KAS-CUT&Tag for direct mapping of transcription bubbles

Key results

1. ShownBubble density (the ratio of IP to total) varies widely among active promoters that have similar Pol II occupancy, and it tracks nascent transcription measured by PRO-seq. It did not follow R-loop signal at active TSSs.

   Of 3,859 active TSSs in K562 (the top 20% of 19,294 by Pol II occupancy), 674 (17%) were bubble-rich (log2 > 1) and 194 (5%) bubble-poor (log2 < -1). PRO-seq medians: 6.70 rich, 5.49 moderate, 3.55 poor, 0.982 inactive (all pairwise BH-adjusted p ≤ 3.10 × 10^-11). R-loops from three HEK293T methods were more frequent at active TSSs but did not correlate with bubble density. · KAS-CUT&Tag maps transcription bubbles; Figure legends

2. ShownThe bubbles come from engaged Pol II. Blocking initiation lowers bubble density at TSSs, while blocking pause release or elongation raises it. TFIIS knockout lowers it at TSSs, gene bodies and post-TES regions without matching losses of occupancy.

   K562, 1 h: triptolide reduced Pol II occupancy and bubble density at TSSs; flavopiridol and actinomycin D increased both (two-sided KS, Holm-adjusted P ≤ 1.039 × 10^-224 for bubble density). HEK293T TFIIS KO: TSS bubble density P = 1.35 × 10^-219 despite higher occupancy; gene-body Pol II-Ser2p density p < 9.99 × 10^-30 with occupancy p = 0.062; post-TES density P = 3.5 × 10^-51. · KAS-CUT&Tag maps transcription bubbles; Transcription bubble density varies across gene…; Human cell culture; Figure legends

3. ShownReplication-coupled (RC) histone genes have the highest bubble density in K562 and GM12878. In K562, KAS-CUT&Tag and CUT&RUN.ChIP show NPAT on these genes together with Pol II. Colocalized NPAT and Pol II-Ser5p foci appear throughout interphase in K562 but mainly in S phase in GM12878.

   In K562, 51/57 active RC histone genes ranked in the top 10% of 3,859 active genes by bubble density (47/57 by PRO-seq). CUT&RUN.ChIP median ratios to Pol II-Ser5p input: NPAT 2.59 vs IgG 2.03 at histone TSSs, 1.15 vs 1.00 at other active TSSs (RPB3 6.48, 1.63). Cells with colocalized foci: GM12878 about 90% in S phase vs 21% in gap phase; K562 75% vs 70%. · Transcription factor NPAT is juxtaposed to…; Figure legends

4. ShownGene bodies with dense Pol II-Ser2p bubbles carry more H3K36me3 and more chromatin-bound U2AF2. The bubble-rich, U2AF2-high genes include RNA-splicing genes.

   K562, 3,754 active genes: H3K36me3 medians 4.66 (rich, n = 342), 2.47 (moderate, n = 3,349), 0.20 (poor, n = 63), BH p ≤ 2.77 × 10^-17. High U2AF2 in 63% (217/342) of rich, 26% (872/3,349) of moderate and none of the poor gene bodies; GO linked 18% of the 217 to RNA splicing. In HEK293, U2AF2 was about 10-fold (rich) and 4-fold (moderate) above poor. · Transcription bubbles are enriched at genes…; Figure legends

5. ShownN3-kethoxal blocks Tn5 insertion into labeled single-stranded DNA just downstream of TSSs. This depletes short fragments in kethoxal-treated CUT&Tag and in published KAS-ATAC data, while Pol II-Ser2p profiles over gene bodies are largely unaffected.

   Kethoxal-treated Pol II-Ser5p CUT&Tag lost signal from the TSS to about +500 bp, where K562 KAS-seq signal is enriched; V-plots over 12,397 protein-coding TSSs showed a loss of subnucleosomal fragments. Kethoxal-treated HEK293T ATAC-seq from two laboratories showed the same loss, plus depleted Tn5 insertion sites downstream of TSSs. IP signal did not track GC content. · KAS-CUT&Tag detects single-stranded DNA bound by…; Transcription bubble density varies across gene…; Data analysis and visualization; Figure legends