Effect of Mediator on transcriptional bursting

Mediator is a universal transcriptional coactivator (Jeronimo and Robert, 2017; Soutourina, 2018). It is recruited to enhancers by sequence-specific transcription factors and allows communication between the enhancers and their target promoters. How this communication is established in not fully understood but a generally accepted view is that the enhancer-bound Mediator physically reaches the promoter (probably via a looping mechanism) and stabilises the pre-initiation complex (PIC) formed by RNA polymerase II (RNAPII) and other general transcription factors. This model, however, raises a conundrum: stabilising the PIC would lead to transcription activation only if PIC formation was rate limiting but, at most genes in metazoans, the rate limiting step is not PIC formation but rather escape from a promoter-proximal pause site (Adelman and Lis, 2012). Hence, stabilising the PIC should not lead to transcription activation. Previous work suggests that Mediator can stimulate promoter-proximal pause release (Jeronimo and Robert, 2017), perhaps explaining how it regulates transcription, but evidence described below suggests this may not explain the full range of Mediator coactivator capacities.
Single molecule RNA fluorescence in situ hybridization and live-imaging have revealed that transcription occurs in burst (Brouwer and Lenstra, 2019). During a burst (“on” state), several initiation events lead to convoys of RNAPII actively transcribing the gene whereas between bursts (“off” state) the gene is kept silent. Recent work from the Raj lab showed that cellular stimuli led to increased burst initiation but not to increased polymerase recruitment rates within bursts (Bartman et al., 2019). This suggests that transcription is mainly regulated by modulating the time a gene is in the “on” state rather than by regulating the different transcription steps (PIC formation, initiation, pausing, elongation) within a burst. Moreover, using allele-specific RNA-seq from single cells, the Sandberg lab recently showed that enhancers regulate burst frequency whereas promoters regulate burst size (duration) (Larsson et al., 2019). They further showed that cell-type specific gene expression is primarily regulated by burst frequency. Based on these data, we hypothesise that Mediator (which mediates its effect from enhancers) regulates gene expression in part by regulating burst frequency.
We will test this hypothesis using a combination of allele-specific single-cell RNA-seq, TT-seq and ChIP-seq in mouse fibroblasts modified by CRISPR/Cas9 mediated knocked-ins to enable small molecule-induced protein degradation.