Transcriptional repression of Gata3 is essential for early B cell commitment

Abstract

The mechanisms underlying the silencing of alternative fate potentials in very early B cell precursors remain unclear. Using gain- and loss-of-function approaches together with a synthetic Zinc-finger polypeptide (6ZFP) engineered to prevent transcription factor binding to a defined cis element, we show that the transcription factor EBF1 promotes B cell lineage commitment by directly repressing expression of the T-cell-lineage-requisite Gata3 gene. Ebf1-deficient lymphoid progenitors exhibited increased T cell lineage potential and elevated Gata3 transcript expression, whereas enforced EBF1 expression inhibited T cell differentiation and caused rapid loss of Gata3 mRNA. Notably, 6ZFP-mediated perturbation of EBF1 binding to a Gata3 regulatory region restored Gata3 expression, abrogated EBF1-driven suppression of T cell differentiation, and prevented B cell differentiation via a GATA3-dependent mechanism. Furthermore, EBF1 binding to Gata3 regulatory sites induced repressive histone modifications across this region. These data identify a transcriptional circuit critical for B cell lineage commitment.

Figure 1

Ebf1-Deficient B-Cell-Lineage-Biased Precursors Possess Increased T Lymphoid Potential (A) BM cells from chimeras reconstituted with B6 or B6.Ebf1^−/− fetal liver 12 weeks previously were stained with antibodies to the host-specific determinant CD45^SJL and the antibodies shown in Figure S1. 2 × 10⁶ events were collected on an LSR2 flow cytometer and gated as in Figure S1. (B) 200 WT or Ebf1^−/− B220⁺Ly6D⁻ or B220⁻Ly6D⁺ cells derived from the indicated chimeras were sorted onto pre-established OP9-DL4 cells supplemented with IL-7, FL, and SCF. Seven days later, cells were counted and analyzed for cell surface marker expression. The mean number of viable (DAPI⁻) CD45⁺ early T cell lineage (Thy1.2⁺CD25⁺) cells in triplicate cultures is shown. Error bars indicate SEMs, ^*p < 0.005. Data are representative of three separate experiments. (C) 96 single WT or Ebf1^−/− B220⁺Ly6D⁻ or B220⁺Ly6D⁺ cells were sorted onto pre-established OP9-DL4 cells in flat-bottom 96-well plates with IL-7, FL, and SCF. Wells containing cell growth were counted and analyzed on day 10. Data are representative of two separate experiments. See also Figure S1.

Figure 2

Notch1 and TCF1 Fail to Prevent Inhibition of Early T Cell Lineage Differentiation by EBF1 (A) LSK (Lin⁻Sca1⁺c-kit⁺) cells were sorted from e14.5 Pax5^−/− fetal livers and transduced with control MigR1 or MigR-EBF1 virus. Viable GFP⁺ cells were sorted 24 hr after transduction onto pre-established OP9-DL4 cells with IL-7, FL, and SCF. Seven days later, cultures were stained and analyzed for absolute numbers of DAPI⁻CD45⁺GFP⁺Thy1.2⁺CD25⁺ cells. Data in right-most graph are means and SEMs from each group. (B) e14.5 Ebf1^−/− fetal liver LSKs were isolated and cotransduced with MigY-EBF1 and MigR-ICN1 viruses and plated on OP9 stromal cells. A day later, cells were washed and replated on OP9s in fresh media supplemented with IL-7, FL, and SCF. On day 7, cultures were stained and analyzed for relative contribution of DAPI⁻CD45⁺ single-transduced (GFP⁺YFP⁻ or GFP⁻YFP⁺) or double-transduced (GFP⁺YFP⁺) cells that coexpressed Thy1.2 and CD25. (C) e14.5 Ebf1^−/− fetal liver LSKs were isolated and cotransduced with MigY-EBF1 and TCF1-VEX viruses as in (B). Plots were gated on DAPI⁻CD45⁺ single- or double-transduced cells. All graphs show means ± SEMs of triplicate samples. Data are representative of three separate experiments. See also Figure S2.

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.

