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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 19, 2021 |
| Title |
Phase separation drives aberrant chromatin looping and cancer development [ChIP-Seq] |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains nucleoporin?s IDR, tandemly dispersed phenylalanine-and-glycine (FG) repeats. However, it remains elusive how unstructured IDRs contribute to oncogenesis. We show that IDR harbored within NUP98-HOXA9, a homeodomain-containing transcription factor (TF) chimera recurrently detected in leukemias, is essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukemic transformation. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TFs but is also required for formation of a broad, ?super-enhancer?-like binding pattern, typically seen at a battery of leukemogenic genes, potentiating their transcriptional activation. Artificial HOX chimera (FUS-HOXA9), created by replacing NUP98?s FG repeats with an unrelated LLPS-forming IDR of FUS, had similar enhancement effects on chimera?s genome-wide binding and target gene activation. Hi-C mapping further demonstrated that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin looping enriched at proto-oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish oncogenic TF condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As LLPS-competent molecules are frequently implicated in diseases, this mechanism can potentially be generalized to many malignant and pathological settings.
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| Overall design |
We compared the genome-wide binding of wildtype (WT) versus IDR-mutated NUP98-HOXA9 and FUS-HOXA9 fusion in the 293 cells. Please note that the GLE2-binding sequence (GLEBS) was deleted from NUP98-HOXA9 (also termed as N-IDR/A9 in the study) and that FUS-HOXA9 was created by in-frame fusion between an unrelated IDR of FUS (amino acids 1-215 that can phase separate) and the HOXA9 homeodomain (also termed as F-IDR/A9 in the study). Phase-separation-disrupting mutations of NUP98?s IDR and FUS? IDR are Phenylalanine-to-Serine (F->S or FS) substitutions at all NUP98 FG-repeats and Tyrosine-to-Serine (Y->S or YS) substitutions at all FUS YG/S sites, respectively. ChIP-seq was conducted with either HA or GFP antibodies in the independently derived cells that express either 3xHA-3xFLAG-tagged or GFP-tagged NUP98-HOXA9, and cells expressing empty vector (EV) served as a negative control. The binding of both WT and IDR-mutated NUP98-HOXA9 (GFP-tagged) was assessed upon cell treatment with 10% of 1,6-Hexanediol for one minute, in comparison to mock treatment. We also examined the genome-wide H3K27 acetylation (H3K27ac) and CTCF binding in the 293 cells that express WT or IDR-mutated NUP98-HOXA9 (3xHA-3xFLAG-tagged). Genome-wide binding of NUP98-HOXA9 (GFP-tagged) was examined in primary murine hematopoietic stem/progenitor cells (HSPCs) transformed by this chimera.Comparison of the genome-wide binding of the wild type (WT) or phase-separation-defective mutant form of NUP98-HOXA9 and FUS-HOXA9 chimeric fusion in the 293 stable expression cells. Here, cells with empty vector (EV) are used as negative control, and the mutant forms of NUP98-HOXA9 and FUS-HOXA9 fusion are F-to-S (FS) and Y-to-S (YS), respectively, which disrupt phase-separation ability harbored within the intrinsically disorganized region (IDR) of NUP98 and FUS. As for NUP98-HOXA9 fusion, the used WT and mutant forms are termed as N_IDR_WT and N_IDR_FS, respectively; as for FUS-HOXA9 fusion, the used WT and mutant forms are termed as F_IDR_WT and F_IDR_YS, respectively. In order to disrupt phase separation, cells are treated with 10% of hexanediol for 1 minute. Fusion proteins are tagged with either HA or GFP tag. These cells are examined by ChIP-seq for their genome-wide patterns of fusion proteins (HA or GFP), H3K27 acetylation (H3K27Ac) or CTCF.
