Figures and data in LMO2 is essential to maintain the ability of progenitors to differentiate into T-cell lineage in mice

Figures
Tables
Additional files

6 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement

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LMO2 is critical for the maintenance of T-cell differentiation potential in *Ebf1*-deficient pro-B cells.

(A) Establishment of *Ebf1*-deficient pro-B cell lines with or without differentiation potential to the T-cell lineage. Lineage markers (CD19, Gr1, TER119, NK1.1)-negative, c-kit-positive cells in *Ebf1^−/⁻* FL were cultured on TD7 or OP9 cells, and *Ebf1*-deficient pro-B cell lines were established. Stably growing pro-B cells with or without T-cell potential (pro-B(+) or pro-B(−)) were cultured on OP9-Mock (Mock) or OP9-Dll4 (Dll4) cells with Flt3L, SCF, and IL7 for 6 days and analyzed for the expression of CD44 and CD25 (right panels) in the lymphoid cell gate (FSC vs. SSC, left panels) by flow cytometry. The numbers in the profiles indicate the relative percentages in each corresponding quadrant or fraction. Numbers of CD25⁺ cells (fold expansion/input) are shown with standard deviation (SD) (right). Statistical analysis was performed using the two-tailed Student’s t-test. **p<0.01. Data are representative of three independent experiments with similar results. (B) Reverse transcription (RT)-quantitative PCR (qPCR) detection of *Meis1*, *Hmga2*, *Bcl11a*, or *Lmo2* transcripts in pro-B(−) (closed columns) and pro-B(+) (open columns) cells. Data represent the mean values of three independent biological replicates, and all values are normalized to the expression of *Actb*. Error bars indicate SD. Three independent experiments were performed, and similar results were obtained. (C) Representative intracellular staining profiles of LMO2 and c-Myc in pro-B(−) (open blue line) and pro-B(+) (open red line) cells are shown. Closed lines (orange) represent staining with control rabbit mAb of pro-B(−) and pro-B(+), which were completely merged. The average mean fluorescent intensity (MFI) of LMO2 is shown with SD (right). A two-tailed Student’s t-test was used for statistical analysis. **p<0.01. Three independent experiments were performed with similar results. (D) Introduction of *Lmo2* is sufficient to maintain the T-cell differentiation potential in pro-B cells. Empty vector- or *Lmo2*-transduced pro-B(−) cells (pro-B(−)/Mock or pro-B(−)/LMO2) were cultured on OP9-Dll4 for 6 days and analyzed for the expression of CD44 and CD25 (right panels) in lymphoid cell gate (left panels) and rat CD2⁺ (lentivirus-infected) CD45⁺ fraction. Numbers of CD25⁺ cells (relative expansion/input) are shown with SD (right). **p<0.01 by two-sided Student’s t-test. Six independent experiments were performed with similar results.

Figure 1—source data 1 Raw data used to generate the graph in Figure 1A.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig1-data1-v1.xlsx
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Figure 1—source data 2 Raw data used to generate the graph in Figure 1B.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig1-data2-v1.xlsx
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Figure 1—source data 3 Raw data used to generate the graph in Figure 1C.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig1-data3-v1.xlsx
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Figure 1—source data 4 Raw data used to generate the graph in Figure 1D.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig1-data4-v1.xlsx
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Figure 1—figure supplement 1

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Characterization of pro-B(+) and pro-B(−) cells.

