LDB1 establishes multi-enhancer networks to regulate gene expression

Abstract

How specific enhancer-promoter pairing is established is still mostly unclear. Besides the CTCF/cohesin machinery, only a few nuclear factors have been studied for a direct role in physically connecting regulatory elements. Here, we show via acute degradation experiments that LDB1 directly and broadly promotes enhancer-promoter loops. Most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin, or YY1 as determined via the use of multiple degron systems. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1 occupied sites but may aid the formation of a subset of LDB1 anchored loops. Leveraging the dynamic reorganization of nuclear architecture during the transition from mitosis to G1-phase, we establish a relationship between LDB1-dependent interactions in the context of TAD organization and gene activation. Lastly, Tri-C and Region Capture Micro-C reveal that LDB1 organizes multi-enhancer networks to activate transcription. This establishes LDB1 as a direct driver of regulatory network inter-connectivity.

Figure 1.. LDB1 mediates chromatin contacts between cis-regulatory elements.

(A) Numbers of structural loops (left) and CRE loops (right) that are weakened (log2FC < −0.5), unchanged or strengthened (log2FC > 0.5) upon LDB1 depletion. Loops are stratified by LDB1 occupancy within anchors. (B) Distribution of CRE loop type for weakened CRE loops. Fraction of loops with RAD21/CTCF co-occupied peaks in both anchors (below). (C) Fraction of enhancers and promoters in G1E-ER4 cells occupied by LDB1 (left), YY1 (middle) and CTCF (right). (D) Schematic representing the motif analysis strategy for heterotypic loops and the top 10 most enriched motifs identified using HOMER known motif enrichment analysis. (E) Change in loop strength upon LDB1 depletion for loops categorized based on LDB1 and CTCF occupancy. Whiskers represent 10^th and 90^th percentiles; P-values calculated using a two-sided Mann-Whitney U test. (F) LDB1-dependent homotypic loop (red arrow) and LDB1-dependent heterotypic loop (green arrow).

Figure 2.. LDB1-dependent CRE loops are associated with transcription activation.

(A) Gene expression changes measured by TT-seq upon LDB1 depletion (n=3).

(B) Gene expression changes (TT-seq) for genes categorized by the number of loop anchors overlapping their TSS. Whiskers represent 10^th and 90^th percentiles; P-values calculated using a two-sided Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C) Baseline gene expression measured by TT-seq. Genes categorized by the number of LDB1 dependent or independent CRE loops they interact with. (D) Cumulative frequency distributions for gene distance to nearest LDB1 ChIP-seq peak. (E) Numbers of inter-TAD vs intra-TAD LDB1-dependent CRE loops. (F) Loop lengths for LDB1-dependent inter-TAD and intra-TAD CRE loops. Whiskers represent 10^th and 90^th percentiles. (G) Loop strengths for LDB1-dependent inter-TAD and intra-TAD CRE loops. Loop strength calculated using 5k resolution.

Figure 3.. LDB1 forms fine-scale looped networks at LDB1-dependent genes

(A) Numbers of LDB1-dependent loops detected by Micro-C or RCMC. ChIP-seq tracks for LDB1 are shown in black. (B) Proportions of LDB1 or CTCF ChIP-seq peaks overlapping weakened loop anchors identified by Micro-C (blue) or RCMC (green). For overlaps with RCMC, only peaks within captured regions are considered. Histograms (right) showing the number of LDB1 or CTCF peaks that overlap with increasing numbers of weakened loop anchors identified by RCMC. (C) Examples of LDB1-dependent looped networks. Green arrows indicate LDB1-dependent loops. (D) 5k resolution TRI-C contact maps for MYC proximal and distal regions. Contacts represent multi-way interactions involving the MYC promoter. Capture probe bin indicated by black arrow. (E) Multiway contacts with the MYC promoter and bins occupied by LDB1 or unoccupied by LDB1. Dots represent normalized multiway contacts for each biological replicate. P values calculated using paired t-test.

Figure 4.. LDB1 occupancy is mutually independent of YY1, CTCF and cohesin at most locations.

(A) ChIP-seq peak intersections between LDB1, CTCF, RAD21 and YY1. (B) LDB1-occupied enhancer elements that are occupied by cohesin (RAD21), YY1, or CTCF. (C) ChIP-seq profiles in LDB1-AID cells for RAD21, CTCF and YY1 before/after LDB1 depletion. Heatmaps and profiles are shown for peaks identified for each factor in the LDB1 replete condition. (D) RAD21 ChIP-seq signal at RAD21 ChIP-seq peaks overlapping CTCF peaks or LDB1 peaks. Whiskers are 10^th and 90^th percentile. (E) ChIP-seq profiles in SMC3-AID, CTCF-AID, and YY1-AID cells before/after 4hr auxin treatment.

Figure 5.. LDB1 Can Function in the absence of cohesin

(A) Change in loop strength for LDB1-dependent CRE loops in response to LDB1 depletion (darker colors) or SMC3 depletion (lighter colors). Loops are categorized as LDB1 only loops (red) or dual sensitive loops (blue). (B) H3K27ac ChIP-seq signal at enhancers within LDB1-only loop anchors or dually-sensitive loop anchors. Only mutually exclusive enhancer elements between the two sets are considered. (C) Relative RNA levels for β-globin measured by RT-qPCR in SMC3-AID cells −/+ ZF-SA and −/+ auxin (4hr). P-values calculated using One-way ANOVA. *p < 0.05, **p < 0.01. (D) Lengths of LDB1 only and LDB1/cohesin dual sensitive loops. (E) Distance to encompassing structural loop anchors for LDB1-only loops and dually sensitive loops. Only loops with an encompassing structural loop are shown. (F) APA plots for LDB1-dependent CRE loops stratified by their response to SMC3 depletion and whether they are encompassed by a structural loop. Numbers represent raw center pixel values.

Figure 6.. LDB1 chromatin occupancy correlates with loop establishment during G1-phase entry

(A) ChIP-seq profiles for LDB1 at each cell cycle stage at all LDB1 peaks identified in asynchronous cells. (B) APA plots from 10k resolution Hi-C data at each cell cycle stage for each category of LDB1-dependent CRE loops. Average ChIP-seq profiles are shown for each loop type for LDB1 peaks within loop anchors. (C) Loop strength (top) and observed contacts between loop anchors (bottom) for each category of LDB1-dependent CRE loops and for structural loops at each cell cycle stage. Median loop strength and observed contacts normalized to prometaphase are shown for each loop category (right). (D) Examples of an LDB1/cohesin dually sensitive loop (top) and LDB1-only loop (bottom). Green arrow indicates the LDB1-dependent loop, blue arrow indicates an encompassing structural loop.