Lineage-specific 3D genome organization is assembled at multiple scales by IKAROS

Abstract

A generic level of chromatin organization generated by the interplay between cohesin and CTCF suffices to limit promiscuous interactions between regulatory elements, but a lineage-specific chromatin assembly that supersedes these constraints is required to configure the genome to guide gene expression changes that drive faithful lineage progression. Loss-of-function approaches in B cell precursors show that IKAROS assembles interactions across megabase distances in preparation for lymphoid development. Interactions emanating from IKAROS-bound enhancers override CTCF-imposed boundaries to assemble lineage-specific regulatory units built on a backbone of smaller invariant topological domains. Gain of function in epithelial cells confirms IKAROS' ability to reconfigure chromatin architecture at multiple scales. Although the compaction of the Igκ locus required for genome editing represents a function of IKAROS unique to lymphocytes, the more general function to preconfigure the genome to support lineage-specific gene expression and suppress activation of extra-lineage genes provides a paradigm for lineage restriction.

Keywords: 3D genome organization; Hi-C; HiChIP; IKAROS; Igκ locus contraction; enhancer loops; inter-compartmental loops; lineage-specific genome organization; lymphocyte development; superTADs.

Figure 1.. IKAROS controls spatial organization of large pre-B cells

(A) Schematic of early B-cell differentiation and conditional deletion of the IKAROS DNA binding domain (IKDN). (B) Experimental strategy to profile WT and IKDN large pre-B cells. Schematic (bottom) illustrates the classification of loops, anchors and differential loops (n=2 replicates). (C) Hi-C and SMC1, H3K27ac, CTCF and IKAROS HiChIP experiments shown at 50kb and 10kb resolution around Nrxn1 in WT (upper triangle) and IKDN (lower triangle). Pixel intensity represents the observed number of contacts/valid read pairs. (D) The number and percentage of Hi-C and HiChIP interactions (WT, IKDN) and differential loops (Down, Up) identified at 10kb resolution stratified by anchor classification. (E) Aggregate peak analysis (APA) of differential chromatin loops. Top labels indicate contact maps from which the aggregate pattern is derived. (F) Distance distributions of all significant chromatin loops identified by Hi-C and HiChIP in WT or IKDN. **** p < 0.0001. (G) The percentage of differential HiChIP loops that overlap with IKAROS ChIP-seq peaks at both (IK_A1A2), only one (IK_A1, IK_A2) or none of the anchors (No IK). Model of long-distance interactions between regulatory sites supported by IKAROS-bound dimers. (H) Distribution of IKAROS peaks that overlap anchors of differential loops identified by HiChIP and Hi-C. (I) Average IKAROS ChIP-seq enrichment (RPM) at enhancer and promoter anchors (10kb) from differential H3K27ac loops as in Figure S1H. Source: TableS1-3.

Figure 2.. IKAROS-dependent 3D chromatin organization controls B cell identity

(A) Change in gene expression between WT and IKDN (DEGs) and overlap with H3K27ac HiChIP loops that are up (red), down (blue) or unchanged (grey). (B) DEGs associated with differential (Down and Up) and non-differential (ND) loops identified by H3K27Ac HiChIP. **** p < 0.0001. (C) GO pathway enrichments for all genes associated with differential H3K27ac loops or DEGs. Statistical significance (−log10p-value) is shown by color scale and gene ratio by circle size. (D) K-means clustering of expression during WT hemo-lymphopoiesis and B cell differentiation of all genes associated with downregulated loop anchors. Examples downregulated in IKDN pre-B cells are highlighted in blue. (E) Examples of down-regulated and (F) up-regulated genes with changes in long-distance interactions. Hi-C contact maps (Observed/Expected, 5kb resolution). Compartment affiliation (PCA): euchromatin-blue, heterochromatin-orange. Normalized histone modification and transcription factor ChIP-seq tracks are shown. Interactions identified by FitHiChIP for IKAROS, H3K27ac, SMC1 and CTCF are shown. Highlights: blue -genes, yellow-re-directed loop anchors. Source: Tables S2-4.

Figure 3.. IKAROS regulates TAD and sub-TAD organization

(A) Size distribution of common and differential TADs. Red dashed line shows the median size of common TADs. (B) Differential aggregate TAD analysis (ATA) of TADs classified into split (1,2) and contracted (4) in IKDN or split in WT (3). WT-IKDN score is shown in all panels. A schematic of TADs in WT and IKDN is shown with the perspective employed for analysis shaded yellow. (2) subset of TADs in (1) with (Z1 > Z0). (3) Note absence of signal in the Z0 sector. (4) Filter X1>X0 reveals the subset of contracted TADs where loss of border-spanning contacts drives the shift. (C) Merged and split TADs at the Igκ locus. O/E Hi-C contacts and TAD calls for WT and IKDN. (D) Loss in CTCF hybrid loops leading to TAD boundary shift around the Blk locus (yellow stripe). (E) Aggregate regional analysis (ARA) of WT and IKDN O/E Hi-C signal at anchors of downregulated hybrid and structural CTCF HiChIP loops. Schematics illustrate the insulation and conduction properties of each anchor type. (F) ARA of WT and IKDN O/E Hi-C signal at enhancer and promoter anchors of downregulated H3K27ac HiChIP loops with or without CTCF (+ or −) binding. Schematics illustrate interactions between adjacent domains and potential mechanisms involved. (G) Aggregate WT and IKDN Hi-C signals of downregulated and upregulated hybrid and structural CTCF HiChIP loops. Difference between WT and IKDN aggregate signals is shown on the right. (H) Average ChIP-seq enrichment (RPM) at downregulated structural loop anchors and at internal decommissioned enhancers. (I) Model by which IKAROS organizes TADs. Source: Tables S2-3, S5.

