Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells

Abstract

Malignant transformation usually inhibits terminal cell differentiation but the precise mechanisms involved are not understood. PU.1 is a hematopoietic-specific Ets family transcription factor that is required for development of some lymphoid and myeloid lineages. PU.1 can also act as an oncoprotein as activation of its expression in erythroid precursors by proviral insertion or transgenesis causes erythroleukemias in mice. Restoration of terminal differentiation in the mouse erythroleukemia (MEL) cells requires a decline in the level of PU.1, indicating that PU.1 can block erythroid differentiation. Here we investigate the mechanism by which PU.1 interferes with erythroid differentiation. We find that PU.1 interacts directly with GATA-1, a zinc finger transcription factor required for erythroid differentiation. Interaction between PU.1 and GATA-1 requires intact DNA-binding domains in both proteins. PU.1 represses GATA-1-mediated transcriptional activation. Both the DNA binding and transactivation domains of PU.1 are required for repression and both domains are also needed to block terminal differentiation in MEL cells. We also show that ectopic expression of PU.1 in Xenopus embryos is sufficient to block erythropoiesis during normal development. Furthermore, introduction of exogenous GATA-1 in both MEL cells and Xenopus embryos and explants relieves the block to erythroid differentiation imposed by PU.1. Our results indicate that the stoichiometry of directly interacting but opposing transcription factors may be a crucial determinant governing processes of normal differentiation and malignant transformation.

Figure 1

PU.1 and GATA-1 interact in vitro. (A) ³⁵S-Labeled proteins, indicated above each panel, were prepared and tested for binding to GST or GST–PU.1 by a ‘pull-down’ assay using proteins immobilized on glutathione–Sepharose beads as described in Materials and Methods. Bound proteins were released and analyzed by SDS–PAGE and autoradiography. An autoradiogram of 10% of each ³⁵S-labeled protein added to the binding reactions is shown in each panel (10% input). Arrowheads indicate the position of the ³⁵S-labeled proteins on the gel. (B) PU.1 or GATA-1, as indicated above each panel, were prepared from the respective GST fusion proteins by thrombin cleavage as described in Materials and methods. The purified proteins were tested for binding to GST, GST–GATA-1 or GST–PU.1 by a pull-down assay. Bound proteins were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. An immunoblot of 25% of the purified PU.1 or GATA-1 added to the binding reactions is shown in each panel (25% Input). (C) As in A except that where indicated 100 μg/ml of ethidium bromide (EtBr) was added to the reaction.

Figure 2

PU.1 and GATA-1 interact in vivo. (A) Nuclear extracts from 293 cells transfected with expression vectors encoding either PU.1 or both PU.1 and GATA-1 were prepared and immunopreciptated with an anti-GATA-1 antibody as described in Materials and Methods. Immunoprecipitates were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. An immunoblot of 15% of the amount of extract that was immunoprecipitated is shown (Input). Arrowheads indicate the positions of the immunodetected proteins. The asterisked (*) arrowhead indicates the position of Ig chains, which are used in immunoprecipitation. (B) Nuclear extracts of MEL cells were prepared and immunoprecipitated with either anti-GATA-1 (α-GATA-1) and anti-p16 (α-control) antibodies or anti-PU.1 (α-PU.1) and anti-cyclin E (α-control) antibodies as described in Materials and Methods. Input lanes are shown as in A, except that 3% of the extract was analyzed for the immunoprecipitation of PU.1 and Western of PU.1 lane. Analysis of immunoprecipitates and other details were as in A. (C, D) Analysis of the effect of mutations in GATA-1 (C) and PU.1 (D) on the GATA-1–PU.1 interaction in vivo. Whole cell extracts from 293 cells transfected with expression vectors encoding the indicated GATA-1 and PU.1 proteins (see Fig. 3B,D) were immunoprecipitated and analyzed as in A. Input lanes are shown as in A.

Figure 3

The PU.1–GATA-1 interaction is mediated by the zinc finger region of GATA-1 and the Ets region of PU.1. Binding of ³⁵S-labeled PU.1 to wild-type and mutant GST–GATA-1 fusion proteins (A, left), or ³⁵S-labeled wild-type and mutant GATA-1 proteins to GST–PU.1 (A, middle and far right), or ³⁵S-labeled wild-type and mutant PU.1 proteins to GST–GATA-1 (C) was tested as described in Fig.1. GST–GUK is a GST fusion protein of the human LIN2A guanylate kinase. (B,D) Schematic diagram of the mutant proteins used for the binding studies in A and C, respectively. The positions of amino acid residues at the termini of regions present in the mutant proteins are shown above the diagrams. (TAD) transactivation domain; (PEST) region rich in PEST residues; (DBD) DNA-binding domain. Binding of ³⁵S-labeled GATA-1, GATA-2, and GATA-3 to GST or GST–PU.1 (E) or ³⁵S-labeled PU.1, Fli-1, and Spi-B to GST or GST–GATA-1 (F) was tested as described in Fig. 1.

