Conserved cis-enhancer fragment regulates the expression of FOG-1 in zebrafish blood and heart. Cis-regulatory modules in the mouse FOG-1 locus on chromosome 8 are compared with other vertebrates (A). The high regulatory potential (hi-RP) and GATA-binding sites (GATA-1_BS) are represented as black boxes (hiRP&GATA-1_BS), the exons as blue boxes, the FOG-1 enhancer candidates (FE1-FE4) as red boxes, and the conservation score as “CS.” These 4 FE fragments were interrogated for in vivo GATA-1–binding activity using ChIP assay and real-time PCR (B). The relative occupancy levels of GATA-1 are indicated by the fold enrichment at each of the sites shown, normalized to levels at the negative control region (2 kb 5′ to the GATA-1 gene enhancer HS1). The bar graphs represent quantification (mean ± SD) for GATA-1 binding (3 independent experiments) using nuclear extracts from differentiated MEL cells. The mouse GATA-2 (GATA-2, −2.8 kb) and the c-kit (c-kit, + 5 kb) promoters were used as positive controls for GATA-1 occupancy.30 *The only significant GATA-1 occupancy in the FOG-1 locus at the FE2 site (P < .001). PI indicates the preimmune sera control. WISH shows endogenous expression of FOG-1 mRNA in the intraembryonic blood island (ICM, red arrow) and Rohon-Beard neurons (yellow arrow) (C). The FE1 from mouse (D,F-G) and zebrafish (H-I) robustly drives the expression of eGFP in the ICM (red arrow), but only FE2 from either zebrafish or mouse is expressed in Rohon-Beard paraspinal neurons (E,J-K and yellow arrows in E,J). The developmental stages are properly indicated.