Figure 4.. Higher-order chromatin compartmentalization is controlled by IKAROS

(A) Bins with different compartment affiliation scores between IKDN and WT classified by affiliation shift. (B) Distributions of differential compartmental bins with (colored) or without (black outline) overlap with differential loop anchors identified by indicated HiChIP and numbers are shown. (C) Differential H3K27ac regulatory loops classified in WT as intra-compartmental, inter-compartmental and consecutive. (D) Examples of compartmental changes associated with intra-(left) and inter-(right) compartmental regulatory loops downregulated in IKDN. Source: Tables S2-3, S6.

Figure 5.. IKAROS maintains lineage-specific chromatin organization

(A) Cultured large pre-B cells harvested at indicated time points after Cre-ERT2 induction and used for Hi-C, HiChIP and ChIP-seq. Detection of IKAROS WT (IK1, IK2) and mutant isoforms (ΔIK1, 2, 3, 7) is shown. (B) The number and percentage of loops (d0, d3, d12) and differential loops identified by H3K27ac HiChIP (d3 vs. d0 and d12 vs. d0), stratified according to anchor classification. (C) Contact counts for the differential H3K27ac HiChIP loops identified after in vitro deletion (d12 vs d0) are displayed for d0, d3, d12 and for WT and IKDN (in vivo). (D) Contact counts for the differential H3K27ac HiChIP loops identified after in vivo deletion (WT vs. IKDN) displayed as in (C). (E) Sites of differential H3K27ac interactions identified in vivo analyzed after in vitro deletion and segregated into clusters of slow, intermediate, and fast changes in contact strength. Average H3K27ac enrichment at the loop anchors and flanking regions is shown across the time course for each group. (F) Downregulated H3K27ac loops at d3 classified according to change of H3K27ac ChIP-seq signal at their anchors (HD high difference, LD low difference, ND no difference). (G) Z-score transformed compartment scores derived from Hi-C data of d0-d18 after in vitro IKAROS deletion were plotted for the differential compartment bins identified in vivo. Positive z-scores = stronger euchromatic affiliation in WT. (H, I) Steady-state (WT, IKDN upper tracks) and in vitro deletion data (d0, d3, d12, lower tracks) are compared for the Nrxn1 (H) and Igλ (I) locus. Source: Tables S2-3, S6-7.

Figure 6.. Regulation of chromatin organization and locus contraction at the Igκ locus

(A) Hi-C at the Igκ locus for WT and IKDN large pre-B cells (10kb). TADs (A-C) and FISH probes are marked. Orientation of Igκ-V segments relative to the J segments is indicated (red-forward and blue-reverse). (B) Genomic tracks as in Figure 5H. (C) The 3’ boundary domain is shown with ChIPseq peaks for indicated chromatin marks, B lineage TFs and other factors. Regulatory (iEκ, 3’Eκ, Ed, HS10) and structural (Cer, Sis) elements and the RCSE (green) that spans them are highlighted. (D) Igκ locus DNA-Immuno FISH using the Vκ (green) and Cκ (red) probes in WT and IKDN pre-B cells (pSer2 RNA Pol II (blue). (E) Distance between Vκ-Cκ regions in WT and IKDN large pre-B cells, **** p < 0.0001). Source: Tables S2-3, S6-7.

Figure 7.. Extra-lineage chromatin organization induced by IKAROS in skin epithelial cells

(A) Experimental strategy and Western blot of IKAROS induction. (B) Number and percentage of loops and differential loops (Lost, Induced (Ind.)) from H3K27ac HiChIP at d3 stratified according to anchor classification and presence/absence of IKAROS ChIP-seq peaks in at least one anchor (IK(+), IK(−)). (C) De novo TF-binding motif enrichment of IKAROS peaks within upregulated H3K27ac loop anchors. (D) Size distribution of induced H3K27ac loops at d3 with (+IK) or without (-IK) IKAROS at their anchors. **** p <= 0.0001. (E) Compartmental changes between Luciferase- and IKAROS-expressing cells at d2. (F) Distributions of differential compartmental regions with and without overlap with IKAROS peaks or induced H3K27ac or CTCF loop anchors. (G-H) Examples of IKAROS-mediated induction of chromatin topology. The IGK RC (red) and iEκ (green) are highlighted. Source: Tables S3-4, S6-7.