Molecular cloning of FOG-2: a modulator of transcription factor GATA-4 in cardiomyocytes

Abstract

GATA transcription factors are important regulators of both hematopoiesis (GATA-1/2/3) and cardiogenesis (GATA-4) in mammals. The transcriptional activities of the GATA proteins are modulated by their interactions with other transcription factors and with transcriptional coactivators and repressors. Recently, two related zinc finger proteins, U-shaped (USH) and Friend of GATA-1 (FOG) have been reported to interact with the GATA proteins Pannier and GATA-1, respectively, and to modulate their transcriptional activities in vitro and in vivo. In this report, we describe the molecular cloning and characterization of a third FOG-related protein, FOG-2. FOG-2 is an 1,151 amino acid nuclear protein that contains eight zinc finger motifs that are structurally related to those of both FOG and USH. FOG-2 is first expressed in the mouse embryonic heart and septum transversum at embryonic day 8.5 and is subsequently expressed in the developing neuroepithelium and urogenital ridge. In the adult, FOG-2 is expressed predominately in the heart, brain, and testis. FOG-2 associates physically with the N-terminal zinc finger of GATA-4 both in vitro and in vivo. This interaction appears to modulate specifically the transcriptional activity of GATA-4 because overexpression of FOG-2 in both NIH 3T3 cells and primary rat cardiomyocytes represses GATA-4-dependent transcription from multiple cardiac-restricted promoters. Taken together, these results implicate FOG-2 as a novel modulator of GATA-4 function during cardiac development and suggest a paradigm in which tissue-specific interactions between different FOG and GATA proteins regulate the differentiation of distinct mesodermal cell lineages.

Figure 1

Predicted amino acid sequence and subcellular localization of FOG-2. (A) Predicted amino acid sequence of FOG-2. Zinc finger motifs are underlined. (B) Schematic illustration of the structures of FOG-2, FOG, and USH. Zinc fingers are numbered. (C) Subcellular localization of FOG-2. COS-7 cells transfected with pcDNAFlag/FOG-2 were assayed by indirect immunofluorescence by using an anti-FLAG antibody (Right) or stained with 4′,6-diamidino-2-phenylindole to visualize nuclei (Left).

Figure 2

Northern analysis of FOG-2 expression. A Northern blot containing 2 μg of Poly(A)⁺ RNA from different adult mouse tissues was hybridized to a radiolabeled FOG-2 cDNA probe (Upper) or a β-actin cDNA probe (Lower). RNA size in kb is shown to the left of the autoradiogram. Sk muscle = skeletal muscle.

Figure 3

In situ hybridization analysis of FOG-2 expression in mouse embryos. Sense and antisense FOG-2 cRNA probes were hybridized to sections of E8.5 to E16.5 mouse embryos. The positive signals seen in the livers, atrial cavities, and dorsal aorti of the E11.5 embryos with both sense and antisense probes represent epifluorescence of erythrocytes. Heart (Ht), Septum Transversum (ST), Neural Epithelium (NE), Urogenital Ridge (Ur), and Gonad (Gn) are shown by arrows.

Figure 4

A physical association between FOG-2 and GATA-4. (A) FOG-2 interacts with GST-GATA-4 in vitro. ³⁵S-labeled in vitro translated FOG-2 was incubated with purified GST or GST-GATA-4 fusion proteins and the complexes were affinity purified by using glutathione-Sepharose beads and resolved by SDS/PAGE. Size markers in kDa are shown to the left of the autoradiogram. (B) FOG-2 interacts with GATA-4 in mammalian cells. COS-7 cells were transfected with an Express epitope-tagged FOG-2 expression construct (FOG-2), a GATA-4 expression construct (GATA4), or both (FOG-2 + GATA4). Nuclear extracts prepared from the transfected cells were immunoprecipitated with an α-GATA-4 antibody. Immune complexes were resolved by SDS/PAGE and analyzed by Western blot by using an α-Express antibody. Molecular masses in kDa are shown to the left of the autoradiogram. (C) FOG-2 interacts with GATA-4 in yeast. Yeast 2 hybrid assays using expression vectors encoding the N-terminal (pG4 Zn N), C-terminal (pG4 Zn C), or both (pG4 Zn N + C) zinc fingers of GATA-4 fused to the DNA binding domain of GAL4 and an expression vector encoding FOG-2 fused to the activation domain of GAL4. Note that all transformants grew under nonselective conditions (No selection). Positive interactions are indicated by yeast growth under selection conditions (Selection) and blue staining indicating β-galactosidase activity (β-galactosidase).

Figure 5

FOG-2 represses GATA-4-mediated transcription from the BNP promoter. (A) NIH 3T3 cells were transfected with a BNP promoter–hGH reporter construct along with GATA-4 and FOG-2 expression constructs as indicated. (B) Primary neonatal rat cardiomyocytes were cotransfected with the BNP promoter–hGH reporter construct along with increasing amounts of a FOG-2 expression plasmid. The data are presented as relative promoter activity normalized to the activity of the reporter plasmid alone in each cell type. Each point represents the mean ± SEM from at least three independent transfections.