Enhancers predominantly control transcription initiation [mNET-seq]

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol 2). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in-vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs). When analysed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol 2 pausing or elongation. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol 2 recruitment and/or initiation rather than via promoter proximal pause release. Using genome-wide analysis we show that enhancers, in general, control gene expression at the stage of Pol 2 recruitment and initiation rather than via pausing. Overall design: To characterise nascent RNA engaged with Pol2 we performed mNET-seq on K562 cells, primary mouse erythroid cells from wildtype or HS2HS3, R1R2 lines. HS2HS3 genotype: HS2 and HS3 enhancers of beta globin are homozygously deleted in combination R1R2 genotype: A compound homozygous deletion of the R1 and R2 (a.k.a -26 and -31) transcriptional enhancers of the alpha globin gene (first reported in Hay et al 2016, Nature Genetics).