Fig 2: Identification of Lmo2-interacting molecules in LPs.A, Myc- and Flag-tagged Lmo2 was retrovirally transduced into Ebf1-deficient LPs. Total extracts from Myc-Flag-Lmo2-expressing LPs were subjected to two-step affinity purification followed by SDS-PAGE and silver staining. All of the visible bands were subjected to mass spectrometry analysis. B, representative Lmo2-binding molecules in LPs are shown with Mascot scores. The full list of the Lmo2-binding molecules is shown in Table S1. C, Gene Ontology (GO) annotation was performed using the DAVID analysis tool (http://david.ncifcrf.gov/). Top six GO terms for Lmo2-interacting molecules in Ebf1-deficient LPs are shown. D, total extracts from Mock or Myc-Flag-Lmo2 transduced LPs were subjected to immunoprecipitation (IP) with mouse control Ig or anti-Flag mAbs followed by immunoblotting with anti-Zbtb1, or anti-Cbfa2t3 Abs. Nuclear lysates (input) were also subjected to immunoblotting with anti-Zbtb1, anti-Cbfa2t3, anti-Myc (Lmo2), and anti-LaminB Abs. E, Lmo2 and Myc-Flag-tagged Zbtb1 mutants (WT, dBTB, or dZn) were transiently transduced into 293T cells. Total extracts from Lmo2 and Zbtb1 transduced 293T cells were subjected to immunoprecipitation with anti-Flag mAb followed by immunoblotting with anti-Lmo2 mAb. Nuclear lysates (input) were also subjected to immunoblotting with anti-Lmo2, anti-Myc (Zbtb1), and anti-LaminB Abs. Data are representative of two (D and E) or three (A) independent experiments. LP, lymphoid progenitor.

Fig 3: Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs.A, experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP+hNGFR+ LP cells were sorted, and subjected to QuantSeq 3' mRNA sequencing. B, GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs (p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1-deficient LPs are shown. The full lists of the DEGs in Lmo2-, Zbtb1-, Cbfa2t2-, or Cbfa2t3-deficient LPs are shown in Table S2. Data are based on three independent biological replicates. C, heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages (10) in response to the deletion of Lmo2, Zbtb1, or Cbfa2t3. Data are based on the average of three biological replicates. D, five days after sgRNA introduction, GFP+hNGFR+ LP cells were sorted (A). Relative expression levels of Tcf7, Bcl11b, Bcl11a, and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. **p < 0.01, *p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million.

Fig 4: Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs.A, specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2, Zbtb1, Cbfa2t2, or Cbfa2t3 was introduced into the Cas9-expressing (GFP+) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP+hNGFR+ cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B, an experimental scheme for the deletion of Lmo2, Zbtb1, Cbfa2t3, and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1-deficient LPs is shown. C, retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP+) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP+hNGFR+ sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D, the percentage of CD25+ cells among GFP+hNGFR+ sgRNA transduced cells (C) is shown with standard deviation (SD). E, ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP+hNGFR+ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F, the percentage of CD25+ cells among GFP+hNGFR+ sgRNA transduced cells (E) is shown with SD. Data are representative of two (A) or three (C and E) independent experiments. Data represent the mean values of three independent biological replicates (D and F). **p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.

Fig 5: 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. Figure 1—source data 2.Raw data used to generate the graph in Figure 1B. Figure 1—source data 3.Raw data used to generate the graph in Figure 1C. Figure 1—source data 4.Raw data used to generate the graph in Figure 1D.