Figure 4

EBF1 Binds to the Gata3 Locus and Induces Repressive Histone Modifications (A) Ebf1^−/− lymphoid progenitors were transduced with a construct encoding an EBF1-Engrailed fusion protein, wild-type EBF1, or control virus. After 12 hr, GFP⁺ cells were harvested, cDNA prepared, and Igll1 or Gata3 mRNA levels determined with Taqman primer-probe sets. The data are normalized to transcript levels for either Igll1 or Gata3 in cells transduced with MigR1 control virus. (B) ChIP via anti-EBF1 was performed with the indicated cell types including CD19⁺ BM lymphocytes and CD3ε⁺ thymocytes. qRT-PCR was performed to amplify immunoprecipitated DNA with flanking primers to sites a or b; detection signals were normalized to input DNA. Data are expressed as fold enrichment over Ebf1^−/− cells, which served as negative control. (C) H3K27me3 modifications within sites 1–5 in CD19⁺ BM lymphocytes relative to Ebf1^−/− cells. (D) Ebf1^−/− cells were transduced with control MigR1 or MigR-EBF1 virus and plated on OP9 cells in presence of IL-7, FL, and SCF. 24 hr later, DAPI⁻GFP⁺ cells were sorted, fixed, and processed for ChIP. Relative H3K27me3 enrichment at sites 1–5 in EBF1 versus control-transduced samples is shown. Data are means ± SEMs of triplicate samples. Error bars indicate SEMs, ^*p < 0.01, ^**p < 0.001. Data are representative of four separate experiments. See also Figures S3 and S4 and Table S1.

Figure 5

Inhibition of EBF1 Binding to the Gata3 Locus Rescues T Cell Differentiation (A) ChIP experiments with HA antibodies were performed on GFP⁺ progenitors transduced 7 days previously with pMX-6ZFP or pMX (control) by site-specific PCR primers. (B) Sorted e14.5 Pax5^−/− fetal liver LSKs were cotransduced with pMX-ZF and MigY-EBF1 viruses and then added to OP9 stromal cells. pMX and MigY-EBF1 cotransduced cells served as control. After 24 hr, cells were washed and replated on OP9-DL4 stromal cells in fresh media supplemented with IL-7, FL, and SCF. On day 7, cultures were stained and analyzed for frequencies of Thy1.2⁺CD25⁺ cells among DAPI⁻CD45⁺ single- or double-transduced cells. (C) Sorted e14.5 Ebf1^−/− fetal liver LSKs were cotransduced with pMX and MigY-EBF1 or pMX-ZF and MigY-EBF1 viruses, added to OP9 stromal cells. After 7 days, viable YFP⁺GFP⁺ cells were sorted, fixed, and subjected to ChIP with either EBF1 or HA antibodies. Relative enrichment of EBF1 versus 6ZFP in transduced Ebf1^−/− cells at “site a” is shown, with ChIP results for “site b” included as controls for EBF1 and 6ZFP occupancy. Data are expressed as fold enrichment over nontransduced Ebf1^−/− cells. (D) Single- or double-transduced cells from pMX and MigY-EBF1 transduced samples as well as pMX-ZF and MigY-EBF1 transduced samples were sorted for RNA 24 hr after infection and analyzed for relative expression of Gata3 by qRT-PCR. Data are means ± SEMs of triplicate samples. Error bars indicate SEMs, ^*p < 0.001. Data are representative of two separate experiments. See also Figure S5 and Table S2.

Figure 6

Decreased GATA3 Expression Is Essential for Early B Cell Development (A) Sorted e14.5 WT fetal liver LSKs were transduced with only pMX-6ZFP or MigY-EBF1 or both viruses, plated on OP9s, and analyzed for frequencies of viable B220⁺CD19⁺ cells on day 7. Double-transduced cells were identified as viable (DAPI⁻) GFP⁺YFP⁺ cells. (B) Sorted CD45^B6+Flt3⁺ LSKs and CLPs from B6.CD45^SJL adults previously reconstituted with BM cells from C57BL/6 (CD45^B6) backcrossed Vav^Cre or Vav^CreGata3^fl/fl mice were transduced with pMX or pMX-6ZFP. Equal numbers of GFP⁺ cells were sorted into triplicate cytokine supplemented OP9 stromal cultures, and B cell differentiation was assessed 7 days later. Graphical data are means ± SEMs of triplicate samples. p < 0.001.