FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors

Abstract

Members of the GATA family of zinc finger transcription factors have been shown to play important roles in the control of gene expression in a variety of cell types. GATA-1, -2, and -3 are expressed primarily in hematopoietic cell lineages and are required for proliferation and differentiation of multiple hematopoietic cell types, whereas GATA-4, -5, and -6 are expressed in the heart, where they activate cardiac muscle structural genes. Friend of GATA-1 (FOG) is a multitype zinc finger protein that interacts with GATA-1 and serves as a cofactor for GATA-1-mediated transcription. FOG is coexpressed with GATA-1 in developing erythroid and megakaryocyte cell lineages and cooperates with GATA-1 to control erythropoiesis. We describe a novel FOG-related factor, FOG-2, that is expressed predominantly in the developing and adult heart, brain, and testis. FOG-2 interacts with GATA factors, and interaction of GATA-4 and FOG-2 results in either synergistic activation or repression of GATA-dependent cardiac promoters, depending on the specific promoter and the cell type in which they are tested. The properties of FOG-2 suggest its involvement in the control of cardiac and neural gene expression by GATA transcription factors.

FIG. 1

Homology between FOG-2, FOG, and Ush. (A) Amino acid alignment of FOG-2, FOG, and Ush proteins. Positions of zinc fingers in FOG-2 are overlined. (B) Schematic representation of FOG and FOG-2.

FIG. 2

Expression of FOG-2 mRNA in adult mouse tissues. FOG-2 transcripts were detected in adult tissues by Northern blotting (top). GAPDH transcripts were measured as a control for RNA loading (bottom).

FIG. 3

Expression of FOG-2 mRNA during mouse development. Dark-field images of in situ hybridization of sagittal sections of E9.5 (A), E11.5 (B to D), E13.5 (E and F), and E15.5 (G and H) embryos illustrate FOG-2 expression. Panels C and D are higher magnifications of panel B. At E9.5 (A), signal predominates in the septum transversum. At E11.5, signal is evident in the undifferentiated wall of the foregut (D) and in the ventricle and atrium (C). At E13.5, distinct foci of expression are evident in the brain (F) and to a lesser degree in the heart (E). At E15.5, focal expression in the telencephalon, tectum tegmentum, and pons (G) is evident. Strong expression is evident in the mesentery of the midgut (H). Whole-mount in situ hybridization of an E10.5 embryo reveals FOG-2 transcripts in the heart (I). Aq, aqueduct; BG, basal ganglia; G, gut; H, heart; L, liver; Lu, lung; M, mesentery; S, stomach; ST, septum transversum; T, telencephalon; 4V, fourth ventricle. Size bars in panels A to H represent 200 μm.

FIG. 4

Interaction assays between GATA-4 and FOG-2. (A) Two-hybrid assays demonstrate interaction of FOG-2 with GATA factors. Activation of an integrated GAL4-dependent lacZ reporter was assayed in liquid culture as described in Materials and Methods. Yeast cells were transformed with pAS1-GATA-4 and/or pACT-FOG-2, as indicated. (B) Coimmunoprecipitation of GATA-4 and FOG-2. GATA-4 and FOG-2 were translated in vitro in the presence of [³⁵S]methionine, and reaction products were immunoprecipitated (Immunoppt.) with anti-GATA-4 antibody, as indicated.

FIG. 5

Localization of FOG-2 to the nucleus. Cos cells were transiently transfected with an expression vector encoding FOG-2 with a FLAG epitope tag at the amino terminus. Staining with mouse anti-FLAG antibody followed by fluorescein isothiocyanate-conjugated horse anti-mouse antibody showed that FOG-2 was localized exclusively to the nucleus.

FIG. 6

Effects of FOG-2 on cardiac muscle promoters. 10T1/2 cells, Cos cells, and neonatal rat cardiomyocytes were cotransfected with luciferase reporter constructs driven by sequences in the α-MHC, BNP, or ANF promoter (200 ng) in the absence or presence of the indicated expression vectors (300 ng). In all cases, transfection mixtures included a CMV-LacZ expression plasmid (200 ng) to normalize for transfection efficiency, and total input DNA was normalized by using empty pcDNA3.1 (Invitrogen). Reporter gene expression in panels A to E is depicted as the fold change in expression relative to the reporter gene without added GATA-4 or FOG-2. Panel F shows a titration of increasing amounts of FOG-2 expression plasmid, which results in dose-dependent inhibition of BNP-luciferase expression. Values in panel F are expressed as relative light units (RLU).