Figure 4

PU.1 inhibits GATA-1-dependent transcriptional activity with synthetic and natural promoters. M1α–GH reporter (15 ng) with and without either 150 ng (A) or 60 ng (B) of pXM–GATA-1 were cotransfected with pEBB or the indicated amounts (nanograms) of pEBB expression constructs encoding PU.1, reverse transcripts of PU.1 (AS–PU.1), or the indicated PU.1 mutants (see Fig. 3D) into NIH 3T3-cells as described in Materials and Methods. (C) αD3-LUC reporter (15 ng) with and without 30 ng of pXM–GATA-1 were cotransfected with pEBB or the indicated amounts of plasmids as in B into HeLa cells. (D) p45 NF-E2-GH reporter (10 ng) with and without 100 ng of pXM–GATA-1 and 100 ng pMT2–FOG were cotransfected with pEBB or the indicated amounts pEBB–PU.1 into NIH-3T3 cells as in A. (E) UAS–LUC reporter (100 ng) were cotransfected into NIH-3T3 with and without 100 ng of SP6–GAL4–Sp1 and 180 ng of pEBB–PU.1. The levels of growth hormone (A,B,D) or luciferase activity (C,E) were determined 48 hr after transfection as described in Materials and Methods. (Insets in A,B,C) The immunoblots with anti-GATA-1 antibody (A,C) and anti-HA antibody (B) of cell extracts corresponding to the indicated transfected samples. Arrowheads indicate the positions of GATA-1 and HA-tagged full-length and mutant PU.1 proteins; the asterisked (*) arrowhead indicates a nonspecific band detected with anti-HA antibody in 293 extracts. Numbers represent the average of at least two independent transfections. Error bars represent the s.e.m.. Each type of experiment was performed between three and seven times with two or three different DNA preparations and produced similar results.

Figure 5

Detection of wild-type and mutant epitope-tagged PU.1 proteins in MEL cell transfectants. MEL cells were transfected with expression constructs encoding both resistance to puromycin and an influenza hemagglutinin (HA)-epitope tagged wild-type or mutant PU.1 proteins as described in Materials and Methods. Puromycin resistant clones were isolated and screened for expression of PU.1 proteins by immunoblotting of cell extracts with anti-HA antibody. The levels of PU.1 proteins (arrowhead) in two representative clones of each type of PU.1 transfectant are shown. PU.1-ΔDBD–HA is deleted for residues 201–272 and PU.1–ΔTAD–HA is deleted for residues 33–100 (see Fig. 3D).

Figure 6

Restoration of hemoglobinization and terminal growth arrest in PU.1-transfected MEL cells by a GATA-1/ER fusion protein. MEL cells (broken line) and PU.1-transfected MEL cells, clone 605 (A) and two representative GATA-1/ER supertransfectants of clone 605, clones 12 (B) and 13 (C) (solid lines), were grown without (♦) or with 5 mm HMBA (▴), 5 mm HMBA with 10⁻⁷m β-estradiol (●) or 10⁻⁷ m β-estradiol (█). At the indicated times, the percentage of hemoglobinized cells was determined by benzidine staining as described in Material and Methods. Similar results were obtained with four other GATA-1/ER supertransfectants. (D) Clone 605 and clone 12 were treated for 72 hr with 10⁻⁷ m β-estradiol, 5 mm HMBA, or both as indicated above the panel. β-Globin mRNA levels were determined by Northern blot hybridization of total cellular RNA with ³²P-labeled β major globin DNA. The lower panel shows the ethidium bromide-stained gel before transfer. MEL cells (broken line) and cells of clones 605 (E) and clone 13 (F) (solid lines) were cultured as in A for the indicated times and plated in plasma clots with β-estradiol as described in Materials and Methods. After 7 days clots were removed, fixed, and stained with hematoxylin and the percentage of colonies with <50 cells was determined by microscopy. At least 200 colonies were counted for each determination.

Figure 7

PU.1 inhibits and GATA-1 restores erythroid differentiation in Xenopus embryos and explants. (A–D) Shown are representative 2.5-day Xenopus embryos following benzidine staining to visualize primitive erythropoiesis in the ventral blood island region (indicated by arrows in A and C). The embryos were injected with 125 pg reverse-strand control RNA (A) or mRNA encoding PU.1 (B) in both blastomeres at the two-cell stage. Embryos shown in C (1 ng) and D (125 pg) had been injected similarly, except that the mRNA was targeted into the two ventral blastomeres at the four-cell stage. (E,F) Globin expression in VMZ explants was analyzed by Western blotting using a monoclonal antibody to embryonic α-globin. (E) Explants were cultured for the times (in hr) indicated above the lanes. Lysates were derived from embryos injected into both blastomeres at the two-cell stage with 25 pg of either a control (C) CMV expression plasmid, or the same plasmid containing the PU.1 cDNA (PU). The lysate in lane WE was from an uninjected 80-hr whole embryo. Each sample contains proteins from six VMZ explants, derived from two independent experiments. (F) For each sample lysates were prepared from 10 VMZ explants isolated from embryos previously injected at the four-cell stage with RNA into both ventral blastomeres. Embryos were injected with various combinations of PU.1, GATA-1, reverse-strand PU.1 (PU.1rs), or a frameshift mutant form of GATA-1 that encodes a nonfunctional protein (G1fs). To control for any nonspecific effects of RNA injection, a total of 350 pg mRNA was injected into each blastomere, supplemented with PU.1rs or G1fs as required. (Lane 1) 250 pg PU.1 + 100 pg G1fs; (lane 2) 250 pg PU.1 + 100 pg GATA-1; (lane 3): 250 pg GATA-1 + 100 pg G1fs; (lane 4): 250 pg PU.1rs + 100 pg G1fs; (lane 5) the same as lane 1 from an independent experiment. (Lower) Coomassie Blue staining of the gel demonstrates equal loading of total protein for each lane.