(A) The stromal cell line TD7 can support B-cell differentiation. BM progenitor cells from C57BL/6 mice were co-cultured with the TD7 or OP9 thymic stromal cells for 6 days. Representative CD19 profiles in lymphocytes are shown. Results represent two independent biological replicates. (B) Lin⁺ cells are generated more frequently on TD7 than on OP9 monolayers. Fetal liver-derived HSCs from *Ebf1*-deficient mice were cultured on TD7 or OP9 monolayers for 3 days. Representative CD11b/Gr1 profiles among the CD45⁺ lymphocytes are shown. Results are representative of two independent biological replicates. (C) Pro-B(−) and pro-B(+) cells express intermediate levels of B220. B220 expression in pro-B(−) and pro-B(+) cells is shown. Results represent three independent replicates. (D) Pro-B(+) cells were co-cultured with OP9-Dll4, 1 ng/ml of IL-7, and 5 ng/ml of Flt3L for 3 weeks. Thereafter, the cells were subjected to flow cytometric analysis. A representative CD4/CD8 profile in lymphocytes is shown. Results represent two independent biological replicates. (E) Pro-B(+) cells (5 × 10⁶, CD45.2) were mixed with BM cells obtained from wild-type congenic mice (2 × 10⁵, CD45.1) and transplanted into the lethally irradiated (9 Gy) *Rag2*/Cg-deficient hosts. The recipient mice were analyzed after 5 weeks of transplantation. Flow cytometric analysis of thymocytes and splenocytes was performed. Representative CD4/CD8 profiles among the CD45.2⁺ thymocytes (left) and splenocytes (right) are shown. Results are representative of two independent biological replicates. (F) Pro-B(−) and pro-B(+) cells express comparable levels of Notch receptors on their surface. Representative Notch1, Notch2, Notch3, and Notch4 profiles on pro-B(−) and pro-B(+) cells are shown. Results are representative of two independent biological replicates. (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of the differentially expressed genes in pro-B(+) and pro-B(−) cells (Supplementary file 1, FC > 10, n = 387) was performed using the DAVID analysis tools (https://david.ncifcrf.gov/). Top three KEGG pathway annotations are shown. Data are based on two independent biological replicates.

Figure 2 with 1 supplement

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Loss of *Lmo2* leads to the differentiation arrest in pro-B(+) cells.

(A) An experimental scheme for the deletion of *Lmo2* using the CRISPR/Cas9 system in pro-B cell lines is shown. (B) Retroviral vectors encoding sgRNA against luciferase (sgCont.) or LMO2 (sgLMO2) were introduced into Cas9-expressing (GFP⁺) pro-B(+) cells. Five (cultured on OP9-Mock for 5 days, left panels) or 10 days (cultured on OP9-Mock for 10 days, right panels) after co-cultured on OP9-Mock cells following the infection, pro-B cells were cultured again on OP9-Mock (Mock) or OP9-Dll4 (Dll4) stromal cells for 3 days. GFP⁺hNGFR⁺ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression (left). The percentages and numbers of CD25⁺ cells among GFP⁺hNGFR⁺ sgRNA-transduced cells, cultured on OP9-Dll4, are shown with SD (right). The data represent the mean values of three independent biological replicates. Each value is indicated by a closed circle. **p<0.01 by two-sided Student’s t-test.

Figure 2—source data 1 Raw data used to generate the graph in Figure 2B.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig2-data1-v1.xlsx
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Figure 2—figure supplement 1

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CRISPR/Cas9-mediated deletion of *Lmo2* in BM progenitors.

(A) Experimental scheme for *Lmo2* deletion in primary BM progenitor cells using the CRISPR/Cas9 system is shown. (B) Flow cytometric analysis of BM progenitors transduced with sgRNA and co-cultured with OP9-Dll1 for 4 days. Representative CD45/hNGFR (sgRNA) profiles in single cells, FSC/Lin profiles in CD45⁺hNGFR⁺ cells, and CD44/CD25 profiles in CD45⁺hNGFR⁺Lin^- cells are shown. Results are representative of three biological replicates. Percentages of CD25⁺ cells among the CD45⁺hNGFR⁺Lin^- cells are shown with SD (right). (C) Specific depletion of targeted LMO2 protein. Four days after sgRNA transduction in Figure 2A, lysates from retrovirus-infected GFP⁺hNGFR⁺ Pro-B(+) cells were subjected to immunoblotting for LMO2. Two independent experiments were performed with similar results.

Figure 2—figure supplement 1—source data 1 Original data used to generate the panels in Figure 2—figure supplement 1C.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2 Raw data used to generate the graph in Figure 2—figure supplement 1B.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig2-figsupp1-data2-v1.xlsx
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Figure 3 with 1 supplement

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LMO2 regulates survival of *Ebf1*-deficient pro-B cells via Bcl11a/Bcl2 pathway.

