Abstract
The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR—tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98–HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid–liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98–HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad ‘super-enhancer’-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98–HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4,5,6,7, this mechanism can potentially be generalized to many malignant and pathological settings.
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Data availability
Next-generation sequencing datasets including those of ChIP–seq, RNA-seq and Hi-C used in this current study are deposited in the NCBI GEO under the accession number GSE144643. The mass spectrometry-based proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023548 and 10.6019/PXD023548. Source data are provided with this paper.
Code availability
The scripts for genomic data analyses and all other data are available from the corresponding author upon request.
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Acknowledgements
We thank M. Kamps, B. Strahl, B. Fahrenkrog, J. Schwaller, J. van Deursen, J. Song and J. Hao for providing reagents used in the study and the Wang laboratory members and J. Bear for discussion and technical support. We thank J. Lippincott-Schwartz for help with lattice light sheet microscopy and J. Rowley and A. Gladfelter for discussion and input. We thank UNC for facilities, including Imaging Core, High-Throughput Sequencing Facility (HTSF), Bioinformatics Core, Flow Cytometry Core, Tissue Culture Facility and Animal Studies Core, for their professional assistance of this work. We thank S. Pattenden for use of the Covaris LE220 instrument which was provided by the North Carolina Biotechnology Center Institute Development Program grant 2017-IDG-1005. The cores affiliated to UNC Cancer Center are supported in part by the UNC Lineberger Comprehensive Cancer Center Core Support Grant P30-CA016086 and UNC Neuroscience Microscopy Core supported, in part, by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124. This work was supported by NIH grants (R01-CA215284 and R01-CA218600 to G.G.W.; R35-GM128645 to D.H.P.; DP2GM136653 to W.R.L.; P20GM121293, R24GM137786, R01CA236209, S10OD018445, and TL1TR003109 to A.J.T; R01HL148128 and R01HL153920 to D.Z.), a Kimmel Scholar Award (to G.G.W.), Gabrielle’s Angel Foundation for Cancer Research (to G.G.W.), Gilead Sciences Research Scholars Program in haematology/oncology (to G.G.W.), When Everyone Survives (WES) Leukemia Research Foundation (to G.G.W.) and UNC Lineberger Stimulus Awards (to D.H.P. and to L.C.). E.S.D. was supported by the NIH-NIGMS training grant T32-GM067553. W.R.L. is a Searle Scholar, a Beckman Foundation Young Investigator, and a Packard Fellow for Science and Engineering. G.G.W. is an American Cancer Society (ACS) Research Scholar, an American Society of Hematology (ASH) Scholar in basic science, and a Leukemia and Lymphoma Society (LLS) Scholar.
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Contributions
J.H.A. designed the research, performed experiments, interpreted data and wrote the manuscript. J.H.A., Y-H. T., H.U., J. L., L. C., D.Z. and G.G.W. performed genomic data analysis. J.H.A. and S.Z. performed in vitro phase separation assays. D.P.K. conducted imaging quantification analysis. A.J.S., S.G.M., R.D.E. and S.D.B. performed proteomic analysis under the supervision of A.J.T. T.A.D. and J.H.A. performed single molecule tracking studies under the supervision of W.R.L. J.H.A. and J.L. performed murine leukaemia assays. E.S.D., I.Y.Q. and J.H.A. performed Hi-C mapping, data analysis and interpretation under the supervision of D.H.P. G.G.W. conceived the idea, supervised and designed the research, interpreted data, and wrote the manuscript with the inputs from all authors.
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Extended data figures and tables
Extended Data Fig. 1 IDR retained within the leukaemia-related NUP98–HOXA9 chimera forms phase-separated condensates in vitro and is essential for establishing phase-separated chimeric transcription factor assemblies in the nucleus.
