Arnold PR , Wells AD , Li XC .

R01 AI106200 NIAID NIH HHS , R01 AI129906 NIAID NIH HHS

	FIGURE 1. eRNA transcription, elongation, and termination. (A) Activated transcription factors bind the enhancer locus and promote nucleosome remodeling. They recruit other transcription factors, cofactors, complexes such as Mediator, and histone modifiers such as P300/CBP. (B) P300/CBP acetylates H3K27, which further opens the enhancer locus and recruits additional proteins such as BRD4 and RNAP II. (C) The CTD of RNAP II is phosphorylated at Ser5. WDR82 binds and recruits MLL methyltransferases, which act as cofactors to initiate RNA transcription. 5′ capping machinery is also recruited to Ser5P and caps the nascent RNA strand. (D) BRD4 and other cofactors facilitate RNAP II transition to elongation, which results in an increase in Ser2P marks on the CTD of RNAP II and methylation of H3K4. However, PASs shortly downstream of the TSS are recognized by WDR82. This leads to recruitment of polyadenylation machinery and Integrator to terminate RNA transcription. Additionally, the RNA exosome is recruited and binds the 5′ cap, leading to rapid degradation of RNA. KDM2A recruits NEDD4 to RNAP II leading to its ubiquitination and dismissal.
	FIGURE 2. eRNA mechansims of action. (A) Promoter-enhancer looping. eRNA interacts with Cohesin complex subunits RAD21 and SMC3 to recruit them to the enhancer locus and promote enhancer-promoter looping. eRNA also interacts with other complexes such as Mediator to promote looping. (B) Histone modifications. eRNA increases H3K27ac by binding P300/CBP and promoting its affinity for H3K27. eRNA also inhibits the H3K27me complex PRC2 by binding the EZH2 subunit with a guanine quadruplex. (C) Interactions with transcriptional machinery. eRNA is brought into close contact with NELF and paused RNAP II by Integrator complex. It interacts with the CDK9 subunit of p-TEFb which phosphorylates NELF and Ser2 of RNAP II’s CTD and increases the affinity of transcription factors for DNA. It then replaces the nascent mRNA strand in binding NELF-E, which allows RNAP II escape and transition to productive elongation. eRNA also increases affinity of BRD4 for acetylated histones.
	FIGURE 3. Super enhancer as phase-separated structure. Phase separated structures such as super enhancers are composed of scaffolding nucleic acids and proteins with intrinsically disordered regions (IDRs). Thermodynamically, they are favored to condense together at greater concentration than found in the cytoplasm, leading to formation of a membraneless phase separated structure. This allows the concentration of clientele proteins with similar biophysical properties and exclusion of other dissimilar proteins. By altering the stoichiometry of scaffolding or clients, cells can control the makeup of biomolecular condensates. In super enhancers, eRNA sequence motifs, secondary structures, or post-translational modifications such as m6A may be bound by Med1, BRD4, or other proteins with IDRs to promote organization, structure, and function of super enhancers.

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