(A) RT-qPCR detection of *Meis1*, *Hmga2*, and *Bcl11a* transcripts in *Lmo2*- (red column), *Meis1*-transfected (green column) pro-B(−) cells, parent pro-B(−) (black column), and pro-B(+) (white column) cells, as shown in Figure 1B. Two independent experiments were performed with similar results. (B) Overexpression of *Bcl2* improves cell survival of pro-B(−) cells. Pro-B(+) cells (open column) and empty vector (mock) or human *BCL2* (*Bcl2*)-transduced pro-B(−) cells were cultured on OP9-Mock or OP9-Dll4 for 2 days. After culturing, the dead cells were detected by staining for Annexin V and 7-AAD in CD45⁺ and hNGFR⁺ (retrovirus-infected) cell populations (Figure 3—figure supplement 1). The cell death index was calculated as the difference in the percentage of dead cells in pro-B cells after co-culturing with OP9-Mock and OP9-Dll4. The data represent the mean values of three independent biological replicates with SD. **p<0.01 by two-sided Student’s t-test. (C) Bcl2 overexpression does not provide differentiation potential in pro-B(−) cells. Pro-B(−) or pro-B(+) cells with human *BCL2* (pro-B(−)/Bcl2, pro-B(+)/Bcl2) were cultured on OP9-Dll4 as shown in Figure 1D. After culturing, the live cells were analyzed for the expression of CD44 and CD25 (left). The data represent the mean values of percentages of CD25⁺ cells in three independent biological replicates with SD (right). ***p<0.001 by two-sided Student’s t-test.

Figure 3—source data 1 Raw data used to generate the graph in Figure 3A.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig3-data1-v1.xlsx
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Figure 3—source data 2 Raw data used to generate the graph in Figure 3B.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig3-data2-v1.xlsx
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Figure 3—source data 3 Raw data used to generate the graph in Figure 3C.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig3-data3-v1.xlsx
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Figure 3—figure supplement 1

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Overexpression of *Bcl2* improves the cell survival in pro-B(−) cells.

Pro-B(+) cells and empty vector (mock) or human BCL2-transduced pro-B(−) cells (pro-B(−)/Mock or pro-B(−)/Bcl2) were cultured on OP9-Mock (Mock) or OP9-Dll4 (Dll4) for 2 days. After the cultures, the dead cells were detected by the staining of both AnnexinV and 7-AAD in CD45⁺ and hNGFR⁺ (retrovirus-infected) cell population. Numbers in the profiles indicate the relative percentages for each corresponding fraction. Three independent experiments were performed with similar results. The ratio of AnnexinV⁺7-AAD⁺ cells in pro-B cells co-cultured with OP9-Mock and OP9-Dll4 (OP9-Dll4/OP9-Mock) is shown with SD (right).

Figure 3—figure supplement 1—source data 1 Raw data used to generate the graph in Figure 3—figure supplement 1.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig3-figsupp1-data1-v1.xlsx
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Figure 4 with 1 supplement

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LMO2 is required for activation of *Tcf7* after Notch signaling.

(A) Expression levels of *Tcf7* and *Gata3* in pro-B(−) and pro-B(+) cells with exogenous BCL2, at 0–5 days after the culture on OP9-Dll4, were analyzed by RT-qPCR. The relative expression (/*Actb*) is shown with SD. *p<0.05, **p<0.01 by two-sided Student’s t-test. (B) Intracellular staining of TCF1 or GATA3 in pro-B(+)/Bcl2 (upper panels) and pro-B(−)/Bcl2 (lower panels) was performed at day 7 after Notch stimulation; representative expression profiles of CD44 and CD25 are also shown (left). Results are representative of three independent experiments. (C) Introduction of *Tcf7* provides differentiation potential for T-cell lineage to pro-B(−)/Bcl2. Pro-B(−)/Bcl2 cells were infected with either empty control or *Tcf7*-containing lentivirus, and the cells were co-cultured on OP9-Mock (upper panels) or OP9-Dll4 (lower panels) for 3 days. Lentivirus-infected cells were analyzed for the expression of CD44 and CD25. Three independent experiments were performed with similar results. The percentages of CD25⁺ cells are shown with SD (right). ***p<0.001 by two-sided Student’s t-test.

