Abstract
Long-range enhancers of transcription are a key component of the genomic regulatory architecture. Recent studies have identified bi-directionally transcribed RNAs emanating from these enhancers known as eRNAs. However, it remains unclear how tightly coupled eRNA production is with enhancer activity. Through our systematic search for long-range elements that interact with the interferon-β gene, a model system for studying inducible transcription, we have identified a novel enhancer, which we have named L2 that regulates the expression of interferon-β. We have demonstrated its virus-inducible enhancer activity by analyzing epigenomic profiles, transcription factor association, nascent RNA production and activity in reporter assays. This enhancer exhibits intimately linked virus-inducible enhancer and bidirectional promoter activity that is largely dependent on a conserved Interferon Stimulated Response Element and robustly generates virus inducible eRNAs. Notably, its enhancer and promoter activities are fully retained in reporter assays even upon a complete elimination of its associated eRNA sequences. Finally, we show that L2 regulates IFNB1 expression by siRNA knockdown of eRNAs, and the deletion of L2 in a BAC transfection assay. Thus, L2 is a novel enhancer that regulates IFNB1 and whose eRNAs exert significant activity in vivo that is distinct from those activities recapitulated in the luciferase reporter assays.
Highlights
Transcriptional enhancers stimulate transcription rate or transcription probability of their target genes [1,2] and can do so from distances of over a megabase (Mb) [3]
Because IFNB1 expression is induced by virus infection, each of these experiments was performed in both uninfected and Sendai Virus (SeV)-infected IMR90 primary human fibroblast cells
If the 3C-based proximal ligation between the IFNB1 promoter and an interrogated site produced any detectable product in the majority of Real-time quantitative PCR (qPCR) reactions in either the uninfected or infected state, we selected the site for subsequent validation steps
Summary
Transcriptional enhancers stimulate transcription rate or transcription probability of their target genes [1,2] and can do so from distances of over a megabase (Mb) [3] They exert very specific effects on developmental patterning [3,4], are involved in human disease [5] and are broadly reflective of cell-type-specific gene expression [6,7]. These functional properties make them an essential component of the genomic regulatory architecture. The functional relationships of these various characteristics remain unclear
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