A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination.

Interactions between transcription and cohesin-mediated loop extrusion can influence 3D chromatin architecture. However, their relevance in biology is unclear. Here, we report a direct role for such interactions in the mechanism of antibody class switch recombination (CSR) at the murine immunoglobulin heavy chain locus (Igh). Using Tri-C to measure higher-order multiway interactions on single alleles, we find that the juxtaposition (synapsis) of transcriptionally active donor and acceptor Igh switch (S) sequences, an essential step in CSR, occurs via the interaction of loop extrusion complexes with a de novo topologically associating domain (TAD) boundary formed via transcriptional activity across S regions. Surprisingly, synapsis occurs predominantly in proximity to the 3' CTCF-binding element (3'CBE) rather than the Igh super-enhancer, suggesting a two-step mechanism whereby transcription of S regions is not topologically coupled to synapsis, as has been previously proposed. Altogether, these insights advance our understanding of how 3D chromatin architecture regulates CSR.

Regulation of RNA polymerase II processivity by Spt5 is restricted to a narrow window during elongation.

Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in Spt5-depleted cells, a fraction of Pol II molecules are dislodged during elongation, thus decreasing the number of Pol II complexes that complete the transcription cycle. Most strikingly, this decrease is restricted to a narrow window between 15 and 20 kb from the promoter, a distance which coincides with the stage where accelerating Pol II attains maximum elongation speed. Consequently, long genes show a greater dependency on Spt5 for optimal elongation efficiency and overall gene expression than short genes. We propose that an important role of Spt5 in mammalian elongation is to promote the processivity of those Pol II complexes that are transitioning toward maximum elongation speed 15-20 kb from the promoter.

Spt5-mediated enhancer transcription directly couples enhancer activation with physical promoter interaction.

Active enhancers are frequently transcribed, yet the regulatory role of enhancer transcription remains debated. Here, we depleted the RNA polymerase II pausing and elongation factor Spt5 in activated mouse B cells and found that approximately 50% of enhancer-gene pairs showed co-regulated transcription, consistent with a potential functional requirement for enhancer transcription. In particular, Spt5 depletion led to loss of super-enhancer-promoter physical interaction and gene expression at the immunoglobulin heavy-chain locus (Igh), abrogating antibody class switch recombination. This defect correlated strictly with loss of enhancer transcription but did not affect acetylation of histone H3 at lysine 27, chromatin accessibility and occupancy of Mediator and cohesin at the enhancer. Strikingly, CRISPRa-mediated rescue of enhancer transcription in Spt5-depleted cells restored Igh gene expression. Our work suggests that Spt5-mediated enhancer transcription underlies the physical and functional interaction between a subset of active enhancers and their target promoters.