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Summary Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to identify and subcategorize enhancers. Here we identify a distinct subset of enhancers in leukemia cells that are functionally dependent upon H3K79me3. Using the DOT1L inhibitor, EPZ-5676, we show that loss of H3K79me3 at these H3K79me3 enhancer elements (KEEs) leads to reduced chromatin accessibility, histone acetylation and transcription factor binding. We then use Capture-C, a high-resolution chromosome conformation capture technique, to show that H3K79me3 is required for KEE interactions with the promoter as well as transcription of the associated genes. Together these data implicate H3K79me3 in having a functional role at a subset of active enhancers where it helps maintain histone acetylation and chromatin accessibility, potentially by promoting phase-separated condensates.

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