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.1999 Jul 20;96(15):8705-10.
doi: 10.1073/pnas.96.15.8705.

Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1

Affiliations

Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1

P Zhang et al. Proc Natl Acad Sci U S A..

Abstract

The process through which multipotential hematopoietic cells commit to distinct lineages involves the induction of specific transcription factors. PU.1 (also known as Spi-1) and GATA-1 are transcription factors essential for the development of myeloid and erythroid lineages, respectively. Overexpression of PU.1 and GATA-1 can block differentiation in lineages in which they normally are down-regulated, indicating that not only positive but negative regulation of these factors plays a role in normal hematopoietic lineage development. Here we demonstrate that a region of the PU.1 Ets domain (the winged helix-turn-helix wing) interacts with the conserved carboxyl-terminal zinc finger of GATA-1 and GATA-2 and that GATA proteins inhibit PU.1 transactivation of critical myeloid target genes. We demonstrate further that GATA inhibits binding of PU.1 to c-Jun, a critical coactivator of PU.1 transactivation of myeloid promoters. Finally, PU.1 protein can inhibit both GATA-1 and GATA-2 transactivation function. Our results suggest that interactions between PU.1 and GATA proteins play a critical role in the decision of stem cells to commit to erythroid vs. myeloid lineages.

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Figures

Figure 1
Figure 1
PU.1 specifically interacts with GATA-1 and GATA-2. (a) Schematic of PU.1 and GATA-2 protein structures is shown. Functional domains are designated according to amino acid residue. (b–d) GST fusion protein interaction studies.35S-methionine-labeled GATA-1 or GATA-2 was incubated with equal amounts of GST or GST-PU.1 fusion proteins. Truncated GST-PU.1 fusion proteins are marked according to amino acid residue. TAD, transactivation domain; PEST, PEST domain; wHTH, winged helix–turn–helix motif; N-f, N finger; C-f, C finger.
Figure 2
Figure 2
Coimmunoprecipitation of PU.1 and GATA proteins. (a) Coimmunoprecipitation of PU.1 and GATA-1 from whole-cell lysates of transfected CV-1 cells and endogenous PU.1 and GATA-1 from K562 cells with anti-PU.1 antibody (P) or with normal IgG (N). Western blot analysis was performed by using anti-GATA-1 antibody. (b) Coimmunoprecipitation of PU.1 and GATA-2 from whole-cell lysates of transfected CV-1 cells. Western blot analysis was performed by using anti-GATA-2 antibody. (c) Coimmunoprecipitation of PU.1 and GATA-2 with anti-GATA-2 antibody (G2) or with normal IgG in transfected CV-1 cells. Western blot analysis was performed with anti-PU.1 antibody. CV-M, mock-transfected CV-1 cells; CV-T, CV-1 cells transfected with PU.1 and GATA-1 (a) or PU.1 and GATA-2 (b andc).
Figure 3
Figure 3
GATA-1 and GATA-2 repress PU.1 transactivation. (a) CV-1 cells were transfected with 100 ng of the multimerized PU.1-binding-site reporter (4XPU-TK), 10 ng of PU.1 expression vector PU.PECE (31), and GATA-1 or GATA-2 expression vectors as indicated. Luciferase activity is shown as fold activation above the level of activity seen with the luciferase reporter in the absence of any added PU.1 expression vector (±SD;n = 4). Western blot analysis was performed by using anti-PU.1 antibody to detect the expression level of PU.1 protein in transfected CV-1 cells as shown, with antitubulin antibody as a loading control. (b) GATA-1 and GATA-2 inhibit PU.1 transactivation of the M-CSF receptor promoter. CV-1 cells were transfected with 100 ng of M-CSF receptor promoter-luciferase, 10 ng of PU.PECE, and GATA-1 or GATA-2 expression vectors as indicated. Luciferase activity is shown as fold activation (±SD;n = 4). GATA-1 or GATA-2 alone did not repress the basal activity of either the 4XPU-TK (a) or the