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 and c).

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.

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.

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).

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.

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) and in 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 of in 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 vitro ³⁵S-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.