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Figure 4—figure supplement 1

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Down-regulation of *Tcf7* in *Lmo2*-deficient pro-B(+) cells.

Expression levels of *Tcf7* in Cas9-expressing pro-B(+) cells co-cultured with OP9-Mock for 5 or 10 days after sgRNA transduction (Figure 2A) were analyzed using RT-qPCR.

Relative expression of *Actb* is shown with SD. *p<0.05, **p<0.01 by two-sided Student’s t-test. Data are based on three biological replicates.

Figure 4—figure supplement 1—source data 1 Raw data used to generate the graph in Figure 4—figure supplement 1.: https://cdn.elifesciences.org/articles/68227/elife-68227-fig4-figsupp1-data1-v1.xlsx
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Figure 5 with 1 supplement

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DNA methylation status of the *Tcf7* locus is maintained by LMO2.

(A) A CpG island at the transcriptional start sites (TSSs) of the *Tcf7* locus, which contains 25 potential CpG methylation sites. (B) The DNA methylation status of CpG island at the TSSs of the *Tcf7* locus was determined by bisulfite sequencing in pro-B(−), pro-B(+), and LMO2-transduced pro-B(−) cells (pro-B(−)/LMO2). Bisulfite-converted genomic DNA around the TSSs of *Tcf7* was amplified using PCR, and each PCR product was sequenced. The 25 horizontal circles each represent a CpG sequence derived from a single PCR product (17 clones from pro-B(−) cells, 9 clones from pro-B(+) cells, and 16 clones from pro-B(−)/LMO2 cells). Closed and open circles indicate methylated and demethylated CpG sites, respectively. The frequencies of the methylated CpGs are shown with SD. Data are based on two independent pooled experiments.

Figure 5—figure supplement 1

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H3K4-3Me levels around the *Tcf7* locus in *Lmo2*-deficient cells.

Cas9-expressing pro-B(+) cells were co-cultured with OP9-Mock for 5 or 10 days after sgRNA transduction (Figure 2A) and then purified.

H3K4-3Me levels around the *Tcf7* locus were determined using the ChIP assay and qPCR analysis. The mean values (% input) are shown with SD. Data are based on three independent experiments. *p<0.05, **p<0.01 by two-sided Student’s t-test.

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Figure 6 with 1 supplement

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LMO2 binds to the upstream region of the *Tcf7* locus.

(A) LMO2 ChIP-seq analyses were performed using the pro-B(+) cell line. The top three enriched sequence motifs among the 1585 reproducible LMO2 peaks are shown. Data are based on ChIP-seq peaks scored as reproducible in two replicate samples. (B) ChIP-seq tracks showing two replicates of LMO2 binding profiles around the *Lyl1*, *Erg*, *Hhex*, *Tcf7*, and *Bcl11a* loci in pro-B(+) cell line with tracks for 1% input.

Figure 6—figure supplement 1

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LMO2 binds to one of the RBPJ and Runx1 binding sites at the *Tcf7* locus.

