HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

Abstract

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of β-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced β-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

Keywords: AML leukemogenesis; CTCF chromatin boundary; HOTTIP lncRNA; R-loops; TAD formation; canonical Wnt transcription; cohesin complex; enhancer/promoter interactions.

Figure 1.. HOTTIP interactome isolated from AML cells contains CTCF/cohesin complex and R-loop-associated proteins

(A) Schematic of HOTTIP ChIRP-LC-MS/MS/WB workflow. (B) Left: one-step blue protein gel staining of proteins isolated from MOLM13 cells by ChIRP using HOTTIP or control LacZ titling probes. Right: partial list of unique polypeptides identified in the HOTTIP, but not in the LacZ ChIRP-LC-MS/MS. (C) Overrepresentation analysis of enriched HOTTIP-interacting protein classes using the DAVID database. The ratio of each protein class represented in the HOTTIP-associated proteome was calculated and statistical significance was ranked according to the Benjamini-Hochberg-corrected p value (p ≤ 0.05). (D) ChIRP-WB validation of HOTTIP-associated proteins. (E) Biotin pull-down of CTCF mutants by biotinylated HOTTIP.

Figure 2.. HOTTIP directly binds a subset of CTCF boundaries by formation of R-loops in AML genome

(A) Heatmap of CTCF binding globally (top) and at HOTTIP/CTCF co-bound promoters (middle) and intergenic regions (bottom) in WT and HOTTIP^−/−; MOLM13 cells. p value calculated by Kolmogorov-Smirnov (K-S) test. (B) Top enriched TF-binding motifs in HOTTIP/CTCF co-occupied peaks according to de novo motif analysis. (C) Histogram of the distribution of HOTTIP and TF motifs within HOTTIP/CTCF co-occupied peaks. (D) Heatmap of R-loop peaks identified by DRIP-seq globally (left), at HOTTIP-bound regions (middle) and at HOTTIP/CTCF co-bound regions (right) in WT and HOTTIP^−/−; MOLM13 cells. p value calculated by K-S test. (E) Overlap of HOTTIP-binding peaks identified by ChIRP-seq and CTCF/cohesin co-occupied sites (left) or cohesin independent CTCF sites (right) identified by CTCF and RAD21 ChIP-seq in MOLM13 cells. (F) Overlap of total reduced HOTTIP peaks and all decreased CTCF/cohesin co-bound sites in the genome comparing WT and HOTTIP^−/−; MOLM13 cells. (G) Heatmap of RAD21, SA1, and SA2 binding identified by ChIP-seq at HOTTIP/CTCF co-bound sites in WT and HOTTIP^−/−; MOLM13 cells. p value calculated by K-S test. (H) Heatmap of H3K27me3 and H3K4me3 profiles identified by ChIP-seq at HOTTIP/CTCF co-bound sites in WT and HOTTIP^−/−; MOLM13 cells.

Figure 3.. HOTTIP regulates CTCF-defined TADs and transcription at canonical Wnt loci in AML genome

(A) Heatmap of ≥2-fold downregulated genes in MOLM13 cells upon HOTTIP^−/−; as determined by RNA-seq. (B) GSEA of downregulated genes after HOTTIP^−/−;. (C) Overlap of TADs identified by Hi-C in WT and HOTTIP^−/−; MOLM13 cells. The domain score of an altered TAD was normalized (quantile-normalization) by subtracting the mean of all TAD Hi-C signals. ANOVA was used to identify significantly altered TADs (Bonferroni-corrected p value < 0.05). (D) GO analysis of genes encompassed by the decreased TADs upon HOTTIP^−/−;. (E) Hi-C interaction map at the CTNNB1 locus comparing WT and HOTTIP^−/−; MOLM13 cells. (F) ATAC-seq analysis of CTNNB1 in WT and HOTTIP^−/−; MOLM13 cells. (G and H) NG Capture-C analysis of CTNNB1 (G) or MYC (H) promoter interactions, CTCF ChIP-seq and HOTTIP ChIRP-seq in WT and HOTTIP^−/−; MOLM13 cells. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively.

