Interaction between FOG-1 and the corepressor C-terminal binding protein is dispensable for normal erythropoiesis in vivo

Abstract

The hematopoietic, zinc-finger protein FOG-1 is essential for the development of the erythroid and megakaryocytic lineages. FOG-1's function in hematopoiesis is dependent on its ability to interact with the transcription factor GATA-1. FOG-1 has also been observed to interact with the corepressor molecule C-terminal binding protein (CtBP) through a peptide motif shared by all FOG family members. In this study, we confirmed that FOG-1 and CtBP interact by coimmunoprecipitation. We further demonstrate that a FOG-1 mutant unable to interact with CtBP has increased erythropoietic (but not megakaryocytic) rescue (relative to the wild type) of a FOG-1(-/-) cell line. To analyze further the physiological role of the FOG-1-CtBP interaction, we generated knock-in mice that express a FOG-1 variant unable to bind CtBP. Surprisingly, these mice are normal and fertile. Furthermore, erythropoiesis at all stages of development is normal in these mice. Erythrocyte production is similar in mutant and wild-type mice even under conditions of erythropoietic stress stimulated by either exogenously added erythropoietin or phenylhydrazine-induced anemia. Thus, despite conservation of the FOG-CtBP interaction site, the in vivo function of FOG-1 in erythroid development is not affected by its inability to interact with the corepressor CtBP.

FIG. 1.

Interaction of FOG-1 with CtBP family members in mammalian cells. (A) Schematic depiction of conserved PIDLS motif from adenovirus E1A in FOG family members and the mutation in FOG-1 that disrupts CtBP binding. Boxes represent zinc fingers; shaded boxes represent GATA-binding zinc fingers. XFOG, Xenopus FOG; USH, Drosophila U-shaped; Ad2 243R, adenovirus E1A. (B) Coimmunoprecipitation of HA-FOG-1 and HA-mutCtBP-FOG-1 (FOG-1 ΔCtBP) with FLAG-tagged human CtBP2 or murine CtB2 in transfected COS cells. Cells were transfected with plasmids expressing the indicated proteins, and nuclear lysates were immunoprecipitated (IP) with anti-FLAG antibody. Western blot (WB) analysis was performed with anti-HA antibody or anti-FLAG antibody.

FIG. 2.

Mutation of the FOG-1-CtBP interaction motif enhances FOG-1-mediated rescue of erythropoiesis but not megakaryopoiesis of a FOG-1^−/− hematopoietic cell line. (A) Benzidine stains of FOG-1^−/− cells rescued with each of the constructs. Magnification, ×400. Positive (hemoglobinized) cells appear black or brown. (B) Percent benzidine-positive cells obtained from at least 10 independent rescue experiments, given as means ± standard errors of the means. (C) Western blot analysis of rescued cells for expression of each construct. Nuclear extracts were prepared from a portion of the rescued cells 1 day after sorting for GFP expression. Equivalent amounts of total protein were separated by SDS-PAGE and immunoblotted with an anti-HA antibody. (D) Semiquantitative RT-PCR analysis of cells rescued with each construct. RT-PCR using vWF- and HPRT-specific primers was performed with [³²P]dCTP for radioisotope incorporation, and the products were separated by PAGE. (E) Bands from panel D were quantified with a PhosphorImager (Molecular Dynamics, Inc.). Data are shown as the vWF signal normalized to the HPRT control signal.

FIG. 3.

Targeting the FOG-1-CtBP interaction in mice. (A) Partial restriction map of the murine Fog-1 wild-type locus, the Fog-1 knock-in targeting vector, and targeted homologous recombination before and after Cre-mediated excision of the selection cassette. The targeting construct contains the HSV-TK and neomycin resistance (NEO) cassettes under the control of the mouse phosphoglycerate kinase promoter. Homologous recombination results in replacement of wild-type Fog-1 with genomic DNA harboring a mutation of aspartic acid 814 to alanine and leucine 815 to serine (PIDLS to PIASS), as well as the incorporation of the neomycin resistance cassette. Fog-1 coding exons are shown as empty boxes, with positions of zinc fingers (zf) underneath. Solid triangles are LoxP sites. The probe used in Southern blotting is shown as a black line. K, KpnI; Xb, XbaI; N, NotI; Hd, HindIII; S, SpeI. (B) Southern blot analysis of ES cell DNA or yolk sac DNA showing the presence of wild-type (+/+), heterozygous (KI/+), and homozygous mutant (KI/KI) genotypes. (C) Sequencing chromatograms obtained with PCR fragments generated from genomic DNA from a homozygous mutCtBP-FOG-1 mouse and from a wild-type littermate.

FIG. 4.

Normal primitive and definitive erythropoiesis in mice lacking FOG-1-CtBP interaction. (Top) May-Grunwald-Giemsa staining of blood cells from wild-type and mutant yolk sacs at embryonic day 10.5 (E10.5). Magnification, ×1,000. (Bottom) May-Grunwald-Giemsa staining of blood cells from wild-type and mutant adult mice. Magnification, ×400.

FIG. 5.

Percent change in hematocrit in wild-type and mutant mice due to EPO administration. Hematocrits measured before and after EPO treatment were subtracted, divided by the initial value, and multiplied by 100. Data are means ± standard errors of the means.

FIG. 6.

Hematocrits of wild-type and mutant mice during recovery from anemia induced by PHZ (two consecutive injections, indicated by arrows). Results are means ± standard errors of the means.