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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 19, 2023 |
| Title |
DNMT3A-coordinated splicing governs the stem state switch toward differentiation in embryonic and hematopoietic stem cells [ChIP-seq] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Upon stimulation by extrinsic stimuli, stem cells initiate a program that enables differentiation or self-renewal. Disruption of the stem-state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem-state switch are known, major regulatory mechanisms remain unclear. Here, we show this switch involves a global increase in splicing efficiency coordinated by DNMT3A, an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and hematopoietic stem cells depends on mRNA processing influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem-state towards differentiation.
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| Overall design |
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for flag-tagged DNMT3A in ATRA-treated mouse es cells
Lysates were harvested with 0.5% trypsin. Cells were washed and crosslinked with formaldehyde for 20 mins (final concentration of 1%). Chromatin from 30x10^6 cells was sheared to 200-500bp fragments with a Covaris M220 sonicator for 8 mins. Antibody was added into lysate and incubated overnight at 4°C. 10ul of pre-blocked Protein G dynabeads were added to each IP tube and incubated for 3 hours. After washes, DNA was eluted in Elution buffer (10ml of 1M Tris pH7.5, 10ml of 0.5M EDTA, 20ml of 5M NaCl, 100ml of 10% SDS, 1ml of Proteinase K, 859ml of Sterile Water). DNA was purified with MinElute PCR purification kit (Qiagen). Libraries were prepared using Thruplex DNA-seq kit (Takara) following manufacturer's protocol. Libraries were sequenced on an Illumina NextSeq 500 instrument (paired-end 75bp reads).
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| Contributor(s) |
Ramabadran R, Reyes J |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Jan 14, 2023 |
| Last update date |
Apr 20, 2023 |
| Contact name |
Margaret Goodell |
| Organization name |
Baylor College of Medicine
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| Street address |
One Baylor Plaza, N1030
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (6)
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| GSM6935037 |
embryonic stem cells, ATRA, rep1 [J1_FL-DNMT3A_control_1] |
| GSM6935038 |
embryonic stem cells, ATRA, rep2 [J1_FL-DNMT3A_control_2] |
| GSM6935039 |
embryonic stem cells, ATRA/RNase, rep1 [J1_FL-DNMT3A_RNase_1] |
| GSM6935040 |
embryonic stem cells, ATRA/RNase, rep2 [J1_FL-DNMT3A_RNase_2] |
| GSM6935041 |
embryonic stem cells, ATRA, rep1 [J1_input_control] |
| GSM6935042 |
embryonic stem cells, ATRA/RNase, rep1 [J1_input_RNase] |
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| This SubSeries is part of SuperSeries: |
| GSE222906 |
DNMT3A-coordinated splicing governs the stem state switch toward differentiation in embryonic and hematopoietic stem cells. |
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| Relations |
| BioProject |
PRJNA923815 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE222904_RAW.tar |
2.5 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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