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| Contributor(s) |
Ahn JH, Wang G, Uryu H |
| Citation(s) |
34163069 |
| Submission date |
Jan 22, 2020 |
| Last update date |
Jul 19, 2021 |
| Contact name |
Gang Greg Wang |
| E-mail(s) |
greg_wang@med.unc.edu
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| Phone |
919-9665952
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| Organization name |
UNC Lineberger Comprehensive Cancer Center
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| Department |
Dept of Biochemistry and Biophysics
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| Lab |
gregwanglab
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| Street address |
450 West Drive
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-7295 |
| Country |
USA |
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| Platforms (3) |
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| Samples (30)
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| GSM5269322 |
HEK293 EV control cell Input (matched for HA IP) |
| GSM5269323 |
HEK293 EV control cell HA IP |
| GSM5269324 |
HEK293 FS mutant NUP98-HOXA9 (HA) Input (matched for HA IP) |
| GSM5269325 |
HEK293 FS mutant NUP98-HOXA9 (HA) HA IP |
| GSM5269326 |
HEK293 WT NUP98-HOXA9 (HA) Input (matched for HA IP) |
| GSM5269327 |
HEK293 WT NUP98-HOXA9 (HA) HA IP |
| GSM5269328 |
HEK293 WT NUP98-HOXA9 (GFP; mock-treated) Input |
| GSM5269329 |
HEK293 WT NUP98-HOXA9 (GFP; mock-treated) GFP IP |
| GSM5269330 |
HEK293 WT NUP98-HOXA9 (GFP; Hexanediol-treated) Input |
| GSM5269331 |
HEK293 WT NUP98-HOXA9 (GFP; Hexanediol-treated) GFP IP |
| GSM5269332 |
HEK293 FS mutant NUP98-HOXA9 (GFP; mock-treated) Input |
| GSM5269333 |
HEK293 FS mutant NUP98-HOXA9 (GFP; mock-treated) GFP-IP |
| GSM5269334 |
HEK293 FS mutant NUP98-HOXA9 (GFP; Hexanediol-treated) Input |
| GSM5269335 |
HEK293 FS mutant NUP98-HOXA9 (GFP; Hexanediol-treated) GFP-IP |
| GSM5269336 |
HEK293 EV control cell (matched for H3K27ac IP) |
| GSM5269337 |
HEK293 EV control cell H3K27ac |
| GSM5269338 |
HEK293 FS mutant NUP98-HOXA9 (HA) H3K27ac Input |
| GSM5269339 |
HEK293 FS mutant NUP98-HOXA9 (HA) H3K27ac IP |
| GSM5269340 |
HEK293 WT NUP98-HOXA9 (HA) H3K27ac Input |
| GSM5269341 |
HEK293 WT NUP98-HOXA9 (HA) H3K27ac IP |
| GSM5269342 |
HEK293 FS mutant NUP98-HOXA9 (HA) Input (matched for CTCF IP) |
| GSM5269343 |
HEK293 FS mutant NUP98-HOXA9 (HA) CTCF IP |
| GSM5269344 |
HEK293 WT NUP98-HOXA9 (HA) Input (matched for CTCF IP) |
| GSM5269345 |
HEK293 WT NUP98-HOXA9 (HA) CTCF IP |
| GSM5269346 |
HEK293 WT FUS-HOXA9 (GFP) Input |
| GSM5269347 |
HEK293 WT FUS-HOXA9 (GFP) GFP IP |
| GSM5269348 |
HEK293 YS mutant FUS-HOXA9 (GFP) Input |
| GSM5269349 |
HEK293 YS mutant FUS-HOXA9 (GFP) GFP IP |
| GSM5269350 |
mouse leukemia WT NUP98-HOXA9 (GFP) input |
| GSM5269351 |
mouse leukemia WT NUP98-HOXA9 (GFP) GFP IP |
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| This SubSeries is part of SuperSeries: |
| GSE144643 |
A phase separation mechanism underlies development of cancer and aberrant organization of three-dimensional chromatin structure |
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| Relations |
| BioProject |
PRJNA602701 |
| SRA |
SRP243925 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE144076_HEK293_CTCF_CHIP_N_IDR_FS_A9_cell.bw |
435.1 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_CTCF_CHIP_N_IDR_WT_A9_cell.bw |
462.6 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_EV_HA_CHIP.bw |
792.7 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_F_IDR_FS_A9_GFP_CHIP.bw |
491.0 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_F_IDR_WT_A9_GFP_CHIP.bw |
515.9 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_H3K27Ac_CHIP_Empty_vector.bw |
936.9 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_H3K27Ac_CHIP_N_IDR_FS_A9_cell.bw |
780.6 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_H3K27Ac_CHIP_N_IDR_WT_A9_cell.bw |
735.8 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_N_IDR_FS_A9_GFP_CHIP_Hexanediol_treated.bw |
528.4 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_N_IDR_FS_A9_GFP_CHIP_mock-treated.bw |
644.9 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_N_IDR_FS_A9_HA_CHIP.bw |
928.6 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_N_IDR_WT_A9_GFP_CHIP_Hexanediol_treated.bw |
538.1 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_N_IDR_WT_A9_GFP_CHIP_mock-treated.bw |
537.2 Mb |
(ftp)(http) |
BW |
| GSE144076_HEK293_N_IDR_WT_A9_HA_CHIP.bw |
804.7 Mb |
(ftp)(http) |
BW |
| GSE144076_HPSC_N_IDR_WT_A9_GFP_CHIP.bw |
446.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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