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. 2008 Oct 1;112(7):2738-49.
doi: 10.1182/blood-2008-03-146605. Epub 2008 Jul 14.

Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation

Isla Hamlett  1 , Julia Draper, John Strouboulis, Francisco Iborra, Catherine Porcher, Paresh Vyas
Affiliations

Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation

Isla Hamlett et al. Blood. .

Abstract

The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL.

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Figures

Figure 1
Figure 1
Isolation of GATA1-containing complexes in L8057 megakaryocyte cells. (A) Scheme of in vivo protein biotinylation and purification by streptavidin beads. (B) Nuclear extracts from wild-type (UnT), BirA-expressing (BirA), biotag-GATA1–expressing (+ ve), and 6 independent L8057 clones expressing biotag GATA1 and BirA biotin ligase were tested by Western blot with anti-GATA1 antibody (top panel). The top band corresponds to the slower migrating biotag GATA1; bottom band, endogenous GATA1. The blot was probed with antistreptavidin-HRP antibody, which confirms biotinylation of GATA1 in extracts from clones 3, 4, 5, and 6. (C) Crude nuclear extracts from cells transfected with either BirA alone (BirA) or BirA/biotag GATA1 (BirA/biotag GATA1) were incubated with streptavidin-coated beads. Precipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PD lane) and stained with Coomassie blue. Approximately 20 μg crude extract was loaded as input (In lane). formula image indicates biotinylated GATA1 (biotag GATA1), as determined by mass spectrometry. (D) Table of proteins and the number of peptides precipitated by streptavidin beads and identified by mass spectrometry. (E) Gel filtration analysis (top). An example of fractionation of crude nuclear extracts from L8057 cells transfected with BirA/biotag GATA1 on a Superose 6 column. Similar results were obtained from wild-type nuclear extracts. formula image indicates position where protein molecular markers elute. The UV profile indicates that proteins elute in a broad fractionation profile. Fractions were taken from the Superose 6 column, precipitated, and analyzed by Western blotting for GATA1 and several potential protein partners (bottom panels). The antibody used is indicated on the lefthand side of the panel. Note that endogenous GATA1 and biotag-GATA1 have a similar elution profile. Vertical line(s) have been inserted to indicate a repositioned gel lane.
Figure 2
Figure 2
Validation of interaction between GATA1 and partner proteins in L8057 cells and primary megakaryocytes. (A) Nuclear extracts from BirA biotin ligase expressing (BirA) and BirA and biotag GATA1 expressing (BirA/biotag GATA1) cells were studied. In indicates input lane (20 μg crude nuclear extract); PD, pull-down lane (nuclear extracts precipitated with streptavidin beads); and Un, unbound supernatant lane (proteins not bound to streptavidin). The antibodies used in the Western blot analysis are indicated on the right of the panels. Vertical line(s) have been inserted to indicate a repositioned gel lane. Biotag GATA1 is pulled down from BirA/bioGATA1-transfected cells but is absent from BirA-only cells (top panel). The positions of endogenous (bottom band) and biotag GATA1 (top band) are indicated. (B) Nuclear extracts from untransfected L8057 cells were immunoprecipitated (IP) with αGATA1 antibody. In indicates 20 μg crude nuclear extract; IP, proteins immunoprecipitated by αGATA1 antibody; Un, unbound proteins left in the supernatant after immunoprecipitation; and IgG, control was immunoprecipitation performed with the corresponding normal IgG. Antibodies used in Western blot analysis are indicated on the right of the panels. FOG1, all members of the pentameric complex, several members of the NuRD complex, as well as ETO2, GFI1B, and ZFP143 all coimmunoprecipitate with GATA1. (C-E) Reverse coimmunoprecipitation experiments. L8057 cell nuclear extracts were immunoprecipitated with antibodies against ETO2 (C), GFI1B (D), or ZFP143 (E). Lanes are marked as in panel A. Antibodies used in Western blot analysis are marked on the right of the panels. (F) Mouse bone marrow cells were cultured for 3 days with thrombopoietin. Percentage of CD41 expressing primary megakaryocytes was assessed by fluorescence-activated cell sorter analysis (left). May-Grunwald-Giemsa staining (right) shows that the morphology of the cells used for immunoprecipitation experiments were a mixture of immature and mature megakaryocytes. (G) Nuclear extracts prepared from primary megakaryocytes shown in panel F were immunoprecipitated with αGATA1 antibody. Coimmunoprecipitated proteins were detected by Western blot analysis. Antibodies used in the Western blot analysis are indicated on the right of the panels. (H) Analysis of Eto2 mRNA expression during megakaryocyte maturation. Mouse bone marrow cells were cultured with thrombopoietin for 3 days. CD41 expressing cells were isolated on each day of culture. May-Grunwald-Giemsa staining shows the morphology of the cells isolated on each day (top panel). All panels were photographed at 40× magnification. These cells were used to extract RNA and make cDNA. Eto2 mRNA levels were quantitated relative to GAPDH levels by Taqman real-time RT-PCR, at each time point (bottom panel).
Figure 3
Figure 3
GATA1 colocalizes with partner proteins in L8057 cells and primary megakaryocytes. Degree of colocalization (shown on the y-axis) is revealed by antibody blocking in L8057 (A) and primary megakaryocyte nuclei (B). αFOG1, -GFI1B, -ETO2, and -ZFP143 antibodies, but not αSC35 antibody, block access to αGATA1 antibody (A,B). Conversely, αGATA1 antibody blocks access of αFOG1, -GFI1B, -ETO2, and -ZFP143 antibodies, to their respective epitopes (B).
Figure 4
Figure 4