ChIP-seq tracks show binding profiles of LMO2 in pro-B(+) cells, and RBPJ and Runx1 in DN1 cells (GSE148441, GSE110020) around the *Tcf7* locus. The conservation track is also shown at the bottom. The −31 and −35 kb RBPJ binding sites are labeled with rectangles. Data are representative of two independent experiments.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Mus musculus)	Ebf1^+/-	Pongubala et al., 2008		Provided by Dr. Grosschedl, Max Planck Institute of Immunobiology and Epigenetics
Genetic reagent (Mus musculus)	B6.Cg-Tg(BCL2)25Wehi/J	Jackson Laboratory	Stock# 002320
Genetic reagent (Mus musculus)	B6.Gt(ROSA)26^{Sortm1.1(CAG-cas9*,-EGFP)Fezh}/J	Jackson Laboratory	Stock# 024858
Cell line (Mus musculus)	OP9	Yokoyama et al., 2013	Stromal cell line derived from fetal murine calvaria (B6 x C3H, op/op)
Cell line (Mus musculus)	TD7	This paper		Mouse fetal thymus (B6, E15.5)-derived mesenchymal cell line
Cell line (Mus musculus)	Pro-B(+)	This paper		Ebf1-deficient fetal liver-derived hematopoietic progenitor cell line
Cell line (Mus musculus)	Pro-B(−)	This paper		Ebf1-deficient fetal liver-derived hematopoietic progenitor cell line
Cell line (Mus musculus)	OP9-Dll4	Hirano et al., 2020
Cell line (Homo sapiens)	HEK293T	Hirano et al., 2020	RRID:CVCL_0063
Cell line (Homo sapiens)	PLAT-E	Hirano et al., 2020	RRID:CVCL_B488
Antibody	FITC anti-mouse CD4 (Rat monoclonal)	BD Biosciences	Cat# 561835 RRID:AB_10894386	FC (1:500)
Antibody	PE anti-mouse CD4 (Rat monoclonal)	BD Biosciences	Cat# 561829 RRID:AB_10926205	FC (1:500)
Antibody	APC anti-mouse CD8a (Rat monoclonal)	BioLegend	Cat# 100711 RRID:AB_312750	FC (1:500)
Antibody	APCCy7 anti-mouse CD8a (Rat monoclonal)	BioLegend	Cat# 100713 RRID:AB_312752	FC (1:500)
Antibody	PerCpCy5.5 anti-mouse CD11b (Rat monoclonal)	BioLegend	Cat# 101227 RRID:AB_893233	FC (1:500)
Antibody	PECy7 anti-mouse CD19 (Rat monoclonal)	Tonbo Biosciences	Cat# 60-0193 RRID:AB_2621840	FC (1:250)
Antibody	PerCpCy5.5 anti-mouse CD25 (Rat monoclonal)	eBioscience	Cat# 45-0251-82 RRID:AB_914324	FC (1:1000)
Antibody	APC-e780 anti-mouse CD25 (Rat monoclonal)	eBioscience	Cat# 47-0251-82 RRID:AB_1272179	FC (1:200)
Antibody	FITC anti-mouse CD44 (Rat monoclonal)	BioLegend	Cat# 103005 RRID:AB_312956	FC (1:500)
Antibody	APCCy7 anti-mouse CD44 (Rat monoclonal)	BioLegend	Cat# 103027 RRID:AB_830784	FC (1:500)
Antibody	PECy7 anti-mouse CD45 (Rat monoclonal)	eBioscience	Cat# 25-0451-82 RRID:AB_2734986	FC (1:400)
Antibody	APC anti-mouse CD45 (Rat monoclonal)	BioLegend	Cat# 103111 RRID:AB_312976	FC (1:1000)
Antibody	PE anti-mouse CD45.1 (Mouse monoclonal)	BioLegend	Cat# 110727 RRID:AB_893348	FC (1:250)
Antibody	PerCpCy5.5 anti-mouse CD45.2 (Mouse monoclonal)	BioLegend	Cat# 109807 RRID:AB_313444	FC (1:250)
Antibody	PerCpCy5.5 anti-mouse B220 (Rat monoclonal)	BioLegend	Cat# 103235 RRID:AB_893356	FC (1:200)
Antibody	APCCy7 anti-mouse Thy1.2 (Rat monoclonal)	BioLegend	Cat# 105327 RRID:AB_10613280	FC (1:1000)