Figure 4.. Hottip activation perturbs β-catenin and its target TADs leading to aberrant HSC activities

(A) Heatmap of ≥2-fold upregulated genes in BM LSK cells from Hottip-Tg mice. (B) GSEA of upregulated genes in LSK cells upon Hottip activation. (C) Heatmap of CTCF binding, from ChIP-seq, and Hottip binding, from ChIRP-seq, at CTCF/Hottip co-occupied sites in BM LK cells from WT and Hottip-Tg mice. (D) CTCF ChIP-seq and Hottip ChIRP-seq binding profiles at the Ctnnb1 locus in WT and Hottip-Tg LK cells. (E) Hi-C interaction maps at the Ctnnb1 locus in WT and Hottip-Tg BM LK cells. CTCF-bound TAD boundaries indicated by red arrows. (F) Overlap of WT and Hottip-Tg Hi-C signals from (E). (G) ATAC-seq analysis of Ctnnb1 in WT and Hottip-Tg BM LSKs. (H) tSNE visualization of BM LK cell subsets from Hottip-Tg (red) and WT (blue) mice by scRNA-seq. LT-HSC, ST-HSC, and MPP populations encompassed by blue circle. (I) Trajectory inference branches/clusters were generated based on the expression levels of lineage-associated genes in cell clusters (left) from WT and Hottip-Tg BM LK cells. Sub-population cell density analysis (right) correlated with the enriched cell number of each population. Higher cell densities shown in dark red. (J) The levels of Myc and Hoxa9 in each cell subset along HSC to MEP differentiation in WT and Hottip-Tg BM LK cells by scRNA-seq. The FDR-corrected p value ≤0.05 by binomial and hypergeometric test. (K) Relative cell numbers in each cell subset along HSC to MEP differentiation in WT and Hottip-Tg BM LK cells by scRNA-seq. The FDR-corrected p value ≤0.05 by binomial and hypergeometric test. (L) GO analysis of upregulated genes in LT- and ST-HSC populations upon Hottip activation by scRNA-seq.

Figure 5.. HOTTIP recognizes and accesses specific HOTTIP/CTCF co-occupied motifs via sequence complementarity to form R-loop structure

(A) CTCF ChIP-seq and HOTTIP ChIRP-seq-binding profiles at the upstream CBSs of CTNNB1 in WT and HOTTIP^−/−; MOLM13 cells. (B) Schematic of probes used in EMSA. The G-4 sequence in HOTTIP, the C-rich HOTTIP-binding motif in the CBS-u2 sequence, and the CTCF-binding motif are shown in red, green, and yellow highlighting, respectively. (C) EMSA of a Cy5-labeled HOTTIP RNA probe (red) and a Cy3-labeled CTNNB1 CBS-u2 probe (green). Orange indicates R-loop formation. RNase A, RNase H, DNase I, and S9.6 antibody added as indicated. (D) Schematic of RIDP procedure (left). RIDP RT-qPCR of HOTTIP precipitated by probes targeting the template or non-template strand of the CBS-u2 site (in relation to the direction of CTNNB1 transcription) or targeting the negative control CBS at ACTB in WT, HOTTIP^−/−; or RNase H treatment (right). Data presented as mean ± SD; ***p ≤ 0.001. (E) CTCF and cohesin-binding ChIP-seq, R-loop DRIP-seq, HOTTIP-binding ChIRP-seq and nascent RNA GRO-seq profiles at the CTNNB1 locus in WT and HOTTIP^−/−; MOLM13 cells. (F) H3K4me3 and H3K27me3 ChIP-seq enrichment profiles at the CTNNB1 locus in WT and HOTTIP^−/−; MOLM13 cells.

