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| Status |
Public on May 27, 2015 |
| Title |
Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B-cell progenitors |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Expression profiling by high throughput sequencing
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| Summary |
In order to investigate how transcription factor dose impacts B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. While combined reduction of Pax5 and Ebf1 dose had minimal impact on the development of the earliest CD19+ progenitors, these cells displayed an increased T-cell potential in vivo and in vitro. Alteration in lineage fate depended on a Notch1 mediated conversion process while no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5+/-Ebf1+/- pro-B cells was reflected in the transcriptional response because even though cells of both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, only the Pax5+/-Ebf1+/- pro-B cells down-regulated genes central for the preservation of stable B-cell identity. This report stresses the importance of transcription factor dose in lymphocyte development and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling after the generation of activated intracellular Notch1. This provides an insight to how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.
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| Overall design |
EBF-1 ChIP-seq: Cultivated CD43+IgM- cells (ProB) cells from Wt, EBF-1 +/-, PAX-5 +/- and EBF-1 +/- PAX-5 +/- (TH) were assessed for EBF-1 binding by ChIP-seq. Replicate Ebf1 ChIP-seq runs on each genotype (Wt, TH, Ebf1+/- and Pax5+/-) and corresponding inputs were pooled into one dataset and analyzed as one combined sample per genotype.
RNA-seq no treatment: Briefly, ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4), WT (n=4), Ebf1+/- (n=2) and Pax5+/- (n=2) were sorted and RNA extracted with Qiagen RNeasy Micro Kit. RNA was sent to UCLA Microarray Core for library preparation and were subsequently for 50 cycles of HiSeq 2000 SBS sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)).
RNA-seq 1 day on OP9DL1 and OP9: In short, in vitro expanded ProB-cells from C57BL/6J Ebf1+/-Pax5+/- (n=4) and WT (n=4) were exposed either on OP9 or OP9-DL1 stromal cells for 24 hours and RNA extracted with Qiagen RNeasy Micro Kit. Due to low reads, two Wt and Ebf1+/-Pax5+/- were sequenced twice. Libraries were constructed using Nugen's Ovation Ultralow Library systems and were subsequently for 50 cycles of NextSeq500 sequencing generating 20-30 million reads/sample. Data analysis was performed with Arraystar® (DNASTAR)).
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| Contributor(s) |
Ungerbäck J, Åhsberg J, Strid T, Somasundaram R, Sigvardsson M |
| Citation(s) |
26056231 |
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| Submission date |
May 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Rajesh Somasundaram |
| E-mail(s) |
rajesh.somasundaram19@gmail.com
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| Phone |
0046708890787
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| Organization name |
Linkoping University
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| Department |
Microbiology and Molecular Medicine
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| Lab |
Lab 1, Floor- 13 Dept of Clinical and Experimental Medicine (IKE)
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| Street address |
Dept of Clinical and Experimental Medicine (IKE)
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| City |
Linkoping |
| ZIP/Postal code |
SE-58185 |
| Country |
Sweden |
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| Platforms (2) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (36)
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| GSM1695667 |
Ebf1 Chip-seq Ebf1+/- ProB no treatment |
| GSM1695668 |
DNA for Ebf1 ChIP-seq WT-ProB no treatment |
| GSM1695669 |
DNA for Ebf1 ChIP-seq TH-ProB no treatment |
| GSM1695670 |
DNA for Ebf1 ChIP-seq Pax5+/- ProB no treatment |
| GSM1695671 |
DNA for Ebf1 ChIP-seq Ebf1+/- ProB no treatment |
| GSM1695672 |
RNA-seq WT-ProB no treatment, rep1 |
| GSM1695673 |
RNA-seq WT-ProB no treatment, rep2 |
| GSM1695674 |
RNA-seq WT-ProB no treatment, rep3 |
| GSM1695675 |
RNA-seq WT-ProB no treatment, rep4 |
| GSM1695676 |
RNA-seq TH-ProB no treatment, rep1 |
| GSM1695677 |
RNA-seq TH-ProB no treatment, rep2 |
| GSM1695678 |
RNA-seq TH-ProB no treatment, rep3 |
| GSM1695679 |
RNA-seq TH-ProB no treatment, rep4 |
| GSM1695680 |
RNA-seq Pax5+/- ProB no treatment, rep1 |
| GSM1695681 |
RNA-seq Pax5+/- ProB no treatment, rep2 |
| GSM1695682 |
RNA-seq Ebf1+/- ProB no treatment, rep1 |
| GSM1695683 |
RNA-seq Ebf1+/- ProB no treatment, rep2 |
| GSM1695684 |
RNA-seq WT-ProB cultured 1 day on OP9, rep1 |
| GSM1695685 |
RNA-seq WT-ProB cultured 1 day on OP9, rep2 |
| GSM1695686 |
RNA-seq WT-ProB cultured 1 day on OP9, rep3 |
| GSM1695687 |
RNA-seq WT-ProB cultured 1 day on OP9, rep4 |
| GSM1695688 |
RNA-seq TH-ProB cultured 1 day on OP9, rep1 |
| GSM1695689 |
RNA-seq TH-ProB cultured 1 day on OP9, rep2 |
| GSM1695690 |
RNA-seq TH-ProB cultured 1 day on OP9, rep3 |
| GSM1695691 |
RNA-seq TH-ProB cultured 1 day on OP9, rep4 |
| GSM1695692 |
RNA-seq WT-ProB cultured 1 day on OP9DL1, rep1 |
| GSM1695693 |
RNA-seq WT-ProB cultured 1 day on OP9DL1, rep2 |
| GSM1695694 |
RNA-seq WT-ProB cultured 1 day on OP9DL1, rep3 |
| GSM1695695 |
RNA-seq WT-ProB cultured 1 day on OP9DL1, rep4 |
| GSM1695696 |
RNA-seq TH-ProB cultured 1 day on OP9DL1, rep1 |
| GSM1695697 |
RNA-seq TH-ProB cultured 1 day on OP9DL1, rep2 |
| GSM1695698 |
RNA-seq TH-ProB cultured 1 day on OP9DL1, rep3 |
| GSM1695699 |
RNA-seq TH-ProB cultured 1 day on OP9DL1, rep4 |
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| Relations |
| BioProject |
PRJNA284927 |
| SRA |
SRP058715 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE69227_RAW.tar |
2.5 Gb |
(http)(custom) |
TAR (of BED) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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