Fig 2: 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 3: Zbtb1-deficient BM progenitors have attenuated ability to differentiate into T-lineage.A, experimental scheme for the deletion of Zbtb1 and Cbfa2t3 using the CRISPR/Cas9 system in BM progenitor cells is shown. BM progenitors obtained from Cas9;Bcl2 Tg mice were transduced with sgRNA against Zbtb1 and Cbfa2t3 and cultured without stroma cells for 2 days. Then, they were transferred onto OP9-DLL1 and cocultured for 4 days. hNGFR+ sgRNA-transduced cells were gated and analyzed for Lin markers, CD45, CD44, and CD25 expression. B, flow cytometric analysis of sgRNA-transduced BM progenitors cocultured with OP9-DLL1 for 4 days. Representative Lin/CD45 profiles in hNGFR+ sgRNA-introduced cells and CD44/CD25 profiles in hNGFR+Lin-CD45high cells are shown. The results are representative of three independent experiments. C, the number of CD25+ cells among hNGFR+ sgRNA transduced cells (B) is shown with SD. The data represent the mean values of three independent biological replicates. **p < 0.01 by two-sided Student’s t test. BM, bone marrow; sgRNA, single-guide RNA.

Fig 4: Lmo2/Cbfa2t3/Zbtb1 complex binds to the -35 kb region of the Tcf7 locus.A, chIP-seq analyses against Zbtb1 and Cbfa2t3 were performed using Ebf1-deficient LPs without Notch stimulation. The top three enriched sequence motifs among the 5898 reproducible Zbtb1 peaks are shown. B, top three enriched sequence of motifs among the 6077 reproducible Cbfa2t3 peaks are shown. C, Venn diagrams show the numbers of reproducible Zbtb1 and Cbfa2t3 ChIP peaks with the previously reported Lmo2 peaks (31) in Ebf1-deficient LPs. D, representative ChIP-seq tracks for Lmo2, Zbtb1, and Cbfa2t3 in Ebf1-deficient LPs without Notch stimulation, and RBPJ in DN1 cells (10) around the Tcf7 locus are shown. The -31 and -35 kb RBPJ-binding sites are labeled with rectangles. Data are based on ChIP-seq peaks scored as reproducible in two replicate samples (A–C), or representative of two independent experiments (D). ChIP-seq, chromatin immunoprecipitation followed by deep sequencing; DN, double-negative; LP, lymphoid progenitor.

Fig 5: 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.