Mature erythrocyte membrane homeostasis is compromised by loss of the GATA1-FOG1 interaction

Abstract

GATA1 plays essential roles in erythroid gene expression. The N-terminal finger of GATA1 (GATA1-Nf) is important for association with FOG1. Substitution mutations in GATA1-Nf, such as GATA1 V205M that diminish the GATA1-FOG1 association, have been identified in human thrombocytopenia and anemia cases. A mouse model of human thrombocytopenia has been established using a transgenic complementation rescue approach; GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of GATA1 V205G , but rescued adult mice suffered from severe thrombocytopenia. In this study, we examined GATA1-deficient mice rescued with GATA1 V205G at a comparable level to endogenous GATA1. Mice rescued with this level of GATA1 V205G rarely survive to adulthood. Rescued newborns suffered from severe anemia and jaundice accompanied with anisocytosis and spherocytosis. Expression of Slc4a1, Spna1, and Aqp1 genes (encoding the membrane proteins band-3, ␣-spectrin, and aquaporin-1, respectively) were strikingly diminished, whereas expression of other canonical GATA1-target genes, such as Alas2, were little affected. Lack of these membrane proteins provoked perturbation of membrane skeleton. Importantly, the red cells exhibited increased reactive oxygen species accumulation. These results thus demonstrate that the loss of the GATA1-FOG1 interaction causes a unique combination of membrane protein deficiency and disturbs the function of GATA1 in maintaining erythroid homeostasis. IntroductionGATA1 is a founding member of the GATA family of transcription factors. Because GATA1, GATA2, and GATA3 are highly expressed in hematopoietic lineages, these factors are collectively referred to as hematopoietic GATA factors. 1 The expression of hematopoietic GATA factor genes are strictly regulated through distinct molecular mechanisms, consequently each GATA molecule shows specificity in its expression profile. 1 In particular, GATA1 is expressed in lineage-committed cells, such as erythroid, megakaryocytic, eosinophilic, mast, and dendritic cells. 2,3 GATA1 plays key roles in regulating the differentiation and survival of these hematopoietic lineage cells. Targeted knockout of the Gata1 gene leads to in utero lethality of the mutant embryos because of defects in primitive and definitive hematopoiesis. 4,5 Conditional Gata1 gene knockout gives rise to red cell aplasia and severe thrombocytopenia. 6 In addition, GATA1 dysfunction is predictive of specific leukemias in mice and humans. 2 GATA1 contains 3 functional domains, that is an N-terminal transactivation (NT) domain, an N-terminal zinc finger (Nf) domain, and a C-terminal zinc finger (cf) domain. 7 These 3 domains have been shown to be important for GATA1 function. 7 In transgenic complementation rescue analyses, GATA1 lacking either the Nf or cf domain poorly rescues GATA1-deficient mice from embryonic lethality, which indicates the importance of both Nf and cf domains in GATA1 function in vivo. 7 The Nf domain interacts with FOG1 (friend of GATA1), and also stabilizes...

GATA1 Binding Kinetics on Conformation-Specific Binding Sites Elicit Differential Transcriptional Regulation

“…Several families with inherited anemia and thrombocytopenia have been reported to harbor GATA1 mutations lacking the potential to interact with FOG1 (43). In this regard, we have conducted transgenic complementation rescue analyses of GATA1-deficient mice utilizing the GATA1 V205G (G1 V205G ) mutant that shows reduced FOG1-interacting activity and found that the rescued mice are prone to suffer from severe spherocytic hemolysis and thrombocytopenia (29,32). In the rescued mice, expression of GATA1 target genes related to the red cell membrane structure is markedly reduced, while expression of the GATA1 target genes related to the erythroid differentiation is relatively preserved.…”

“…Sample preparations were performed as previously described with minor modifications (29). Briefly, 1 ϫ 10 7 liver cells of E12.5 mouse embryos and 1 ϫ 10 7 cells of MEL cell clones that express biotinylated GATA1 proteins were cross-linked with 1% and 0.4% formaldehyde for 10 min, respectively, and neutralized with glycine.…”

“…E12.5 fetal livers were fixed, sectioned, and stained with hematoxylin and eosin solutions as previously described (29). Peripheral blood smears were stained with WrightGiemsa solutions.…”

N- and C-terminal Transactivation Domains of GATA1 Protein Coordinate Hematopoietic Program

“…Chromatin Immunoprecipitation (ChIP) Assay-Chromatin fixation and purification procedures were performed as described previously (24) with the exception that mouse erythroleukemia (MEL) cells were used. MEL cells were cultured in a same manner as the QT6 cells.…”

Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes

“…Erythrocyte osmotic fragility analysis was carried out according to the method described by Hasegawa et al (2012). Briefly, erythrocytes were incubated in a series of NaCl concentrations ranging from 0.24% to 0.68%.…”