Figure 6.. The CBS-u2 boundary at the CTNNB1 locus is critical to maintain TAD structure, transcription, and β-catenin-driven leukemogenesis

(A) Schematic of the CTNNB1 locus showing the locations of CBSs, sub-TADs, and TAD. (B) CTCF ChIP-qPCR analysis of the indicated sites in WT, CBS-u1^−/−; and CBS-u2^−/−; MOLM13 cells. (C) RT-qPCR analysis of the indicated transcripts in WT, CBS-u1^−/−;, and CBS-u2^−/−; MOLM13 cells. (D) Proliferation of WT, CBS-u1^−/−;, and CBS-u2^−/−; MOLM13 cells. (E) HOTTIP ChIRP-qPCR analysis of the indicated sites in WT, CBS-u1^−/−;, and CBS-u2^−/−; MOLM13 cells. (F) DRIP-qPCR analysis of the indicated sites in WT, CBS-u1^−/−;, and CBS-u2^−/−; MOLM13 cells. (G) NG Capture-C analysis of CTNNB1 promoter interactions in WT and CBS-u2^−/−; MOLM13 cells. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively. Capture-C data were aligned with CTCF, H3K4me3, and H3K27me3 ChIP-seq profiles in the CTNNB1 locus in WT, HOTTIP^−/−;, and CBS-u2^−/−; MOLM13 cells. (H) Kaplan-Meier survival curves of NSG mice transplanted with WT, CTNNB1^+/−, and CBS-u2^−/−; MOLM13 cells. n = 5. (I) hCD45⁺ cells chimerism in the BM and PB of NSG mice transplanted with WT, CTNNB1^+/−, and CBS-u2^−/−; MOLM13 cells. n = 3. (J) Kaplan-Meier survival curves of NSG mice transplanted with WT or CBS-u2^−/−; primary AML cells carrying MLL⁺ (LPP4) or NPM1^C+FLT3-ITD⁺ (#974) mutations. n = 4. (K) hCD45⁺ cell chimerism in the BM of NSG mice transplanted with WT or CBS-u2^−/−; primary AML cells. Data in (B)–(F) and (H)–(K) are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 7.. HOTTIP-mediated R-loop is required for maintaining CTCF boundary, TAD integrity, and AML pathogenesis

(A) Schematic of the CTNNB1 locus. Shown is the CBS-u2 sequence with CTCF (yellow) and HOTTIP (green) motifs and the sgRNA target site (red) indicated. (B) RT-qPCR analysis of β-catenin and its target gene expression upon expression of dCas9-RNase H or dCas9-RNase H^D210N with or without CBS-u2-targeted sgRNA or exogenous β-catenin expression in MOLM13 cells. (C) Proliferation of WT, CBS-u2^RH-WT, CBS-u2^RH-Mut, and β-catenin-rescued CBS-u2^RH-WT MOLM13 cells. (D) CTCF ChIP-qPCR analysis of the indicated sites in WT, CBS-u2^RH-WT, CBS-u2^RH-Mut, and β-catenin-rescued CBS-u2^RH-WT MOLM13 cells. (E) HOTTIP ChIRP qPCR analysis of the indicated sites in WT, CBS-u2^RH-WT, CBS-u2^RH-Mut, and β-catenin-rescued CBS-u2^RH-WT MOLM13 cells. (F) DRIP-qPCR analysis of the indicated sites in WT, CBS-u2^RH-WT, CBS-u2^RH-Mut, and β-catenin-rescued CBS-u2^RH-WT MOLM13 cells. (G) NG Capture-C analysis of CTNNB1 promoter interactions upon expression of CBS-u2^RH-WT. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively. Capture-C data were aligned with CTCF, H3K4me3, and H3K27me3 ChIP-seq profiles in the CTNNB1 locus in WT, HOTTIP^−/−;, and CBS-u2^RH-WT MOLM13 cells. (H) Kaplan-Meier survival curves of NBSGW mice transplanted with WT, CBS-u2^RH-Mut, or CBS-u2^RH-WT MOLM13 cells. (I) Kaplan-Meier survival curves of NSG mice transplanted with CBS-u2^RH-WT or CBS-u2^RH-Mut OCI-AML3 cells. Mice were sacrificed when they were paralyzed due to the disease. Data in (B)–(F) and (H)–(I) are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.