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| Status |
Public on May 23, 2023 |
| Title |
Comparative binding patterns of Chromatin Regulators in normal versus leukemic hematopoiesis [ChIP-Seq] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Cellular differentiation-trajectories require extensive alterations in chromatin structure and function, elicited by a diverse array of chromatin-factors (CFs). Using haematopoiesis as a model-system, we combined bulk ex vivo and single-cell in vivo CRISPR screens to comprehensively characterise the role of CF families in cell-fate decisions. We uncover marked lineage specificities for many CFs, dissecting specific CF-dependencies in vivo at single-cell resolution. This highlights functional diversity among related CFs (i.e. BAF-subcomplexes, MLL-methyltransferases), but also reveals functional redundancy among unrelated Repressive-complexes that restrain excessive myeloid differentiation. Epigenetic-profiling, molecularly explains CF lineage-dependencies, identifying differentiation stage-specific interactions between individual CFs and lineage-determining Transcription Factors (TFs). Intriguingly, non-canonical BAF remodeler disruption abrogates myeloid-TF function generating a pre-leukaemia phenotype. Screening CF functions in leukemia, we show that leukaemia cells abrogate normal CF function, restraining differentiation trajectories by engaging in novel CF-TF interactions that suggest potential therapeutic avenues. Together, our work greatly elucidates the role of CFs and their TF-partners across normal and malignant haematopoiesis.
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| Overall design |
Here we have analysed the binding patterns of the canoncal-BAF subunit Smarcb1 and the H3K4-methyl writers Kmt2d (MLL4) and Kmt2a (MLL1) in normal myelopoiesis and in a murine leukemic model driven by the Npm1c/Flt3-ITD mutations. For the normal hematopoiesis we have profiled early myeloid progenitors (GMPs) FACs-sorted from bone-marrow as Lin-, ckit+, sca1-, FcgR-III+, cD34-), in vivo FACs-sorted bone marrow monocytes (Lin-, CD11b) and exvivo derived monocytes (expanded from LSK prgenitors with Flt3-ITD, G-CSF, IL3 and GM-CSF). Leukemic cells were purified from primary tumours and expanded exvivo for few passages with SCF, IL3 and IL6. Freshly isolated cells were dual-crosslinked with 3 mM EGS+DSG+DMA for 20 minutes followed by 1% Formaldehyde for 5 minutes. Chromatin extracts were prepared by nuclei isolation with an hypotoniic buffer (25 mM Hepes 7.5 M, 10 mM NaCl, 0.2% Igepal + Protease Inhibtors), followed by sonication in 0.5% SDS, 5 mM EDTA. Chromatin extracts were quantified and diluted 5x to reach a concentration of 150 mM NaCl, 0.1% SDS, 1% Tx-100, 1 mM EDTA, 1X Prot Inh. Immunoprecpitation was carried out overnight (10-12 h) with the appropiate amount of the relevant antibody and the immuno-complexes were captured on the following day by incubation with ProtA + ProtG Magna-ChIP beads (Sigma). After that, DNA was purified with SPRI beads and libraries were prepared using the NEB Next Ultra II kit (NEB).
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| Contributor(s) |
Lara-Astiaso D, Goñi-Salaverri A, Mendieta-Esteban J, Narayan N, Del Valle C, Gross T, Giotopoulos G, Navarro-Alonso M, Zazpe J, Marchese F, Torrea N, A Calvo I, K Lopez C, Alignani D, Lopez A, Saez B, Taylor-King JP, Prosper F, Fortelny N, Huntly BJ |
| Citation(s) |
37580596 |
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| Submission date |
Sep 16, 2022 |
| Last update date |
Aug 22, 2023 |
| Contact name |
Brian Huntly |
| E-mail(s) |
bjph2@cam.ac.uk
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| Organization name |
University of Cambridge
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| Department |
Department of Haematology
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| Street address |
Hills Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0AW |
| Country |
United Kingdom |
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| Platforms (1) |
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| Samples (50)
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| This SubSeries is part of SuperSeries: |
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| Relations |
| BioProject |
PRJNA881276 |
| SRA |
SRP397632 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE213507_Bcell_Brd9_ChIP9_peaks.narrowPeak.gz |
77.2 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Bcell_Kmt2a_ChIP12_peaks.narrowPeak.gz |
581.2 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Bcell_Kmt2d_ChIP12_peaks.narrowPeak.gz |
337.4 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_DM_Brd9-merged-sub37618974_peaks.narrowPeak.gz |
93.9 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_DM_Kmt2a-merged-sub101786848_peaks.narrowPeak.gz |
848.8 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_DM_Kmt2d-merged-sub146746296_peaks.narrowPeak.gz |
1.0 Mb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_DM_Smarcb1-merged-sub173331792_peaks.narrowPeak.gz |
999.0 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_DM_Stat5a_Cyn1_peaks.narrowPeak.gz |
994.0 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Ery_Brd9-merged_peaks.narrowPeak.gz |
90.5 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Ery_Kmt2a_ChIP11_peaks.narrowPeak.gz |
473.7 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Ery_Kmt2d-merged_peaks.narrowPeak.gz |
81.4 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Ery_Smarcb1_ChIP9_peaks.narrowPeak.gz |
1.1 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_GMP_Brd9-merged_peaks.narrowPeak.gz |
367.4 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_GMP_Kmt2a_ChIP11_peaks.narrowPeak.gz |
1.4 Mb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_GMP_Kmt2d_ChIP11_peaks.narrowPeak.gz |
1.1 Mb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_GMP_Smarcb1_ChIP11_peaks.narrowPeak.gz |
1.3 Mb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_MEP_Brd9-merged_peaks.narrowPeak.gz |
554.5 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_MEP_Kmt2a_ChIP11_peaks.narrowPeak.gz |
1.4 Mb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_MEP_Kmt2d_ChIP11_peaks.narrowPeak.gz |
264.0 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_MEP_Smarcb1_ChIP12_peaks.narrowPeak.gz |
313.1 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mono_Brd9-merged_peaks.narrowPeak.gz |
469.5 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mono_Kmt2a-merged_peaks.narrowPeak.gz |
683.5 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mono_Kmt2d-merged_peaks.narrowPeak.gz |
313.5 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mono_Smarcb1-merged_peaks.narrowPeak.gz |
76.8 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mye_Brd9-abcam-sub37618974_140421_peaks.narrowPeak.gz |
395.9 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mye_Kmt2a-merged-sub101786848_peaks.narrowPeak.gz |
804.0 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mye_Kmt2d-merged-sub146746296_peaks.narrowPeak.gz |
873.4 Kb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mye_Smarcb1-merged-sub173331792_peaks.narrowPeak.gz |
1.0 Mb |
(ftp)(http) |
NARROWPEAK |
| GSE213507_Mye_Stat5a_Cyn1_peaks.narrowPeak.gz |
622.0 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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