A Novel Interferon-Beta-Associated Enhancer: Establishing a platform for new and future insights dissecting the function of long-range enhancers of transcription

Enhancers of transcription can function from either nearby or great distances to stimulate the transcription of target genes. These enhancers can be active in highly specific contexts, leading them to play critical roles in the control of gene expression in both developmental and stimulus-responsive contexts. The molecular sequence of events at work at short-range enhancers has been extensively dissected, with substantial knowledge about long-range enhancers coming from the study of a related regulatory phenomenon, the Locus Control Region (LCR). However, much remains to be understood about the mechanisms behind long-range enhancers. Key questions in the field center on the relationship between the molecular events at short-range and long-range regulatory elements, the drivers of chromosomal looping, and the relationship of enhancer activity to the newly discovered transcripts emanating from them, known as eRNAs.;Tremendous opportunities exist if a more conducive system can be characterized in which to study the mechanisms of enhancer function. Through a systematic search, I have identified a novel virus-inducible enhancer associated with the key immune-response gene Interferon-beta that I have named L2. The characteristics of Interferon-beta and L2 make them a conducive system for dissecting the molecular basis of long-range enhancer function. Leveraging this, I have demonstrated L2's 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. Thus, L2 is a novel Interferon-beta-associated enhancer that exhibits multiple virus inducible activities, some of which are independent of its endogenous cis-associated eRNAs. This sets the stage for deeper molecular characterizations of L2's activities, and its relationship to Interferon-beta transcriptional regulation.;In addition, I have probed L2's relationship with the surrounding region of the genome. L2's epigenomic profile extends across cell types, raising new questions about the regulatory architecture governing Interferon-beta expression across cell-types. L2 also exhibits chromosomal interactions with a distant gene, CDKN2B, that is involved in tumor suppression, cell cycle control, and lymphoid development. This suggests the potential for coregulation and roles for Interferon-beta in these processes. Finally the process of cloning L2 revealed that a highly conserved paralog of this enhancer resides within the locus, whose epigenomic profile suggests an immune cell-restricted potential far activity.;The L2-Interferon-beta pair has revealed new insights into the molecular basis of long-range enhancer activity, with the discovery of promoter activity offering an explanation for the production of eRNAs observed at this site. In addition, the activity retained in the absence of the endogenously associated eRNA sequences suggest that they are complex elements that may have both eRNA-dependent and eRNA-independent activity. In addition, the discovery of promoter activity offers an explanation for the production of eRNAs observed at this site. Taking advantage of the previous studies of the regulation of Interferon-beta will make this an extremely powerful platform for deeper explorations into the mechanisms of long-range enhancer activity.