a, Schematic showing the domain architecture of normal NUP98 (top), normal HOXA9 (middle) and leukaemic NUP98–HOXA9 chimera (bottom; with either GFP or 3×HA–3×Flag tag fused to C terminus). The GLFG or non-GLFG (xFG) motif contents, which make up IDR, and other important domains are shown. GLEBS represents the GLE2-binding sequence, which directs the NUP98 interaction with GLE2 (also known as RAE1) for mRNA export when NUP98 acts as component of nuclear pore complex60. Red arrows indicate the common breakage point of NUP98 and HOXA9. b, Immunoblotting of NUP98–HOXA9, either full-length (WT) or with GLEBS deleted (Δ132-224; see a), as detected by the indicated antibodies after stable transduction into primary mouse HSPCs. For gel source data, see Supplementary Fig. 1. c, Mouse HSPCs stably transduced with wild-type or GLEBS-deleted NUP98–HOXA9 showed similar proliferation in liquid cultures (n = 3 independent cell cultures per group), in agreement to previous reports17,61. Empty vector (EV)-infected HSPCs served as a control. Data are mean ± s.d. d, Live-cell fluorescence imaging (GFP; with zoomed-in and zoomed-out views shown in the top and bottom panels, respectively) of 293FT cells with stable transduction of GFP-tagged NUP98–HOXA9, wild-type, GLEBS-deleted (also referred to as N-IDRWT/A9; see Fig. 1a) or carrying a DNA-binding-defective mutation in homeodomain (HDN51S) or a Phe-to-Ser mutation that substitutes Phe residues within all FG repeats to Ser (IDRFS, also referred to as N-IDRFS/A9; see Fig. 1a). The right panel shows immunoblotting of endogenous normal NUP98 in 293FT cells, as well as the stably transduced exogenous NUP98–HOXA9, either wild-type (lane 1) or GLEBS-deleted (lane 2), as detected by antibodies against GLEBS of NUP9817. For gel source data, see Supplementary Fig. 1. Scale bars, 10 μm. e, Schematic of the indicated N-IDR fusion domains with a varying number of FG repeats. The IDR portion used for in vitro assay in main Fig. 1d is indicated by a red dotted line. f, SDS–PAGE images showing recombinant N-IDR domain protein with the indicated varying number of FG repeats (His6×-tagged; see e), purified with Ni-column and an additional size exclusion column purification step. The protein size is labelled above the recombinant protein. g, Anti-GFP immunoblotting for GFP-tagged NUP98–HOXA9 chimera with the indicated varying number of FG repeats described in e after stable transduction in 293FT cells. For gel source data, see Supplementary Fig. 1. h, Live-cell fluorescence imaging for the N51S-mutated N-IDR/A9 (GFP-tagged) with either wild-type (top) or the Phe-to-Ser mutated IDR (bottom) in 293FT stable expression lines before (left) and after (right) treatment with 10% 1,6-hexanediol for 1 min. The left panels show zoomed-in images of a representative cell from the right panels of zoomed-out cell images. Scale bar, 10 μm.
Extended Data Fig. 2 IDR contained within chimeric transcription factor is required for leukaemic transformation of primary mouse HSPCs.
a, b, Immunoblotting (a) and fixed cell immunostaining (b; anti-Flag) of the LLPS-competent N-IDRWT/A9 and LLPS-incompetent N-IDRFS/A9 after stable transduction in 293FT cells. The left panel of b shows a zoomed-in view on the right panel. Scale bars, 10 μm. For gel source data, see Supplementary Fig. 1. c, Venn diagram shows significant overlap between the N-IDRWT/A9 and N-IDRFS/A9 interactomes as detected by BioID, with the cut-off value set as the log2-transformed fold change value above 2 compared with control. Examples of the detected interacting proteins are shown below. d–f, Immunostaining (d; anti-GFP), Wright–Giemsa staining (e) and FACS with the indicated surface markers (f) using mouse HSPCs 1 month after transduction of N-IDRWT/A9 (GFP or 3×HA-3×Flag-tagged), which revealed a typical acute myeloid leukaemia phenotype (cKit+, CD34+, MacIhigh, CD19−, B220−). The insert in d shows a zoomed-in view of the representative cell. Scale bars, 5 μm. For FACS gating strategy, see Supplementary Fig. 1. g, H&E-stained spleen section images for the indicated cohort at 10 × magnification. White pulp (WP) is outlined with white line for the sample from mice transplanted with empty vector-infected HSPCs (top). Note that clear demarcation between white pulp and red pulp (RP), as observed in cohorts receiving either empty vector or the mutant forms of fusion (bottom), is lost in those with N-IDRWT/A9 and F-IDRWT/A9 (middle) due to an excessive expansion of transformed leukaemia cells that infiltrated into spleen, leading to splenomegaly observed in i and Fig. 1k. h, Live-cell fluorescence (GFP) imaging of 293FT cells with stable expression of an artificial HOXA9 chimera created by replacing the NUP98 FG repeats with an unrelated IDR of the RNA-binding protein FUS, either wild-type or Tyr-to-Ser mutated (hereafter referred to as the F-IDRWT/A9 and F-IDRYS/A9 fusion, respectively; see Fig. 1a), before and after treatment with 10% 1,6-hexanediol for 1 min. Scale bar, 10 μm. i, Representative image of spleen from mice 7 months after transplantation of mouse HPSCs stably transduced with either F-IDRWT/A9 (left) or F-IDRYS/A9 (right).
