Proximity-dependent recruitment of Polycomb repressive complexes by the lncRNA Airn

Abstract

During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between the Airn lncRNA and chromatin correlated with underlying intensity of PRC recruitment and PRC-directed modifications. Deletion of CpG islands that contact the Airn locus altered long-distance repression and PRC activity in a manner that correlated with changes in chromatin architecture. Our data imply that the extent to which Airn expression recruits PRCs to chromatin is controlled by DNA regulatory elements that modulate proximity of the Airn lncRNA product to its target DNA.

Keywords: Airn; CHART-seq; CP: Molecular biology; ChIP-seq; Hi-C; Xist; chromatin archtecture; epigenetics; long noncoding RNA; polycomb repressive complex; transcription.

Figure 1.. Airn expression induces large-scale changes to chromatin architecture

(A) Hi-C contact heatmaps of allelic observed counts in (i) C/B wild-type, (ii) C/B Airn truncation, and (iii) B/C wild-type TSCs; n = 2 or 3. Allelic heatmaps are partitioned and at 50-kb resolution. (B) Subtraction contact heatmaps of log2-transformed (PAT – MAT) observed counts (i–iii) as in (A). (C) Eigenvectors at 50-kb resolution for “A” and “B” chromosome compartmentalization. In heatmaps: dotted lines, 15-Mb Airn target domain; purple circle, Airn gene; green circles, other loci of interest. Datasets used are listed in Table S4. KR bal., Knight-Ruiz balanced. See STAR Methods for detailed description of analyses. See also Figure S1.

Figure 2.. Airn-dependent changes in chromatin architecture coincide with the presence of PRC-deposited modifications

(A) Tiling density plots of allelic Hi-C Airn viewpoint observed contact counts in (i) C/B wild-type, (ii) C/B Airn truncation, and (iii) B/C wild-type TSCs. Colored blocks in (i), and FISH probes analyzed in (B). (B) Allelic Hi-C Airn viewpoint contacts from (Ai) vs. average distance to Airn measured by RNA/DNA FISH in C/B wild-type TSCs from Schertzer et al. Spearman’s r and p values are shown. (C and D) Tiling density plots of allelic (C) H3K27me3 ChIP-seq and (D) H2AK119ub ChIP-seq signal in C/B wild-type TSCs. Data are from Schertzer et al. n=4 and 2. (E) Tiling density plot of allelic Airn viewpoint (PAT – MAT) observed counts in C/B wild-type TSCs from (Ai). (F) Scatterplots of Airn viewpoint (PAT – MAT) observed counts vs. (left) H3K27me3 and (right) H2AK119ub. Spearman’s r and p values are shown. In tiling plots: yellow/purple bar, Airn viewpoint/gene; green bars, other loci of interest. Datasets used are listed in Table S4. Norm Obs Counts, counts normalized for SNP density; Norm RPM, reads per million total reads normalized for SNP density. See STAR Methods for detailed description of analyses. See also Figure S2.

Figure 3.. Airn-dependent repression centers around regions that form pre-existing contacts with the Airn locus and harbor CGIs bound by vPRC1

(A) Tiling density plots of allelic Hi-C Airn viewpoint observed-over-expected (O/E) contact counts in (i) C/B wild-type, (ii) C/B Airn truncation, and (iii) B/C wild-type TSCs. y axes as in Figure 2. Yellow bar, viewpoint; green bars, other loci of interest. (B) Genome browser graphics of regions harboring peaks of O/E contact. ChIP-seq tracks, non-allelic read density from C/B wild-type TSCs; n = 2 or 3. Red or blue asterisks, significant enrichment on maternal or paternal alleles, respectively (p < 0.05, permutation). Pink rectangles, DNA regions deleted in Figures 5, 6, and 7. Datasets used are listed in Table S4. See STAR Methods for detailed description of analyses. See also Figure S4 and Table S3.

