Transcription processes compete with loop extrusion to homogenize promoter and enhancer dynamics

Abstract

The spatiotemporal configuration of genes with distal regulatory elements is believed to be crucial for transcriptional control, but full mechanistic understanding is lacking. We combine simultaneous live tracking of pairs of genomic loci and nascent transcripts with molecular dynamics simulations to assess the Sox2 gene and its enhancer. We find that both loci exhibit more constrained mobility than control sequences due to stalled cohesin at CCCTC-binding factor sites. Strikingly, enhancer mobility becomes constrained on transcriptional firing, homogenizing its dynamics with the gene promoter, suggestive of their cotranscriptional sharing of a nuclear microenvironment. Furthermore, we find transcription and loop extrusion to be antagonistic processes constraining regulatory loci. These findings indicate that modulating chromatin mobility can be an additional, underestimated means for effective gene regulation.

Fig. 1.. Constrained Sox2 promoter and enhancer mobility.

(A) Overview of the Sox2 locus, showing (top to bottom): ESC Hi-C map, showing the TAD delimited by Sox2 and SCR (positions shown in blue); scaled positions of ANCH1 and ANCH3 labels inserted in the Sox2-SCR_WT and Inter-Down ESC lines; zoomed-in ESC chromatin immunoprecipitation sequencing profiles for acetylated histone H3 lysine-27 (H3K27ac, blue), the cohesin subunit RAD21 (brown), and CTCF (orange). Red and cyan rectangles indicate the exact positions of the ANCH3 and ANCH1 labels, respectively. Positions of elements deleted in this study (SRR107 and SRR111, cyan; CTCF_Prom, red; CTCF_SCR, blue) are indicated. Orientations of major CTCF motifs are denoted by arrows. (B) Representative images of Sox2-SCR_WT (top) and Inter-Down (bottom) nuclei (after segmentation and removal of cytoplasmic signal); scale bar, 2 μm. Insets show zoomed regions around spots corresponding to bound parS sequences. (C) Musculus-specific 4C-seq profiles (mean of two replicates) using the SCR as bait are shown for F1 and Sox2-SCR_WT ESCs before and after transfection with ParB vectors. The two regions consistently called as interactions are denoted in gray, and the minimum P values for compared interaction scores with F1 (two-tailed t test) are denoted. (D) Uncorrected ensemble MSD curves for labeled Sox2 (red), SCR (green), Inter (light gray), and Down (dark gray) sequences, with lines showing median values and shading indicating the MAD. (E) Sample tracks of the 2D trajectories for the same four regions as in (D), plotted on the same scale. Displacements have been corrected for substrate movement, and the plots have been centered on the median positions. (F) Violin plot showing the distributions of apparent anomalous exponents measured from the individual movies for each of the four regions as in (D). Comparisons are made by Wilcoxon rank sum tests with Benjamini-Hochberg multiple testing correction, with q values given.

Fig. 2.. Stalled cohesin loop extrusion at CTCF sites limits chromatin mobility.

(A) Sample tracks of 2D trajectories for labeled SCR and Down regions, at either 0 or 3 days of differentiation, plotted on the same scale. Displacements have been corrected for substrate movement, and the plots have been centered on the median positions. (B) Violin plots showing the distributions of apparent anomalous exponents measured from the individual movies for Sox2, Inter, SCR, and Down regions, comparing 0 and 3 days of differentiation. Comparisons are made by Wilcoxon rank sum tests, with P values given. (C) Sample tracks of 2D trajectories for labeled Sox2 and SCR regions in Sox2-SCR_Rad21 cells after treatment with dimethyl sulfoxide (DMSO) (control) or dTag-13 (to abrogate RAD21). Displacements have been corrected for substrate movement, and the plots have been centered on the median positions. (D) Violin plots showing the distributions of apparent anomalous exponents measured from the individual movies for Sox2, Inter, SCR, and Down regions of Sox2-SCR_Rad21 or Inter-Down_Rad21 cells, comparing DMSO and dTag-13 treatments. Comparisons are made by Wilcoxon rank sum tests with Benjamini-Hochberg correction, with q values given. (E) Sample tracks of 2D trajectories for labeled Sox2 and SCR regions in Sox2-SCR_WT cells or cells where the local CTCF site has been deleted (Sox2-SCR_ΔCTCF-Prom and Sox2-SCR_ΔCTCF-SCR, respectively). Displacements have been corrected for substrate movement, and the plots have been centered on the median positions. (F) Violin plots showing the distributions of apparent anomalous exponents measured from the individual movies for Sox2, comparing Sox2-SCR_WT, Sox2-SCR_ΔCTCF-Prom, and Sox2-SCR_ΔCTCF-SCR cells. Comparisons with wild-type are made by Wilcoxon rank sum tests, with P values given. (G) Violin plots showing the distributions of apparent anomalous exponents measured from the individual movies for SCR, comparing Sox2-SCR_WT, Sox2-SCR_ΔCTCF-Prom, and Sox2-SCR_ΔCTCF-SCR cells. Comparisons with wild-type are made by Wilcoxon rank sum tests, with P values given.

