Afr1: Gene Cloning and its Role in Liver Gene Regulation

The mouse alpha-fetoprotein (AFP) and H19 genes are transcribed at high levels in the fetal liver and repressed at birth, leading to low but detectable levels of APP and H19 mRNA in the adult liver. This repression is regulated, in part, by a locus called Alpha-fetoprotein regulator 1 (Afr1). Afr1 was originally identified by an analysis of serum AFP levels in different mouse strains; this study showed that BALB/cJ mice had roughly 10-fold higher AFP levels that did other mouse strains. Thus, unlike a majority of mammalian factors controlling gene expression that have been identified using biochemical approaches, Afr1 was identified genetically. Using a transgenic mouse approach, we showed that the 250 bp AFP promoter is sufficient for Afr1 control. In contrast to our results, studies by Shirley Tilghman and colleagues indicated that Afr1 controls postnatal AFP and H19 mRNA levels at the posttranscriptionallevel. Based on these data, we have developed a model in which Afr1 acts as a negative regulator of AFP and H19 expression by a mechanism that couples transcription and posttranscriptional events. While a connection between these events has been established in the literature, mechanisms that link these processes are only beginning to be uncovered. Therefore, by studying Afr1 regulation of AFP and H19, we are likely to learn more about the transcriptionallposttranscriptional connections as well as how this may be developmentally regulated to control gene expression. To further explore our hypothesis of Afr1-mediated regulation, we propose the following aims: Aim 1: To use the transient tail-vein injection assay to localize the cis-acting element within the AFP and H19 promoters that are governed by Afr1. Aim 2: To identify the Afr1 gene by positional cloning by 1) developing a high resolution map of the Afr110cus, 2) generating a contig of the Afr1 region and identify candidate Afr1 genes by analysis of human and mouse genomes, and 3) cloning the Afr1 gene by BAC transgene complementation. Aim 3: To determine the mechanism by which Afr1 acts to regulate AFP and H19 in the liver by 1) analyzing posttranscriptional steps that may be affected by Afr1, 2) characterizing Afr1 at the gene, mRNA, and protein level, and 3) analyzing Afr1 function.