Hepatocyte Nuclear Factor 3 Relieves Chromatin-mediated Repression of the α-Fetoprotein Gene

Abstract

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.

A cell-type-specific transcriptional network required for estrogen regulation of cyclin D1 and cell cycle progression in breast cancer

“…We noticed that FoxA1 was expressed in MCF7 (as well as in the other ER␣-positive breast cancer cell lines analyzed) and HepG2 but not in HeLa and MDA-MB-231 cells (Crowe et al 1999;Williamson et al 2005), paralleling the cellspecific functional activity of enh2. As such we tested the hypothesis that FoxA1 could be important in determining an active chromatin structure at enh2 by performing DNase I sensitivity assays with nuclei isolated from MCF7 cells transfected with si LUC or si FoxA1.…”

Transcriptional regulation of liver development

“…It has recently been demonstrated that HNF-3 is such a factor and that its interaction with nucleosomal DNA is more stable than with free DNA (Cirillo and Zaret 1999). Furthermore, in vitro transcription assays using chromatin-assembled templates have shown directly that HNF-3 can relieve chromatin-mediated repression of alpha-fetoprotein gene expression (Crowe et al, 1999). Once bound, HNF-3 has been found to direct nucleosome positioning within the context of the albumin enhancer (McPherson et al, 1993;Cirillo et al, 1998;Shim et al, 1998;Cirillo and Zaret 1999).…”

Sequence and Spacing of TATA Box Elements Are Critical for Accurate Initiation from the β-Phaseolin Promoter

“…In Vitro Transcription-DNA templates were in vitro transcribed upon addition of a RNA polymerase II-containing nuclear HeLa extract at a final protein concentration of 0.5 g/ l and an NTP/salts/energygenerating mix to give final concentrations of 0.7 mM CTP, UTP, GTP, ATP, 5 mM MgCl 2 , 66 mM KCl, 5 mM creatine phosphate, 10 units/ml of creatine kinase, 2.5 mM dithiothreitol, 9.0% glycerol and 18 mM HEPES (pH7.9) as described previously (28). Unless otherwise noted, all in vitro transcription reactions were performed with 500 ng of template DNA.…”