Extended Data Fig. 3 ChIP–seq reveals binding patterns of NUP98–HOXA9 that carries either wild-type or an Phe-to-Ser mutated IDR.
a, Summary of the counts of ChIP–seq read tags for the indicated samples. b, Scatterplots showing correlation of global N-IDRWT/A9 (left) or N-IDRFS/A9 (right) ChIP–seq signals using either HA (x axis) or GFP (y axis) antibodies in two biological replicates of 293FT stable cells. Coefficient of determination (R2) is determined by Pearson correlation. c, Total number of the called HA ChIP–seq peaks in stable 293FT cell lines expressing HA-tagged N-IDRWT/A9 (left) or N-IDRFS/A9 (middle) or empty vector control (right). d, e, Pie chart showing distribution of the indicated annotation feature among the called N-IDRWT/A9 (d) or N-IDRFS/A9 (e) ChIP–seq peaks in 293FT stable expression cells. f, g, Summary of the most enriched motifs identified within the called N-IDRWT/A9 (f) or N-IDRFS/A9 (g) ChIP–seq peaks in 293FT stable expression cells. Motif enrichment was statistically determined by ZOOPS scoring (zero or one occurrence per sequence) coupled with the hypergeometric enrichment calculations. h, Gene Ontology analysis of genes associated with broad super-enhancer-like peaks of N-IDRWT/A9 as identified in 293FT stable cells. P values were determined by Fisher’s exact test.
Extended Data Fig. 4 Enhanced chromatin occupancy, as well as a broad super-enhancer-like binding pattern typically seen at leukaemia-related genomic loci, is characteristic for the LLPS-competent NUP98–HOXA9 (N-IDRWT/A9) and not its LLPS-incompetent IDR mutant (N-IDRFS/A9).
a–e, Integrative genomics viewer (IGV) views for the indicated ChIP–seq signal at the well-known leukaemia-associated loci such as the HOXA (a), HOXB (b) and HOXD (c) gene clusters, MEIS1 (d) and MEIS2 (e). Samples from top to bottom are HA (tracks 1–3) and H3K27ac (tracks 4–6) ChIP–seq signals in the 293FT cells stably expressed with either empty vector (tracks 1 and 4; EV in track 1 acts as a negative control for HA ChIP) or the HA-tagged N-IDRWT/A9 (tracks 2 and 5) or N-IDRFS/A9 (tracks 3 and 6), GFP ChIP–seq signals (tracks 7–12) in the 293FT cells stably expressed with GFP-tagged N-IDRWT/A9 (tracks 7–8 represent samples after treatment with vehicle or 10% 1,6-hexanediol, respectively, for 1 min), N-IDRFS/A9 (tracks 9–10 represent samples after treatment with vehicle or 1,6-hexanediol, respectively), F-IDRWT/A9 (track 11) or F-IDRYS/A9 (track 12), as well as CTCF ChIP–seq in 293FT cells with N-IDRWT/A9 (track 13) or N-IDRFS/A9 (track 14). HA and CTCF ChIP–seq signals were normalized to input signals, whereas GFP ChIP–seq, conducted in the spike-in controlled experiments, normalized to the spike-in Drosophila chromatin signals (those from antibody of a Drosophila-specific histone, H2Av).
