Rev-Erb in macrophage gene expression : a case study of transcriptional regulation through distal regulatory elements

This dissertation contains four divisions represented in three chapters. The first chapter introduces transcription factor Rev-Erb, a transcriptional repressor belonging to the superfamily of nuclear receptors. Chapter 1 then continues with the physiological roles and functional mechanisms of enhancers. These are DNA regulatory elements key to orchestrate temporal and spatial expression of genes. Our work on Rev-Erb in macrophages, described in the second chapter, provides evidences of a novel transcription regulatory mechanism at enhancer elements. We identified novel target genes of Rev-Erb, such as matrix metalloproteases 9 (mmp9) and fractalkine receptor (Cx3cr1), which are negatively regulated by Rev-Erb from distal enhancer elements. In fact, genome-wide binding analysis shows that most Rev-Erb localizes to putative macrophage enhancers. A recent discovery demonstrated that RNAs are actively transcribed at enhancers on a genomic- scale. In line with this finding, Rev-Erb represses RNA transcription at enhancers. The mechanism is similar to its action at promoters by inhibition of RNA polymerase II recruitment and deacetylation of histones. Given that both enhancer-RNAs (eRNAs) and mRNAs are negatively regulated by Rev-Erb, we further test whether eRNA is a functional intermediate for target gene regulation. Using Mmp9 as a model, targeting its eRNA with RNA interference decreases mRNA expression. Furthermore, in Rev-Erb deficient macrophages, both Mmp9 eRNA and mRNA are derepressed; but targeting eRNA is sufficient to reverse Mmp9 mRNA derepression. Our finding suggests a functional role for enhancer-RNA. When acting through enhancers, Rev-Erb regulates target genes by repressing transcription at enhancers as a mechanistic intermediate. The last chapter explores ways to utilize the enormousness of genome-scale data from human populations to study functions of transcription regulatory elements relevant to diseases through forward genetics. Understanding mechanisms of transcriptional regulation, including discoveries of functional and targetable intermediates such as enhancer- RNAs, suggests new methods of disease detection and treatmen