Characterization of GATA1-containing protein complexes in L8057 cells. (A) Scheme of the sequential immunoprecipitation experiments. (B-D) L8057 cell crude nuclear extracts were first immuno-depleted with αSCL (B), αGFI1B (C), or αETO2 (D) antibodies (left panels). The lanes are named as in Figure 2. Coimmunoprecipitated proteins were analyzed by Western blot using antibodies indicated on the right of the panels. A second immunoprecipitation was performed on the depleted supernatant (right panels) using αGATA1 antibodies. Coimmunoprecipitated proteins were analyzed by Western blot using antibodies marked on the right of the panels. (E) Models of the possible composition of protein complexes from L8057 cells derived from experiments in this figure.
Figure 5
Figure 5
Knock down of ETO2 in L8057 cells. (A) Diagram of lentiviral construct used to express Eto2-directed shRNA (shETO2). cPPT indicates central polypurine tract; CTS, central termination sequence; and H1, H1 promoter. EF1α promoter and the eGFP reporter gene are shown. (B) Knock down of ETO2 protein expression (top panels) was assessed in L8057 cells infected with either control shRNA (Control, lefthand lane) or shRNA directed against ETO2 (shETO2, righthand lane) by Western blot analysis in 3 independent experiments. The blots were probed with anti-POL II antibody (bottom panels) to control for protein loading. Percentage knock down of normalized ETO2 protein expression is shown. (C,D) L8057 cells expressing a control shRNA or ETO2 shRNA were induced to undergo megakaryocytic differentiation with TPA. Cells were stained for acetylcholine esterase (AchE) activity after 4 days of induction (C). Images were acquired using an Olympus BX60 microscope with a QImaging camera (Surrey, BC) using an Olympus lens at 10×/0.5 and 40×/0.5 numeric aperture objectives. Openlab version 3 software (Improvision, Coventry, United Kingdom) was used for image acquisition, and images were exported into Adobe Photoshop version CS2 (Adobe Systems, San Jose, CA) for processing. Note the increased number of brown staining (AchE-positive) cells that are larger in size, indicative of greater megakaryocyte maturation, when cells are infected with the ETO2 shRNA. The percentage of AchE-positive cells counted in 3 independent experiments (D). Cells treated with DMSO served as a control for TPA induction. (E) Pf4 (left) and GPIIb (right) mRNA levels were quantitated relative to GAPDH levels by Taqman real-time RT-PCR in L8057 cells infected with virus expressing either control shRNA (▭) or shETO2 (formula image). Data shown in panels D and E are the average of 3 independent experiments, and error bars represent 2 SD.
Figure 6
Figure 6
ETO2 directly represses the Pf4 promoter in transactivation assays via GATA, GAT, and E-box sites. (A) Comparison of the nucleotide sequence of the 5′ upstream region of the Pf4 gene from 6 species. Sequences highlighted in bold or by a box indicate a GAT, E-box, ETS, and GATA motifs. *Conserved nucleotides. Coordinates in nucleotides are with respect to the transcriptional start site. (B) Schematic representation of the luciferase gene reporter construct (pGL4.10) under the control of the enhancer/promoter regions of the rat Pf4 gene. Coordinates in nucleotides are with respect to the Pf4 gene transcriptional start site. Sequence motifs for transcription factors are marked. ↱shows the position of the transcriptional start site. The luciferase gene is drawn as an open box. (C) 293T fibroblasts were transiently transfected with either a promoterless luciferase gene (pGL4.10) or a luciferase gene regulated by 1200 nucleotides 5′ of the transcriptional start site of the rat Pf4 gene (rPF4 1200). Cells were also transfected with expression vectors expressing the indicated transcription factors. The triangle represents transfection with increasing amounts of ETO2 expression vector (50, 150, and 300 ng). Expression of luciferase in cells transfected with rPF4 1200 with expression vectors for all 6 transcriptional regulators (C6: GATA1, SCL, E2A, LDB1, LMO2, and FLI1) was set to 100%. (D) Western blot analysis confirms expression of transfected proteins in 293T cells in the absence (+ C6) or presence (+ C6 + E) of the ETO2 expression plasmid. Expression of mSIN3A protein serves as a loading control. (E) Expression of luciferase protein in 293T cells transfected with either full-length rPF4 1200 or truncated forms of the rat Pf4 promoter (rPF4 151 and rPF4 97) with C6 factors in the presence or absence of 150 ng ETO2 expressing plasmid. (F) Transcriptional effect of point mutations of the GAT motif at −130 and GATA site at −30 and E-box motif at −102 (relative to the transcriptional start site) in the construct rPF4 151 on luciferase gene expression were tested in 293T cells in the presence of the transcription factors indicated at the bottom of the diagram. In panels C, E, and F, the results are the mean plus or minus 2 SD of 3 to 5 independent experiments.
Figure 7
Figure 7
Recruitment of the pentameric complex and ETO2 to the Pf4 promoter in L8057 cells. (A) Schematic representation of the endogenous mouse PF4 locus. Coordinates in nucleotides are with respect to the transcriptional start site. Sequence motifs for transcription factors are marked. The ↱shows the position of the transcriptional start site. The 3 end-to-end arrows above the locus (-ve, P2, and P1) represent the location of the genomic sequences amplified by Taqman real-time PCR in the chromatin immunoprecipitation assay (ChIP) in panel B. (B) ETO2 and the pentameric complex co-occupy the Pf4 locus in L8057 cells. ChIP was performed using chromatin isolated from L8057 cells and antibodies directed against (i) GATA1, (ii) ETO2, (iii) SCL, (iv) E2A, (v) LDB1, and (vi) FLI1. Immunoprecipitated material was analyzed by Taqman real-time RT-PCR. The y-axis represents the fold enrichment normalized with respect to IgG and a control locus, the Gapdh promoter, at specific sites in the Pf4 promoter region (PF4-ve, P2, and P1).
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