Figure 4.. Presence of Airn lncRNA on chromatin correlates with presence of PRC1 and PRC2 and centers around pre-existing contacts with the Airn locus

(A–D) Tiling density plots of allelic (A) Airn CHART-seq, (B) H3K27me3 ChIP-seq, (C) RING1B ChIP-seq, and (D) EZH2 ChIP-seq signal in C/B (i) wild-type, (ii) Airn highly expressing (H-E), and (iii) Airn truncation TSCs; n = 1 or 2. y axes are as in Figure 2. H3K27me3 and RING1B ChIP-seq data are from Schertzer et al. (E and F) Tiling density plots of (E) Airn CHART-seq and (F) H3K27me3 ChIP-seq signal in untreated (Airn OFF) or dox-treated (Airn ON) H-E ESCs; n = 1. (G) Tiling density plot of Hi-C Airn viewpoint O/E counts in ESCs. Yellow/purple bar, Airn viewpoint/gene; green bars, other loci of interest. Datasets used are listed in Table S4. See STAR Methods for detailed description of analyses. See also Figures S2 and S5.

Figure 5.. DNA regulatory element deletions alter levels of PRC-directed modifications and gene repression throughout the Airn target domain

(A–D) Tiling density plots of allelic (left) H3K27me3 and (right) H2AK119ub ChIP-seq signal in C/B (A) ΔSlc22a3, (B) ΔT, (C) ΔPde10a, and (D) ΔCluster TSCs; n = 1 per clonal line. y axes are as in Figure 2. Data from NTG clones were averaged; n = 4. (E) Paternal expression of the 27 Airn target genes across genotypes relative to NTG; n = 1, 2, or 4. Asterisks, significant changes relative to NTG (p < 0.05, Welch two-sample t test). In tiling plots: purple bar, Airn gene; green bars, other loci of interest; pink bars, DNA regions deleted. Datasets used are listed in Table S4. See STAR Methods for detailed description of analyses. See also Figure S6 and Table S2.

Figure 6.. Changes in DNA contacts with Airn mirror changes in PRC activity caused by regulatory element deletion

(A) Hi-C subtraction contact heatmaps of log2-transformed (PAT – MAT) observed counts in C/B (i) NTG, (ii) ΔSlc22a3, and (iii) ΔPde10a TSCs; n = 2. (B) Eigenvectors at 50-kb resolution for “A” and “B” chromosome compartmentalization (i–iii) as in (A). (C) Tiling density plots of allelic Airn viewpoint observed contact counts (i–iii) as in (A). y axes as in Figure 2. (D) Tiling density plots of allelic Airn viewpoint (PAT – MAT) observed counts (i–iii) as in (A). In heatmaps: dotted lines, 15-Mb Airn target domain; purple circles, Airn gene; green circles, other loci of interest. In tiling plots: yellow bar, Airn locus viewpoint; green bars, other loci of interest; pink bar, DNA region deleted. Datasets used are listed in Table S4. See STAR Methods for detailed description of analyses. See also Figure S7.

Figure 7.. Airn expression is coincident with dissolution of DNA loops encasing Slc22a3 and a local increase in PRC-directed modifications

(A) Allelic Hi-C contact heatmaps of the Airn contact domain at 5-kb resolution in (i) C/B wild-type, (ii) C/B Airn truncation, and (iii) B/C wild-type TSCs. Black arrows, SIP-called DNA loops. (B) SMC1A/Cohesin and CTCF ChIP-seq. Percent parental biases at loops (i and ii) as in (A). (C) Allelic contact heatmaps of Airn contact domain at 5-kb resolution in C/B (i) NTG, (ii) ΔSlc22a3, and (iii) ΔPde10a TSCs. Gray arrows, DNA loops of interest from (A). In heatmaps: circles, 5′ end of genes/CGIs; rectangles, gene bodies; purple, Airn gene; green, Slc22a3 gene. (D) Boxplots of average allelic (left) H3K27me3 and (right) H2AK119 ChIP-seq signal over Airn contact domain in NTG, ΔSlc22a3, and ΔPde10a TSCs; n = 2. Asterisks, significant changes relative to NTG (p < 0.05, Welch two-sample t test). Error bars, data points outside the interquartile range. (E) Model: DNA regulatory elements modulate frequency of contact with and repression by Airn. (i) On maternal allele, pre-existing contacts with Airn locus render certain regions more susceptible torepression than others. (ii) On paternal allele, PRCs and PRC-deposited modifications to chromatin increase contacts with Airn locus, which in turn increase intensity of long-distance repression. (iii) Loss of Slc22a3 CGI attenuates repression by reducing local PRC recruitment and contact with distal regions. (iv) Loss of Pde10a CGI increases frequency with which surrounding regions contact Airn. Datasets used are listed in Table S4. See STAR Methods for detailed description of analyses.