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Enhancer–promoter contact formation requires RNAPII and antagonizes loop extrusion

Nature Genetics volume 55, pages 832–840 (2023)Cite this article

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Abstract

Homotypic chromatin interactions and loop extrusion are thought to be the two main drivers of mammalian chromosome folding. Here we tested the role of RNA polymerase II (RNAPII) across different scales of interphase chromatin organization in a cellular system allowing for its rapid, auxin-mediated degradation. We combined Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Gained loops, extrusion of which was antagonized by RNAPII, almost invariably formed by engaging new or rewired CTCF anchors. Lost loops selectively affected contacts between enhancers and promoters anchored by RNAPII, explaining the repression of most genes. Surprisingly, promoter–promoter interactions remained essentially unaffected by polymerase depletion, and cohesin occupancy was sustained. Together, our findings reconcile the role of RNAPII in transcription with its direct involvement in setting-up regulatory three-dimensional chromatin contacts genome wide, while also revealing an impact on cohesin loop extrusion.

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Fig. 1: Micro-C enhances high-resolution views of 3D genome folding.
Fig. 2: Loops forming upon RNAPII degradation engage new CTCF anchors.
Fig. 3: RNAPII depletion selectively affects enhancer–promoter and enhancer–enhancer loops.
Fig. 4: Models of 3D chromatin folding in the presence or absence of RNAPII.

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Data availability

NGS data generated in this study are available via the NCBI Gene Expression Omnibus repository under accession number GSE178593 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE178593). All other data used for analyses come from our previous study39, and are available under accession number GSE160321 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE160321). Source data are provided with this paper.

Code availability

All custom code used for Micro-C analysis is available at https://github.com/shuzhangcourage/Micro-C-CUT-tag/tree/v1.0.0 (ref. 73), and all the code used for our Molecular Dynamics simulations is available at https://zenodo.org/record/7674875#.Y_n5zXbMLBR (ref. 74).

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Acknowledgements

We thank M. Oudelaar, K. Wendt and all members of the Papantonis lab for discussions, and the Maeshima lab (NIG, Japan) for the DLD-1 mAID-RPB1 cells. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) via the SPP2202 (PA 2456/11-2) and SPP2191 Priority Programs (PA 2456/17-1), and the TRR81 TransRegio program (INST 160/697-1), all awarded to A.P. S.Z. is supported by a China Scholarship Council fellowship. S.Z. and N.Ü. are members of the International Max Planck Research School for Genome Science. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. These authors contributed equally: Shu Zhang, Nadine Übelmesser.

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  1. Institute of Pathology, University Medical Center Göttingen, Göttingen, Germany

    Shu Zhang, Nadine Übelmesser, Mariano Barbieri & Argyris Papantonis

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S.Z. performed all bioinformatics analyses; N.Ü. performed all experiments; M.B. performed the computational modeling; and A.P. conceived and supervised the study and compiled the manuscript with input from all co-authors.

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Correspondence to Argyris Papantonis.

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Extended data

Extended Data Fig. 1 Effects of RNAPII depletion on chromatin organization and protein levels.

a, Left: Schematic of the biallelic tagging strategy in the endogenous POLR2A loci. Right: Fractionation blots showing the levels of RPB1 and Ser5-phosphorylated RNAPII, Mediator subunit 24, and Lamin B1 from DLD1-mAID-RBP1 cells treated or not with auxin to deplete RNAPII. HSC70 levels provide a control. Blots have been replicated at least twice. b, Representative tracks of CUT&Tag signal for H3K27me3, H3K27ac, SMC1A, and CTCF from control (left) and auxin-treated DLD1-mAID-RBP1 cells (right) along 0.55 Mbp of chr1. c, Heatmaps of nucleosome occupancy deduced from Micro-C data, of chromatin accessibility deduced from ATAC-seq, and of CTCF and SMC1A occupancy deduced from CUT&Tag around CTCF loop anchors before (ctrl) and after RNAPII degradation (+auxin). d, As in panel c, but showing scaled ATAC-seq signal around gene promoters and enhancers. e, As in panel a, but for Ser5-phosphorylated RNAPII, NIPBL, SMC1A, CTCF, and H3K27me3 levels in the soluble and chromatin fractions of DLD-1 cells. HSC70 levels provide a control. Blots have been replicated at least twice. f, Boxplots depicting the distribution of genes containing (genes with +aux loops) or not (ctrl genes) gained loop anchors upon RNAPII depletion. In the plots, center lines represent the median value, box-limits the 25th and 75th percentiles, and whiskers extend 1.5x each box’s interquartile range. *P < 0.01, two-sided Wilcoxon-Mann-Whitney test.

Source data

Extended Data Fig. 2 Effects of RNAPII depletion on the 3D organization of facultative heterochromatin.

a, Micro-C contact maps from control (left) and auxin-treated cells (right) in two exemplary genomic regions of chr1 at 2-kbp resolution aligned to H3K27me3, H3K27ac, CTCF, and SMC1A CUT&Tag signal tracks. Loops called for each region and condition are also shown by spider plots (bottom). b, Aggregate plots of all H3K27me3-anchored loops emerging in auxin-treated cells. c, Bar plot showing per cent of gained loops with one or two H3K27me3 anchors or with H3K27me3 in the next-door genomic bin (that is, within <10 kbp from the anchor).

Extended Data Fig. 3 Changes in loops and stripes following RNAPII depletion.

a, Micro-C contact maps from control (left) and auxin-treated cells (right) in an exemplary genomic region on chr1 at 4-kbp resolution aligned to H3K27ac, CTCF, and SMC1A CUT&Tag signal tracks. Loops called for each region and condition are also shown by spider plots (bottom). b, Plots of interaction frequency decay as a function of genomic distance from control and auxin-treated cells (top) and their first derivative (bottom). c, Aggregate plots of gene promoter-promoter (P-P) or enhancer- promoter loops (E-P) in control and auxin-treated cells that involve (+CTCF) or not CTCF (wo CTCF) in at least one anchor. d, Line plot showing mean SMC1A CUT&Tag signal from control and auxin-treated cells in the 6 kbp around H3K27ac peaks from 590 super-enhancers. e, As in panel c, but for H3K27ac signal around active gene promoters or enhancers. f, Average plots showing mean signal of stripes with one CTCF and one transcriptional anchor before (ctrl) and after RNAPII depletion (+auxin). Zoom-in: Aggregate plots for loops at the end of the stripes. g, As in panel c, but for shared loops that rewire one anchor by <20 kbp (see cartoon). h, Boxplots depicting the lengths of genes downregulated upon RNAPII depletion that are linked (genes with lost E-P loops) or not (all other genes) to lost E-P loops. In the plots, center lines represent the medians, box-limits the 25th and 75th percentiles, and whiskers extend 1.5x each box’s interquartile range. *: P < 0.01, two-sided Wilcoxon-Mann-Whitney test.

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Zhang, S., Übelmesser, N., Barbieri, M. et al. Enhancer–promoter contact formation requires RNAPII and antagonizes loop extrusion. Nat Genet 55, 832–840 (2023). https://doi.org/10.1038/s41588-023-01364-4

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