Antibody	FITC anti-mouse Gr-1 (Rat monoclonal)	BioLegend	Cat# 108405 RRID:AB_313370	FC (1:500)
Antibody	Biotin anti-mouse Gr-1 (Rat monoclonal)	eBioscience	Cat# 13-5931-86 RRID:AB_466802	FC (1:300)
Antibody	Biotin anti-mouse TER119 (Rat monoclonal)	eBioscience	Cat# 13-5921-85 RRID:AB_466798	FC (1:300)
Antibody	Biotin anti-mouse CD11b (Rat monoclonal)	eBioscience	Cat# 13-0112-86 RRID:AB_466361	FC (1:300)
Antibody	Biotin anti-mouse CD11c (Armenian Hamster monoclonal)	eBioscience	Cat# 13-0114-85 RRID:AB_466364	FC (1:300)
Antibody	Biotin anti-mouse CD19 (Rat monoclonal)	eBioscience	Cat# 13-0193-85 RRID:AB_657658	FC (1:300)
Antibody	Biotin anti-mouse NK1.1 (Rat monoclonal)	eBioscience	Cat# 13-5941-85 RRID:AB_466805	FC (1:300)
Antibody	Biotin anti-mouse CD3ε (Armenian Hamster monoclonal)	eBioscience	Cat# 13-0031-82 RRID:AB_466319	FC (1:300)
Antibody	PE anti-rat CD2 (Mouse monoclonal)	BioLegend	Cat# 201305 RRID:AB_2073811	FC (1:500)
Antibody	PE anti-human NGFR (Mouse monoclonal)	eBioscience	Cat# 12-9400-42 RRID:AB_2572710	FC (1:500)
Antibody	PE Hamster IgG (Armenian Hamster monoclonal)	BioLegend	Cat# 400907 RRID:AB_326593	FC (1:200)
Antibody	PE anti-mouse Notch1 (Armenian Hamster monoclonal)	BioLegend	Cat# 130607 RRID:AB_1227719	FC (1:200)
Antibody	PE anti-mouse Notch2 (Armenian Hamster monoclonal)	BioLegend	Cat# 130707 RRID:AB_1227725	FC (1:200)
Antibody	PE anti-mouse Notch3 (Armenian Hamster monoclonal)	BioLegend	Cat# 130507 RRID:AB_1227733	FC (1:200)
Antibody	PE anti-mouse Notch4 (Armenian Hamster monoclonal)	BioLegend	Cat# 128407 RRID:AB_1133997	FC (1:200)
Antibody	Alexa Fluor 647 Mouse IgG1 (Mouse monoclonal)	BioLegend	Cat# 400130 RRID:AB_2800436	FC (2 µl per test)
Antibody	Alexa Fluor 647 anti-GATA3 (Mouse monoclonal)	BD Biosciences	Cat# 560068 RRID:AB_1645316	FC (8 µl per test)
Antibody	Rabbit-IgG (Rabbit monoclonal)	Cell Signaling Technology	Cat# 3900 RRID:AB_1550038	FC (1:500)
Antibody	Anti-TCF1 (Rabbit monoclonal)	Cell Signaling Technology	Cat# 2203 RRID:AB_2199302	FC (1:100)
Antibody	Anti-LMO2 (Rabbit monoclonal)	Abcam	Cat# ab91652 RRID:AB_2049879	FC (1:200) ChIP (2ug/sample)
Antibody	Anti-c-Myc (Rabbit monoclonal)	Cell Signaling Technology	Cat# 5605 RRID:AB_1903938	FC (1:100)
Antibody	DyLight 488 anti-rabbit IgG (Donkey polyclonal)	BioLegend	Cat# 406404 RRID:AB_1575130	FC (1:250)
Antibody	DyLight 649 anti-rabbit IgG (Donkey polyclonal)	BioLegend	Cat# 406406 RRID:AB_1575135	FC (1:500)
Antibody	Anti-Tubulinα (Mouse monoclonal)	Sigma	Cat# T6199 RRID:AB_477583	WB (1:1000)
Antibody	Anti-LMO2 (Mouse monoclonal)	Novus	Cat# NB110-78626 RRID:AB_1084895	WB (1:1000) ChIP (2 µg/sample)
Antibody	Anti-human LMO2 (Goat polyclonal)	R&D Systems	Cat# AF2726 RRID:AB_2249968	ChIP (2 µg/sample)
Antibody	Anti-H3K4me3 (Rabbit polyclonal)	Millipore	Cat# 07-473 RRID:AB_1977252	ChIP (5 µl/sample)
Recombinant DNA reagent (plasmid)	pCMV-VSV-G-RSV-Rev	RIKEN BRC	Cat# RDB04393	Lentiviral packaging plasmid