Extended Data Fig. 5 Formation of the enhanced and broad super-enhancer-like binding patterns of leukaemia-related chimera transcription factors requires an intact phase-separation-competent IDR.
a, b, Hockey-stick plot shows distribution of the input-normalized ChIP–seq signals of N-IDRWT/A9 (a) or H3K27ac (b) across all enhancers annotated by H3K27ac peaks (transcriptional start site ± 2.5 kb regions were excluded) in 293FT cells. Dotted line indicates the threshold level set by the ROSE algorithm to call super-enhancers. Relative rankings of super-enhancers associated with some example genes are shown. c, Venn diagram illustrates overlap among super-enhancers called based on N-IDRWT/A9 and H3K27ac ChIP–seq signals. d, e, Box plots showing averaged ChIP–seq signals for k-means clustered peaks (see Fig. 2b) of the LLPS-competent N-IDRWT/A9 (WT; d) show a marked reduction in binding after treatment of 293FT stable cells with 1,6-hexanediol (WT+H), relative to treatment with vehicle control (WT+V); this reduction is particularly significant for peak clusters 1–3 shown in Fig. 2b. By contrast, genomic binding of N-IDRFS/A9 (FS; e) shows general insensitivity to the same treatment of 1,6-hexanediol (FS+H) in comparison to mock (FS+V). Right, averaged ChIP–seq signal distribution profiles are shown for N-IDRWT/A9 and N-IDRFS/A9 over a 10-kb region in the indicated peak cluster as an example. Box plots as defined in Fig. 3f. f, Venn diagram to compare genes associated with the broad super-enhancer-like peaks of N-IDRWT/A9 after treatment with 1,6-hexanediol (+H), relative to vehicle control (+V), after treatment for 1 min. g, Hierarchical clustered heat maps for the pairwise correlation of ChIP–seq signals between each of the indicated sample. The coefficients were determined by Pearson correlation. HA and GFP represent ChIP–seq for HA-tagged and GFP-tagged chimera transcription factors, respectively.
Extended Data Fig. 6 The phase-separation-promoting property within F-IDR is sufficient to induce the enhanced binding of the chimeric transcription factor.
a, Heat maps showing the k-mean clustering of ChIP–seq signals for chimeric transcription factors that contain the NUP98 IDR (N-IDRWT/A9 and N-IDRFS/A9, two panels on the left) or FUS IDR (F-IDRWT/A9 and F-IDRYS/A9, two panels on the right) reveal a similarly enhanced binding for the LLPS-competent chimera that carries a wild-type form of IDR, relative to its LLPS-incompetent IDR mutant, in 293FT stable expression cells. Note that, although to a lesser degree, the artificially created F-IDRWT/A9 fusion also displays a broad, super-enhancer-like binding pattern at the same sites observed with the N-IDRWT/A9 fusion. b, Pie chart showing percentage distribution of the indicated genomic annotation feature among the ChIP–seq peaks of GFP-tagged F-IDRWT/A9 (left) or F-IDRYS/A9 (right) in the 293FT stable expression cells. c, Heat maps (left) and its averaged ChIP–seq signal distribution profiles (right) for k-mean clustered peaks of N-IDRWT/A9 in the transformed mouse HPSCs. d, Venn diagram showing overlap between the annotated genes associated with the clusters 1–3 of N-IDRWT/A9 ChIP–seq peaks detected in the transformed mouse HPSCs (left) and the 293FT stable expression cells (right). Examples of the shared oncogenes are shown below. e, IGV views of N-IDRWT/A9 ChIP–seq signals (GFP-tagged) at the indicated loci in mouse HSPCs transformed by this chimera. f, ChIP–qPCR to assess the binding of GFP-tagged N-IDRWT/A9 or N-IDRFS/A9 at CCL15 (a negative control region), PBX3 and HOXA9 in the 293FT stable cells after treatment with 10% 1,6-hexanediol for 1 min (+H), relative to mock (+V). ChIP signals, normalized to those of input, are presented as mean ± s.d. of three replicate experiments. g, ChIP–qPCR to assess the binding of GFP-tagged F-IDRWT/A9 or F-IDRYS/A9 at CCL15 (a negative control region), PBX3 and HOXA9 in the 293FT stable cells. ChIP signals, normalized to those of input, are presented as mean ± s.d. of three replicates.
