Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages

Abstract

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Keywords: Endothelial cell; Etv2; Gata2; Hematopoietic cell.

Fig. 1

Gata2 and Etv2-EYFP are co-expressed during early embryogenesis. (A and B), headfold stage (E7.5) (A), and E8.5 (B) embryos were analyzed by epifluorescence (a) and immunohistochemistry (b–h). Whole mount expression pattern of EYFP protein at E7.5 is shown in panel a. A parasagittal section (b) of an E7.5 embryo and a transverse section of an E8.5 embryo at the heart level (e) were stained with antibodies to GFP (green) and Gata2 (red). Yellow indicates overlap of green and red channels. The boxed areas indicated in panels b and e are enlarged in panels c, d, f–h, in separate and overlayed channels. Arrowheads mark cells that co-express Gata2 and EYFP. Co-expression of Gata2 and EYFP is observed in blood islands (c) and lateral plate mesoderm (d) at E7.5, and dorsal aorta (f), cardinal veins (f), endocardium (g), and yolk sac endothelium (h) at E8.5. Abbreviations are as follows: cv, cardinal vein: da, doral aorta: ec, endocardium: ysv, yolk sac vessel. Bars: 50 μm.

Fig. 2

Gata2 and Etv2 functionally interact with each other. (A) Protein-protein interaction between overexpressed Etv2 and Gata2. HA-tagged Etv2 and Flag-tagged Gata2 are overexpressed in C2C12 cells as shown using Western blot analysis. Etv2 could be co-immunoprecipitated with the Gata2 complex. Gata2 is also detected in the immunoprecipitation complex of Etv2. (B) The ES/EB system, in which HA-tagged Etv2 is engineered to be expressed at a similar level as endogenous Etv2. The endogenous Gata2 can be immunoprecipitated with the HA-tagged Etv2. (C) Transcriptional assays in C2C12 cells demonstrate that Etv2 transactivates the Lmo2 upstream 25K enhancer reporter (Lmo2+25K-Luc) modestly (3-fold), and Gata2 transactivates the reporter approximately 8-fold. Etv2 and Gata2 synergistcally transactivate the reporter up to 18-fold. (D) Tie2 promoter reporter (Tie2 promoter-Luc) can be transactivated by both Etv2 (34-fold) and Gata2 (4-fold). Co-expression of Etv2 and Gata2 results in a synergistic 64-fold transactivation.

Fig. 3

Definition of the protein-protein interaction domain between Etv2 and Gata2. (A) Schematic illustration of Etv2 deletional constructs and their interaction with Gata2. The Ets domain of Etv2, but not the N-terminal domain could bind to Gata2. (B) Coomassie brilliant blue staining of the purified GST-Etv2 deletion fusion proteins. (C) Gata2 protein is synthesized in vitro in the presence of ³⁵S-Methionine. Gata2 could be pulled down by the GST fusion protein harboring the Ets domain, such as the full length and C-terminal proteins, but not the N-terminal protein. (D) Schematic outline of Gata2 deletional constructs and their interaction with Etv2. All of the deletions retaining the GATA domain could interact with Etv2, but not those that lack the GATA domain, such as the N-terminal or C-terminal region. (E) GATA2 deletional proteins are synthesized in vitro and labeled with ³⁵S-Methionine, shown as input (upper lane). These Gata2 deletional proteins were utilized for the pulldown assay with purified GST-Etv2 fusion protein (lower lane).

Fig. 4

Overexpression of Etv2 and Gata2 during ES/EB differentiation. (A) Schematic illustration of the Etv2-Gata2 construct. Etv2 and Gata2 are linked through the 2A peptide sequence. The fusion construct was translated into two proteins, Etv2 and Gata2, through the ribosome skipping mechanism. (B–D) Co-expression of Etv2 and Gata2 in EBs results in enhanced hematopoietic and endothelial lineage differentiation. The EBs were treated with doxycycline (+ Dox) or left untreated (−Dox) from EB day 3 to day 4 and harvested for FACS analysis on Day 6. The hematopoietic lineage is identified as the c-Kit⁺/CD41⁺ cell population (B), and the endothelial lineage is represented as the Flk1⁺/CD31⁺ (C) and the Flk1⁺/Tie2⁺ cell population (D). (E–G) Quantitative summary of the lineage analysis in B–D, respectively.

Fig. 5

Spi1 is a common target gene of Etv2 and Gata2. (A) qRT-PCR analysis of gene expression upon induction of Etv2, Gata2 or Etv2-Gata2 in ES cells. (B) ChIP assay of the Spi1 promoter using anti-HA and anit-Gata2 sera. Primers detecting the Gapdh promoter was used as a control of nonspecific binding. (C–D) EMSA assay of the Spi1 promoter with Etv2 (C) or Gata2 (D), respectively. The specific binding and supershift signals are marked at the left. wt, wildtype competitor, mut, mutated competitor. The ctrl (control) antibody is a rabbit IgG. The ctrl (control) in the protein lysate is the translation lysate with the empty vector plasmid. (E) Schematic illustration of Spi1 promoter constructs. The 0.2 kb proximal promoter of Spi1 harbors 4 Ets motifs and 2 Gata motifs (Ets-m, all of the four Ets motifs mutated; Gata-m, both Gata motifs mutated). (F) Transcriptional assays of the Spi1 promoter-reporter by Etv2 and Gata2. Etv2 can transactivate the Spi1 promoter reporter up to 5-fold. This transactivation is attenuated to a basal level upon mutation of the Ets motifs (Ets-m). Gata2 transactives the Spi1 promoter reporter to approximately 5-fold. Gata2 transactivation is completely abolished when both Gata motifs are mutated (Gata-m). Co-expression of Etv2 and Gata2 synergistically transactivates the reporter up to 20-fold. Mutation of the Ets motifs or the Gata motifs resulted in approximately 6-fold reduction of the synergistic transactivation.