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| Status |
Public on Feb 13, 2017 |
| Title |
BCL11B AND COMBINATORIAL RESOLUTION OF CELL FATE IN THE T-CELL GENE REGULATORY NETWORK |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
T-cell development from hematopoietic progenitors depends on multiple transcription factors, mobilized and modulated by intrathymic Notch signaling. Key aspects of T-cell specification network architecture have been illuminated through recent reports defining the roles of transcription factors PU.1, GATA-3, and E2A, their interactions with Notch signaling, and roles of Runx1, TCF-1, and Hes1, providing the basis for a comprehensively updated model of the T-cell specification gene regulatory network (GRN). However, the role of lineage commitment factor Bcl11b has been unclear. We use Self-Organizing Maps (SOM) on 63 RNA-seq datasets from normal and perturbed T-cell development to identify positive and negative regulation targets of Bcl11b during commitment and relate them to other regulomes. Both activation and repression targets can be bound by Bcl11b in vivo, but both depend strongly on developmental context. The newly clarified role of Bcl11b distinguishes discrete components of commitment, resolving how innate lymphoid cell, myeloid and dendritic, and B cell fate alternatives are excluded by different mechanisms.
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| Overall design |
Ten million of BM-derived DN3 cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by additional 10 min with addition of formaldehyde up to 1%. The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco). Pelleted nucleus were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30sec sonication followed by 30sec rest, with max power. Six μg per 10^7 cells of anti-Bcl11b Abs (a mixture of A300-383A (Bethyl), A300-385A (Bethyl), ab18465 (Abcam) and 12120 (CST)) were hybridized to Dynabeads Protein A/G (Invitrogen) and added to the diluted chromatin complex. They were incubated over night at 4°C, then washed and eluted for 6hr at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 μg proteinase K). Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (NEB #E6240) following manufacturer’s instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample.
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| Contributor(s) |
Longabaugh WJ, Zeng W, Zhang JA, Hosokawa H, Jansen C, Li L, Romero-Wolf M, Liu P, Kueh HY, Mortazavi A, Rothenberg EV |
| Citation(s) |
28584128 |
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| Submission date |
Jan 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Hiroyuki Hosokawa |
| E-mail(s) |
hosokawa.hiroyuki.g@tokai.ac.jp
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| Organization name |
Tokai University School of Medicine
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| Department |
Department of Immunology
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| Street address |
143 Shimokasuya
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| City |
Isehara |
| State/province |
Kanagawa |
| ZIP/Postal code |
2591193 |
| Country |
Japan |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA361158 |
| SRA |
SRP096665 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE93572_BM_DN3_aBcl11b-ChIP_HOMER_IDR_peaks.csv.gz |
576.4 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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