Figure 3.

EBF1 Represses Gata3 Transcript Levels

(A) Ebf1^−/− lymphoid progenitors were transduced with MigR1, MiY-EBF1, or Mig-Pax5 virus alone or cotransduced with MiY-EBF1 and Mig-Pax5 and plated on OP9 cells supplemented with IL-7, FL, and SCF. 50,000 DAPI⁻YFP⁺GFP⁻, YFP⁻GFP⁺, or YFP⁺GFP⁺ cells were sorted 24 hr later for RNA isolation. Transcript levels of the indicated genes were assayed by qRT-PCR. Expression levels in MigR1-transduced samples were set to 1 and sorted pro-B cells (B220⁺CD43⁺AA4.1⁺CD19⁺) were included as an additional control. Error bars indicate SEMs, ^*p < 0.001.

(B) 2 × 10⁷Pax5^−/− pro-B cells were transduced with control MigR1 or MigR-EBF1 virus and assayed for relative levels of Gata3 transcripts 24 hr later by sorting on DAPI⁻GFP⁺ cells as in (A). Starting cell numbers were high due to the refractory nature of these cells to retroviral transduction. Error bars indicate SEMs, ^*p < 0.01.

(C) Ebf1^−/− pre-pro-B cells were single transduced with Mig-ICN1 or TCF1-VEX or cotransduced with MigY-EBF1 and MigR-ICN1 or MigY-EBF1 and TCF1-VEX viruses. Seven days later, RNA was isolated and assayed for relative expression of Gata3 transcripts as in (A).

(D) 50,000 B220⁺Ly6D⁻ or B220⁺Ly6D⁺ BM cells were sorted from chimeras established with WT or Ebf1^−/− fetal liver progenitors as in Figure 1A. cDNA was prepared and qRT-PCR performed as in (A). Expression levels for the indicated genes in WT B220⁺Ly6D⁺ cells were arbitrarily set to 1, except for Igll1 detection where B220⁺Ly6D⁻ cells were employed. ND, signal not detected. Data are means ± SEMs of triplicate samples. Error bars indicate SEMs, ^*p < 0.001, ^**p < 0.01.

All data are representative of three separate experiments. See also Figure S3.