EXPERIMENT_TITLE: GSM4259898: HEK_HiC_NUP_IDR_WT_A9_2.1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C was performed exactly as described by Rao et al(Rao et al., 2014). Five million of cells were crosslinked in 1% of formaldehyde for ten minutes with stirring and quenched by adding 2.5M glycine to a final concentration of 0.2M for 5 min with rocking. Cells were pelleted by spinning at 300g for 5 min at 4°C. The pellet was washed with cold PBS and spun again prior to freezing in liquid nitrogen. Cells were lysed with 10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630 and protease inhibitors (Sigma, P8340) for 15 min on ice. Cells were pelleted and washed once more using the same buffer. Pellets were resuspended in 50μl of 0.5% SDS and incubates for 7 min at 62°C. Next, reactions were quenched with 145μl of water and 25μl of 10% Triton X-100 (Sigma, 93443) at 37°C for 15 min. Chromatin was digested overnight with 25μl of 10X NEBuffer2 and 100U of MboI at 37°C with rotation. Reactions were incubated at 62°C for 20 min to inactivate MboI and then cooled to room temperature. Fragment overhangs were repaired by adding 37.5 μl of 0.4mM biotin-14-dATP, 1.5 μl of 10mM dCTP, 1.5 μl of 10mM dGTP, 1.5 μl of 10mM dTTP, and 8 μl of 5U/ul DNA Polymerase I, Large (Klenow) Fragment and incubating at 37°C for 1.5 hr. Ligation was performed by adding 667 μl of water, 120 μl of 10X NEB T4 DNA ligase buffer, 100 μl of 10% Triton X-100, 12 μl of 10 mg/ml BSA, and 1 μl of 2000 U/ul T4 DNA Ligase and incubating at room temperature for 4 hr with slow rotation. Samples were pelleted at 2500g and resuspended in 432 μl water, 18 μl 20 mg/ml proteinase K, 50 μl 10% SDS, 46 μl 5M NaCl and incubated for 30 min at 55°C. The temperature was raised to 68°C and incubated overnight. Samples were cooled to room temperature. 874 μl of pure ethanol and 55 μl of 3M sodium acetate pH 5.2 were added to each tube which were subsequently incubated for 15 min at −80°C. Tubes were spun at max speed at 2°C for 15 min and washed twice with 70% ethanol. The resulting pellet was resuspended in 130 μl of 10mM Tric-HCl, pH8 and incubated at 37°C for 15 min. DNA was sheared using an LE220 Covaris Focused-ultrasonicator to a fragment size of 300-500 bp. Sheared DNA was size selected using AMPure XP beads. 110 μl of beads were added to each reaction and incubated for 5 min. Using a magnetic stand supernatant was removed and added to a fresh tube. 30μl of fresh AMPure XP beads were added and incubated for 5 min. Beads were separated on a magnet and washed twice with 700 μl of 70% ethanol without mixing. Beads were left to dry and then sample was eluted using 300 μl of 10 mM Tris-HCl, pH 8. 150 of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads were washed resuspended in 300 μl of 10 mM Tris HCl, pH 7.5. This solution was added to the samples and incubated for 15 min at room temperature. Beads were washed twice with 600μl Tween Washing Buffer (TWB; 250 μl Tris-HCl, pH 7.5, 50 μl 0.5 M EDTA, 10 mL 5M NaCl, 25 μl Tween 20, and 39.675 mL water) at 55°C for 2 min with shaking. Sheared ends were repaired by adding 88 μl 1X NEB T4 DNA ligase buffer with 1mM ATP, 2 μl of 25 mM dNTP mix, 5 μl of 10U/μl NEB T4 PNK, 4ul of 3U/μl NEB T4 DNA polymerase I, 1μl of 5U/μl NEB DNA polymerase I, Large (Klenow) Fragment and incubating at room temperature for 30 min. Beads were washed two more times with TWB for 2 min at 55°C with shaking. Beads were washed once with 100 μl 1X NEBuffer 2 and resuspended in 90 μl of 1X NEBuffer 2, 5 μl of 10 mM dATP, 5μl of 5U/μl NEB Klenow exo minus, and incubated at 37°C for 30 min. Beads were washed two more times with TWB for 2 min at 55°C with shaking. Beads were washed once in 50 μl 1X Quick Ligation reaction buffer and resuspended in 50 μl 1X Quick Ligation reaction buffer. 