Dual DNA/protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia
Data files
Nov 03, 2020 version files 5.94 GB
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63x_tp3_k27ac488_tile_Stitch.czi
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Abstract
The architecture of chromatin specifies eukaryotic cell identity by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase/peroxidase approach, integrative DNA And Protein Tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors, and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights with potential therapeutic implications. Our findings demonstrate the power of iDAPT as a discovery platform for both the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.
Methods
ATAC-see was performed similarly as previously described with slight modifications19. Enzyme and transposon DNA were mixed at a 1:1.25 enzyme:MEDS-A/B-AF647 molar ratio and incubated at room temperature for 1 h. Adherent cells were grown on glass coverslips (Fisher Scientific, 12-540A) until 80-90% confluent, washed with 1xPBS, fixed with 1% formaldehyde (Electron Microscopy Services) in 1xPBS for 10 min, and washed twice with ice-cold 1xPBS. Immobilized cells were lysed by incubation with LB1 for 3 min followed by LB2 for 10 min at room temperature. Cells were then subject to tagmentation (20% dimethylformamide, 10 mM MgCl2, 20 mM Tris-HCl pH 7.5, 33% 1xPBS, 0.01% digitonin, 0.1% Tween-20, and 80 pmol enzyme equivalent of enzyme:DNA complex in a total volume of 100 μL) for 30 min at 37 °C in a humidified chamber. Subsequently, cells were washed with 50 mM EDTA and 0.01% SDS in 1xPBS three times for 15 min each at 55 °C, lysed for 10 min with 0.5% Triton X-100 in 1xPBS at room temperature, and blocked with 1% BSA and 10% goat serum in PBS-T (1xPBS and 0.1% Tween-20) for 1 h in a humidified chamber. Primary antibody was added to slides in 1% BSA/PBS-T and incubated at 4 °C overnight; slides were then washed and subjected to secondary antibody staining for 1 h. Slides were washed with PBS-T three times for 15 min each, stained with DAPI (Sigma, 1 μg/mL) for 1 min, washed with PBS for 10 min, and mounted with Fluorescence Mounting Medium (Dako). Confocal microscopy images were taken with an LSM 880 Axio Imager 2 or an LSM 880 Axio Observer at 63x magnification (Zeiss). Images were processed with Fiji/ImageJ v2.0.0.
Primary antibodies used were anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (rabbit, Abcam ab5095, 1:500), anti-H3K27Ac (rabbit, Abcam ab4729, 1:500), anti-H3K9me3 (rabbit, Abcam ab8898, 1:500), anti-SC35 (mouse, SC-35, Abcam ab11826, 1:1000). Secondary antibodies used were Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (Thermo Fisher Scientific A11008, 1:1000) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 conjugate (Thermo Fisher Scientific A11001, 1:1000).