiles.

Gene expression is regulated by numerous elements including enhancers, insulators, transcription factors, and architectural proteins. Regions of DNA distal to the transcriptional start site, called enhancers, play a central role in the temporal and tissue-specific regulation of gene expression through RNA polymerase II. The identification of enhancers and other cis regulatory elements has largely been possible due to advances in next generation sequencing technologies. Enhancers regulate gene expression through chromatin loops mediated by architectural proteins such as YY1, CTCF, the cohesin complex, and LDB1. Additionally, enhancers can be transcribed to produce non-coding RNAs termed enhancer RNAs that likely participate in transcriptional regulation. The central role of enhancers in regulating gene expression implicates them in both normal physiology but also many disease states. The importance of enhancers is evident by the suggested role of SNPs, duplications, and other alterations of enhancer function in many diseases, ranging from cancer to atherosclerosis to chronic kidney disease. Although much progress has been made in recent years, the field of enhancer biology and our knowledge of the cis regulome remains a work in progress. This review will highlight recent seminal studies which demonstrate the role of enhancers in normal physiology and disease pathogenesis.