2 μl of NEB DNA Quick ligase and 3 μl of an illumina indexed adaptor were added and the solution was incubated for 15 min at room temperature. Beads were reclaimed using the magnet and washed two more times with TWB for 2 min at 55°C with shaking. Beads were washed once in 100 μl 10 mM Tris-HCl, pH 8 and resuspended in 50 μl 10 mM Tris-HCl, pH8. Hi-C libraries were amplified for 7-12 cycles in 5 μl PCR primer cocktail, 20 μl of Enhanced PCR mix, and 25 μl of DNA on beads. The PCR settings included 3 min of 95°C followed by 7-12 cycles of 20 s at 98°C, 15 s at 60°C, and 30 s at 72°C. Samples were then held at 72°C for 5 min before lowering to 4°C until samples were collected. Amplified samples were brought to 250 μl with 10 mM Tris-HCl, pH 8. Samples were separated on a magnet and supernatant was transferred to a new tube. 175 μl of AMPure XP beads were added to each sample and incubated for 5 min. Beads were separated on a magnet and washed once with 700 μl of 70% ethanol. Supernatant was discarded. 100 μl of 10 mM Tris-HCl and 70 μl of fresh AMPure XP beads were added and the solution was incubated for 5 min at room temperature. Beads were separated with a magnet and washed twice with 700 μl 70% ethanol. Beads were left to dry until cracking started to be observed and eluted in 25 μl of Tris HCl, pH 8.0.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: 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 largely elusive how unstructured IDRs contribute to oncogenesis. We here show that IDR or FG repeats harbored within NUP98-HOXA9, a HOX transcription factor (TF) chimera recurrently detected in acute leukemia patients, is essential for establishing nuclear liquid-liquid phase separation (LLPS) puncta and for inducing leukemic transformation of primary hematopoietic cells in vitro and in vivo. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TF oncoproteins but is also required for formation of a broad, 'super-enhancer'-like binding pattern, typically seen at a battery of leukemia-related loci exemplified by HOX, MEIS and PBX genes, potentiating their transcriptional activation. An artificial HOX chimera, created by replacing NUP98's FG repeats with an unrelated LLPS-forming IDR of FUS, had similar enhancement effects on chimera's chromatin binding and target gene activation. Via Hi-C mapping, we further demonstrated that the phase-separated NUP98-HOXA9 protein assembly is able to induce de novo formation of CTCF-independent chromatin looping enriched at leukemic oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish multi-molecule assemblies of oncogenic TFs via a phase separation mechanism, which simultaneously enhances their chromatin targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As a range of LLPS-competent molecules are implicated in various human cancers, this mechanism can potentially be generalized to many malignant and diseased settings. Overall design: Constructs of a NUP98-HOXA9 fusion with a wild type intrinsically disordered region (NUP_IDR_WT/A9) and a NUP98-HOXA9 fusion with a phenylalanine to serine (F to S) mutant intrinsically disordered region (NUP_IDR_FS/A9) were retrovirally transduced into the HEK293FT cell line to produce stable NUP_IDR_WT/A9 and NUP_IDR_FS/A9 cell lines. In situ Hi-C was performed as described by Rao et al. 2014 (PMID: 25497547) on each cell line in biological duplicate. To retain library complexity, two Hi-C libraries were generated for each biological replicate (2 cell lines x 2 biological replicates x 2 libraries = 8 libraries total).
SAMPLE_TITLE: HEK_HiC_NUP_IDR_WT_A9_2.1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "NUP_IDR_WT/A9 expressing HEK293FT cell line"}, {"TAG": "cell line background", "VALUE": "HEK293FT"}, {"TAG": "genotype/variation", "VALUE": "expressing NUP_IDR_WT/A9"}, {"TAG": "nha9 idr type", "VALUE": "Wildtype"}, {"TAG": "biological replicate", "VALUE": "2"}, {"TAG": "technical replicate", "VALUE": "1"}]
Pool_Member_@attributes_sample_title: HEK_HiC_NUP_IDR_WT_A9_2.1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HEK_HiC_NUP_IDR_WT_A9_2.1
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