Extended Data Fig. 7 Single-molecule tracking shows that phase-separation-competent N-IDRWT/A9 proteins behave with less dynamic characteristics, compared with phase-separation-incompetent N-IDRFS/A9.
a, Representative images of single-molecule particles identification in an N-IDRWT/A9-expressing cell, either the original captured image (left) or after processing to remove background (right). Scale bars, 5 μm. b, c, Single-particle tracks for mean speed (b) and mean displacement (c) of either N-IDRWT/A9 or N-IDRFS/A9 single molecules within the temporally registered reference frame binned into 1-s intervals. d, e, Displacement (d) and mean velocity (e) of single-particle tracks indicate that N-IDRWT/A9 with the LLPS-competent IDR (WT) is less mobile and navigates nuclear space at a slower rate than its LLPS-incompetent IDR mutant (FS). Dots indicate mean values in a single cell. Line indicates one standard deviation. P values determined by two-sided t-test. f, g, The diffusion coefficient for chromatin-bound (f) and freely diffusing states (g) of N-IDRWT/A9 or N-IDRFS/A9, calculated based on single-molecule tracking studies of its 293FT stable expression cells. P values determined by two-sided t-test.
Extended Data Fig. 8 An LLPS-competent IDR within the leukaemia-related transcription factor chimera is essential for potentiating transcriptional activation of the downstream oncogenic gene-expression program.
a, Fixed cell immunostaining for the 3×HA-3×Flag-tagged N-IDRWT/A9 (left; anti-Flag) and the indicated histone modification (middle) in the 293FT stable expression cells. Top panels show the enlarged images of an example region within the white dotted box shown in the bottom panels, in which the transcription factor chimera is co-localized with H3K27ac (top) and not H3K9me3 (bottom). Scale bars, 10 μm. b, Pearson’s correlation coefficient values between N-IDRWT/A9 and the indicated histone modification. The red dotted line indicates the calculated average value of each plot. The calculated means (red dotted lines) were compared with an independent two-tailed Student’s t-test. n, the number of cells analysed. c, RT–qPCR to assess the effect of phase separation in target gene expression in 293FT cells. All of the tested HOX and MEIS2 genes are direct targets of both N-IDRWT/A9 and N-IDRFS/A9 based on ChIP–seq, whereas MYC is not and serves as a negative control. Note that LLPS-competent N-IDRWT/A9 induces significantly more upregulation of target genes, relative to LLPS-incompetent N-IDRFS/A9. PCR signals were normalized first to those of an internal control (18S RNA) and then to vector-expressing cells and presented as mean ± s.d. of three replicated experiments. ***P < 0.001; ****P < 0.0001; two-sided t-test. n.s., not significant. d, Heat map illustrating relative expression of the 374 genes that show significant upregulation post-transduction of F-IDRWT/A9, compared to empty vector and its IDR-mutant form (F-IDRYS/A9), in 293FT stable expression cells. e, Venn diagrams showing the overlap of the significantly downregulated genes identified 7 days after transduction of the indicated construct into mouse HPSCs. f, Gene set enrichment analysis (GSEA) shows that, compared with that of N-IDRFS/A9, the expression N-IDRWT/A9 in mouse HPSCs is positively correlated with the indicated leukaemia- or HSPC-related gene sets (top) and negatively correlated with the indicated differentiation-related gene sets (bottom). The P value was calculated by an empirical phenotype-based permutation test; the false discovery rate (q) is adjusted for gene set size and several hypotheses testing whereas the P value is not. g, Venn diagrams showing the overlap of the significantly upregulated (left) or downregulated (right) genes identified after transduction of the indicated construct into mouse HPSCs.
Extended Data Fig. 9 Hi-C mapping reveals that a phase-separation-competent IDR within NUP98–HOXA9 is required to induce formation of CTCF-independent chromatin loops at the leukaemia-related genomic loci.
a, Matrix of Pearson correlation coefficients of loop counts among and between biological replicates of N-IDRWT/A9 (WT; n = 4 replicates) or N-IDRFS/A9 (FS; n = 4 replicates) conditions. Numbers following WT or FS indicate biological replicate for that condition. b, Example correlation plots of loop counts between biological replicates and conditions. c, All loops were partitioned into either WT- or FS-specific loops and split into separate loop anchors. Loop anchors were then intersected with ChIP–seq peaks of N-IDR/A9 or CTCF. The percentage of observed (Obs.) overlaps for each feature is shown as a vertical blue line. The red line shows the expected (Exp.) distribution of overlaps as determined by randomly sampling loop anchors and calculating the overlap of each feature 1,000 times. P values were determined by summing the number of expected values greater than (or less than if the observed value was less than the mean) the observed value for that feature. d–g, 3C–qPCR assays measuring the change in crosslinking frequency of either an N-IDRWT/A9-specific loop at the PBX3 locus (d, e) or a CTCF-dependent loop (f, g; at Chr17 (41604677–41883642)) after treatment of 293FT stable cells with 10% 1,6-hexanediol for 1 min (+H), relative to mock (+V). The IGV view panels at d and f show the indicated ChIP–seq signals, with positions of the used 3C–PCR primers labelled under IGV tracks. PCR was performed using the same constant forward primer (C) paired with a differently numbered reverse primer (P1 to P4) at each locus tested. Panels e and g are plotted with signals of 3C–qPCR measuring the relative crosslinking frequency at PBX3 (d, e) or a Chr17 locus with CTCF loop (f, g) before (V) and after (H) treatment with 1,6-hexanediol. Signals in e are normalized to those of the N-IDRFS/A9-expressing cells (n = 3 replicated experiments). P values were determined by two-sided t-test. Data are mean ± s.d. of three or six replicates.