Fig. 3.. Promoter and enhancer dynamics are synchronized on transcriptional firing.

(A) Schematic of triple-label system of Sox2-SCR_MS2 ESCs. The Sox2-SCR_WT line is additionally tagged with 24 copies of the MS2 repeat at Sox2 3′UTR for visualization of nascent RNA with MCP-ScarletI. (B) Left, representative image of transcriptionally active Sox2-SCR_MS2 nucleus, with the OR3-IRFP signal shown in red, the OR1-EGFP signal shown in cyan, and the mScarletI signal shown in yellow; scale bar, 2 μm. Insets show zoomed regions around spots corresponding to bound parS sequences and transcription site. Right, images of the same Sox2-SCR_MS2 nucleus at 30-s intervals, showing each of the three channels individually in monochrome, and the overlaid image with the same color scheme as on the left. Arrows show the position of a transcription site (MCP⁺), which is inactivated by ~3 min. Scale bar, 2 μm. (C) Ensemble MSD curves, corrected for substrate movement, comparing active and inactive alleles at labeled Sox2 (orange and dark red, respectively) and SCR (green and dark green, respectively) regions. Lines indicate median values and shading the MAD. Red arrow indicates vertical shift in MSD plot when comparing Sox2 dynamics, and green/dark green arrows indicate the changes in slope of the SCR MSD plots. (D) Violin plot showing the distributions of apparent anomalous exponents measured from the individual movies for Sox2 and SCR in Sox2-SCR_MS2 cells, comparing transcriptionally active and inactive alleles. Comparisons are made by Wilcoxon rank sum tests, with P values given. (E) Sample tracks of the 2D trajectories for labeled Sox2 and SCR regions in Sox2-SCR_MS2 cells, which are transcriptionally active (left) or inactive (right). Displacements have been corrected for substrate movement, and the plots have been centered on the median positions.

Fig. 4.. Loop extrusion and transcriptional processes compete to constrain chromatin.

(A) Violin plot showing distributions of apparent anomalous exponents measured for Sox2 and SCR in Sox2-SCR_WT and Sox2-SCR_ΔSRR107,111 cells. Comparisons are made by Wilcoxon rank sum tests, with P values given. (B) Sample tracks of 2D trajectories for labeled SCR in Sox2-SCR_WT and Sox2-SCR_ΔSRR107,111 cells. Displacements have been corrected for substrate movement, and the plots have been centered on the median positions. (C) Ensemble MSD curves for the SCR derived from simulations of wild-type ESCs (dark blue) and those with ΔSRR107,111 (cyan) or ΔCTCF-SCR (purple) when accounting for competition between cohesin and RNA polymerase occupancy. Lines indicate median values and shading the MAD. (D) Violin plot showing the distributions of apparent diffusion coefficients derived from simulations for the SCR in Sox2-SCR_WT, Sox2-SCR_ΔSRR107,111, and Sox2-SCR_ΔCTCF-SCR cells. Comparisons are made by Wilcoxon rank sum tests, with P values given. (E) Bar chart showing mean expected cohesin occupancy at the SCR-proximal CTCF site, derived from simulations of Sox2-SCR_WT and Sox2-SCR_ΔSRR107,111 cells. Comparisons are made by two-tailed t test. (F) ChIP-qPCR quantification, performed in triplicate, expressed as fold enrichment over a negative control region, of amount of RAD21 binding at the musculus/129 (colored) or castaneus (white) alleles of the SCR CTCF site in Sox2-SCR_WT and Sox2-SCR_ΔSRR107+111 cells. Comparisons between allelic binding are made by two-tailed t tests, with P values given. (G) Schematic of competing processes dictating dynamics at the SCR. At the top, extruding cohesin complexes (yellow) place temporary local constraints on mobility of the bound sequences. Such constraints are stabilized when the cohesin encounters bound CTCF (orange) in the appropriate orientation. At the bottom, bound RNA polymerase and transcription factors can also coordinate the Sox2 and SCR interaction but are refractory to cohesin recruitment and/or extrusion, hence maintaining a relatively more mobile chromatin locus.