A liver-specific factor and nuclear factor I bind to the rat alpha-fetoprotein promoter

Proteins putatively involved in the transcriptional control of rat (alpha-fetoprotein (AFP) gene expression in the liver were identified by analyzing the in vitro binding of proteins from nuclear extracts of fetal and adult rat livers to the cloned rat AFP promoter region (-197 to +48) using a combination of gel shift and DNase I footprinting assays. Three stable and specific high affinity complexes (I, II, and III) were detected by gel shift analysis of fetal rat liver extracts. Complex II was specific to extracts from fetal liver while the two others (I and III) were also formed with extracts from adult liver. Complex I was highly liver-specific since it was not detected with extracts of kidney, spleen, and brain. DNase I and gel shift competition experiments using a synthetic oligonucleotide indicated that it is formed upon binding of a liver-specific factor to region -65 to -46 of the rat AFP promoter. This region, perfectly conserved in the rat, mouse, and man, had been previously shown to be crucial for liver-specific expression of the mouse AFP gene. The binding of this liver-specific factor to this DNA region may thus represent a key step in the specificity of expression of AFP gene in liver. Gel shift and DNase I footprinting competition experiments showed that complex III was formed by binding of the widely distributed nuclear factor I to the rat AFP promoter in the region -125 to -100. It is potentially significant that, in extracts from fetal liver, nuclear factor is also involved with another binding factor in the formation of the stage-specific complex II.