Extended Data Fig. 10 Hi-C mapping reveals the chromatin loops specific to cells with the LLPS-competent NUP98–HOXA9, compared with the LLPS-competent mutant, at leukaemia-relevant gene loci.
Views for Hi-C mapping, RNA-seq and ChIP–seq for CTCF, N-IDR/A9 and H3K27ac at the HOXB (a), EYA4 (b), and SKAP2-HOXA loci (c) in 293FT stable cells expressing either N-IDRWT/A9 (WT) or N-IDRFS/A9 (FS). Hi-C mapping views (top) show results from the N-IDRWT/A9 or N-IDRFS/A9 expressing cells (bottom and top diagonal, respectively). Corresponding ChIP–seq and gene tracks are shown below each Hi-C plot. N-IDRWT/A9 loops are indicated by red arrows.
Extended Data Fig. 11 Model illustrating requirement of LLPS-competent IDR within NUP98–HOXA9 for leukaemogenesis and activation of the oncogenic gene-expression program.
The LLPS-competent IDR contained with NUP98–HOXA9 is crucial for promoting long-distance chromatin looping between proto-oncogene promoter and enhancers, which thus induces an oncogenic gene-expression program and malignant development.
Supplementary information
Supplementary Information
This file contains the Supplementary Discussion, Supplementary references and Supplementary Figures 1-2 (the gel source data and FACS gating strategy).
Supplementary Table 1
List of protein partners associated with either N-IDRWT/A9 or N-IDRFS/A9 in 293 cells as detected by the BioID approach.
Supplementary Table 2
List of the broad ChIP-seq peaks of N-IDRWT/A9 in 293 stable cells after treatment with 10% of 1,6-hexanediol, relative to mock, for one minute, as well as H3K27ac and N-IDRWT/A9 associated super-enhancers defined by ROSE.
Supplementary Table 3
List of genes directly bound by N-IDRWT/A9 (HA- or GFP-tagged) and F-IDRWT/A9 in 293 stable cells as detected by ChIP-seq.
Supplementary Table 4
RNA-seq reveals transcripts showing the significant expression changes post-transduction of N-IDRWT/A9, in comparison to empty vector (EV) or N-IDRFS/A9, in 293 stable expression cells.
Supplementary Table 5
RNA-seq reveals transcripts showing significant expression changes post-transduction of F-IDRWT/A9, in comparison to EV or F-IDRYS/A9, in 293 stable expression cells.
Supplementary Table 6
RNA-seq reveals transcripts showing significant expression changes post-transduction of N-IDRWT/A9, in comparison to EV or N-IDRFS/A9, into primary murine HSPC cells.
Supplementary Table 7
RNA-seq reveals transcripts showing significant expression changes post-transduction of F-IDRWT/A9, in comparison to EV, into primary murine HSPC cells.
Supplementary Table 8
List of reagents (such as plasmid, primers and antibodies) used in this study.
Video 1
Live cell imaging readily detects the fusion events of aggregates formed by GFP-tagged N-IDRWT/A9N51S upon its induced expression in the HeLa stable cell line. Each frame represents 24 seconds. This video is rendered at 10 frames per second. Example screen shots illustrating fusion events observed in this video are presented in Fig. 1h.
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Ahn, J.H., Davis, E.S., Daugird, T.A. et al. Phase separation drives aberrant chromatin looping and cancer development. Nature 595, 591–595 (2021). https://doi.org/10.1038/s41586-021-03662-5
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DOI: https://doi.org/10.1038/s41586-021-03662-5
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