Recombinant DNA reagent (plasmid)	pCAG-HIVgp	RIKEN BRC	Cat# RDB04394	Lentiviral packaging plasmid
Recombinant DNA reagent (plasmid)	pLVS-EF-IR2	This paper		Lentiviral vector with IRES-rat CD2
Recombinant DNA reagent (plasmid)	mLmo2/pLVS-EF-IR2	This paper		pLVS-EF-IR2 Lentiviral vector encoding mLmo2
Recombinant DNA reagent (plasmid)	mTcf7/pLVS-EF-IR2	This paper		pLVS-EF-IR2 Lentiviral vector encoding mTcf7
Recombinant DNA reagent (plasmid)	GCDN	Hirano et al., 2015		Retroviral vector with IRES-human NGFR
Recombinant DNA reagent (plasmid)	hBCL2/GCDN	This paper		GCDN Retroviral vector encoding hBCL2
Recombinant DNA reagent (plasmid)	mMeis1/GCDN	This paper		GCDN Retroviral vector encoding mMeis1
Recombinant DNA reagent (plasmid)	mHmga2/GCDN	This paper		GCDN Retroviral vector encoding mHmga2
Recombinant DNA reagent (plasmid)	Cas9-GFP	Hosokawa et al., 2018		Retroviral vector to express Cas9 and GFP
Recombinant DNA reagent (plasmid)	E42-dTet	Hosokawa et al., 2018		Retroviral vector to express sgRNA and human NGFR
Recombinant DNA reagent (plasmid)	sgRNA against Luciferase (control)	Hosokawa et al., 2018		5’-ggcatttcgcag cctaccg-3’
Recombinant DNA reagent (plasmid)	sgRNA against LMO2 #1	This paper		5’-tcgatggccgag gacattg-3’
Recombinant DNA reagent (plasmid)	sgRNA against LMO2 #2	This paper		5’-aatgtcctcggc catcgaa-3’
Recombinant DNA reagent (plasmid)	sgRNA against LMO2 #3	This paper		5’-gaaagccatcga ccagtac-3’
Sequence-based reagent	ActB (Forward)	This paper	PCR primers	5’-tacagcccgggg agcat-3’
Sequence-based reagent	ActB (Reverse)	This paper	PCR primers	5’-acacccgccac cagttc-3’
Sequence-based reagent	Meis1 (Forward)	This paper	PCR primers	5’-gacgctttaaag agagataaagatgc-3’
Sequence-based reagent	Meis1 (Reverse)	This paper	PCR primers	5’- catttctcaaa aatcagtgctaaga -3’
Sequence-based reagent	Hmga2 (Forward)	This paper	PCR primers	5’-aaggcagcaaaa acaagagc-3’
Sequence-based reagent	Hmga2 (Reverse)	This paper	PCR primers	5’-gccgtttttctc caatggt-3’
Sequence-based reagent	Bcl11a (Forward)	This paper	PCR primers	5’-ccaaacaggaac acacatagcaga-3’
Sequence-based reagent	Bcl11a (Reverse)	This paper	PCR primers	5’-ggggattagagc tccgtgt-3’
Sequence-based reagent	Gata3 (Forward)	This paper	PCR primers	5’-ttatcaagccca agcgaag-3’
Sequence-based reagent	Gata3 (Reverse)	This paper	PCR primers	5’-tggtggtggtct gacagttc-3’
Sequence-based reagent	Lmo2 (Forward)	This paper	PCR primers	5’-gaggcgcctcta ctacaa-3’
Sequence-based reagent	Lmo2 (Reverse)	This paper	PCR primers	5’-gatccgcttgtcacaggatg-3’
Sequence-based reagent	Tcf7 (Forward)	This paper	PCR primers	5’-cagctcccccatactgtgag-3’
Sequence-based reagent	Tcf7 (Reverse)	This paper	PCR primers	5’-tgctgtctatatccgcaggaa-3’
Sequence-based reagent	Tcf7 promoter region (Forward)	This paper	PCR primers	5’-ttaagtttttattggtgaatgagtt-3’
Sequence-based reagent	Tcf7 promoter region (Reverse)	This paper	PCR primers	5’-aaaaaactccaaaaataaaacccac-3’