M-CSF receptor promoter (b). Western blot analysis was performed by using anti-FLAG antibody to detect the expression levels of FLAG-tagged PU.1, GATA-1, and GATA-2 proteins in transfected CV-1 cells as shown, with antitubulin antibody as a loading control. (c) Deletion mutation of the C finger of GATA-1 loses the ability to inhibit PU.1 function. (Left) CV-1 cells were transfected with the M-CSF receptor promoter and PU.1, ID5, and ΔC-f expression vectors as indicated. Luciferase activity is shown as fold activation (±SD;n = 3). (Right) The reporter is the erythropoietin receptor promoter-driving human growth hormone (EpoR-GH). Four micrograms of EpoR-GH and 9 μg of GATA-1 and GATA-2 were used in these transfection experiments. ID5, N-terminal deletion (amino acids 1–63 were deleted) of GATA-1; ΔC-f, C finger deletion mutation of GATA-1.
Figure 4
Figure 4
GATA proteins inhibit PU.1 transactivation by displacing the PU.1 coactivator, c-Jun. (a) GATA proteins do not inhibit PU.1 binding to DNA. (Left) EMSA using a α-32P-ATP-labeled PU.1-binding-site probe (32) andin vitro transcribed and translated PU.1 and GATA-1 proteins. PU.1 protein (0.2 μl) was used for each reaction, along with increasing amounts of GATA-1 protein as indicated. (Right) SDS/PAGE gel ofin vitro transcribed and translated 2 μl of PU.1 and 2 μl of GATA used in EMSA. (b) GATA-1 and GATA-2 do not inhibit PU.1 activation domain function. CV-1 cells were transfected with 4 μg of multimerized GAL4 DNA-binding sites in front of a minimal TK promoter used as a reporter. One hundred nanograms of PU.1 activation domain/GAL4 DNA-binding domain fusion protein (PUAD/GAL4BD) in pcDNA3 was used as a transactivator, and GATA-1 or GATA-2 was added as indicated. Luciferase activities were corrected by an internal-control Renilla luciferase vector and shown as fold activation (±SD;n = 3). (c) GATA-1 inhibits c-Jun binding to PU.1. (Left)In vitro35S-methionine-labeled c-Jun or GATA-1 was incubated with GST-PU.1 Ets domain (GST-PU.1/ETS) or GST alone (GST). The amounts of GATA-1 and c-Jun added are indicated at the top. (Right) SDS/PAGE gel of c-Jun (1 μl) and GATA-1 (2 μl) used in the GST pull-down experiment. (d) GATA-1 inhibits c-Jun coactivation of PU.1 function. F9 cells were transfected by using Lipofectamine Plus with 0.3 μg of 4XPU-TK, 0.1 μg of PU.PECE, 0.05 μg of pSV-Sport1-c-Jun [a c-Jun expression vector (20)], and 1.3 μg of pcDNA3-GATA-1. Luciferase activates were determined 24 hr after transfection and shown as fold activation (±SD;n = 3). The same experiments were done by using the M-CSF receptor promoter-luciferase reporter as shown (Right) using GATA-1 or GATA-2.
Figure 5
Figure 5
PU.1 inhibits GATA transactivation function. CV-1 cells were transfected with 4 μg of pT81 (minimal TK promoter-driving luciferase gene expression) or multimerized GATA-binding-site reporter (3XGATA-TK), 0.5 μg of GATA-1 or GATA-2 expression vector, and 2.5 μg of PU.1 expression vector PU.pECE as indicated by the CaPO4 method. Luciferase activity is shown as fold activation above the level of activity seen with pT81 in the presence of the expression vector backbone (pECE for PU.1 and pcDNA3 for GATA-1 and GATA-2) (±SD;n = 4).
Figure 6
Figure 6
GATA-1 and GATA-2 inhibit PU.1 function at different stages during hematopoietic cell differentiation to specific lineages through protein—protein interactions. The model hypothesizes that in stem cells, GATA-2 blocks PU.1 and c-Jun interaction and, therefore, inhibits PU.1 activation of its downstream target genes. In erythroblasts, up-regulation of GATA-1 blocks coactivation of PU.1 by c-Jun. But in developing myeloid progenitors, with decreased expression of GATA-1 and GATA-2, PU.1 and c-Jun synergistically activate PU.1 target genes such as the M-CSF receptor (20). G-1, GATA-1; G-2, GATA-2.
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