The ␣-fetoprotein gene (AFP) is tightly regulated at the tissue-specific level, with expression confined to endoderm-derived cells. We have reconstituted AFP transcription on chromatin-assembled DNA templates in vitro. Our studies show that chromatin assembly is essential for hepatic-specific expression of the AFP gene. While nucleosome-free AFP DNA is robustly transcribed in vitro by both cervical (HeLa) and hepatocellular (HepG2) carcinoma extracts, the general transcription factors and transactivators present in HeLa extract cannot relieve chromatin-mediated repression of AFP. In contrast, preincubation with either HepG2 extract or HeLa extract supplemented with recombinant hepatocyte nuclear factor 3 ␣ (HNF3␣), a hepatic-enriched factor expressed very early during liver development, is sufficient to confer transcriptional activation on a chromatin-repressed AFP template. Transient transfection studies illustrate that HNF3␣ can activate AFP expression in a non-liver cellular environment, confirming a pivotal role for HNF3␣ in establishing hepatic-specific gene expression. Restriction enzyme accessibility assays reveal that HNF3␣ promotes the assembly of an open chromatin structure at the AFP promoter. Combined, these functional and structural data suggest that chromatin assembly establishes a barrier to block inappropriate expression of AFP in non-hepatic tissues and that tissue-specific factors, such as HNF3␣, are required to alleviate the chromatin-mediated repression.
The mouse alpha-fetoprotein gene is expressed at high levels in the fetal liver and is transcriptionally silenced at birth. The repression is governed, at least in part, by the 250 base pair (bp) AFP promoter. We show here that the AFP promoter is dramatically repressed by HNF3 in HepG2 hepatoma cells. This repression is governed by the region between −205 and −150. Furthermore, this fragment can confer HNF3-mediated repression on a heterologous promoter. The repression is abolished by a mutation that is centered at −165. EMSA analyses using in vivo and in vitro synthesized proteins indicate that HNF3 proteins do not bind DNA from the −205 to −150 region. We propose that HNF3 represses AFP promoter activity through indirect mechanisms that modulate the binding or activity of a liver-enriched factor that interacts with the −165 region of the AFP promoter.
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