Sequence-based reagent	Tcf7 TSS (Forward)	This paper	PCR primers	5’-gcagcaagggttgcattt-3’
Sequence-based reagent	Tcf7 TSS (Reverse)	This paper	PCR primers	5’-ttgtctgtactgggctgtttacat-3’
Sequence-based reagent	Tcf7 -31kb (Forward)	This paper	PCR primers	5’-ttccatccaccgttttgttt-3’
Sequence-based reagent	Tcf7 -31kb (Reverse)	This paper	PCR primers	5’-ggcgtgtggtgggaatacta-3’
Sequence-based reagent	Tcf7 -35kb (Forward)	This paper	PCR primers	5’-ctgcaagcagctggaagtc-3’
Sequence-based reagent	Tcf7 -35kb (Reverse)	This paper	PCR primers	5’-cactggaagctgtgagtgatg-3’
Sequence-based reagent	Igk 3’UTR (Forward)	This paper	PCR primers	5’-ggcacatctgttgctttcgc -3’
Sequence-based reagent	Igk 3’UTR (Reverse)	This paper	PCR primers	5’-ggggtaggga gcaggtgtat-3’
Peptide, recombinant protein	PerCpCy5.5 streptavidin	BioLegend	Cat# 405214	FC (1:200)
Peptide, recombinant protein	Recombinant Mouse SCF	PeproTech	Cat# 250-03
Peptide, recombinant protein	Recombinant Human FLT3L	PeproTech	Cat# 300-19
Peptide, recombinant protein	Recombinant Mouse IL-7	PeproTech	Cat# 217-17
Commercial assay or kit	Foxp3 / Transcription Factor Staining Set	eBioscience	Cat# 00-5523-00	Used to detect TCF1 and GATA3
Commercial assay or kit	Fixation/Permeabilization Solution Kit with BD GolgiStop	BD Biosciences	Cat# 554715	Used to detect Lmo2 and c-Myc
Commercial assay or kit	Permeabilization Buffer Plus	BD Biosciences	Cat# 561651	Used to detect Lmo2 and c-Myc
Commercial assay or kit	High Capacity cDNA Reverse Transcription Kit	Thermo Fisher Scientific	Cat# 4368814
Commercial assay or kit	NucleoSpin Tissue	TaKaRa Bio	Cat# 740952.50
Commercial assay or kit	MethylEasy Xceed	TaKaRa Bio	Cat# ME002
Commercial assay or kit	TaKaRa EpiTaq HS (for bisulfite-treated DNA)	TaKaRa Bio	Cat# R110A
Commercial assay or kit	Mighty TA-cloning kit	TaKaRa Bio	Cat# 6028
Commercial assay or kit	Anti-Biotin MicroBeads	Miltenyi Biotec	Cat# 130-090-485
Commercial assay or kit	Dynabeads Protein A	Thermo Fisher Scientific	Cat# 10001D
Commercial assay or kit	Dynabeads Protein G	Thermo Fisher Scientific	Cat# 10003D
Commercial assay or kit	Dynabeads M-280 Sheep Anti-Rabbit IgG	Thermo Fisher Scientific	Cat# 11203D
Commercial assay or kit	NE-PER Nuclear and Cytoplasmic Extraction Reagents	Pierce	Cat# 78833
Commercial assay or kit	PCR purification Kit	Qiagen	Cat# 28004
Commercial assay or kit	Fast SYBR Green Master Mix	Thermo Fisher Scientific	Cat# 4385614
Commercial assay or kit	NEBNext Ultra II DNA Library Prep with Sample Purification Beads	NEB	Cat# E7103S
Commercial assay or kit	NEBNext Multiplex Oligos for Illumina	NEB	Cat# E7500S
Chemical compound, drug	Trizol reagent	Thermo Fisher Scientific	Cat# 15596026
Chemical compound, drug	7-AAD	BioLegend	Cat# 420403	FC (1:50)
Chemical compound, drug	Alexa Fluor 647 Annexin V	BioLegend	Cat# 640911	FC (1:40)
Chemical compound, drug	DSG (disuccinimidyl glutarate)	Thermo Fisher Scientific	Cat# 20593	1 mg/ml
Software, algorithm	FlowJo	BD Biosciences	RRID:SCR_008520