Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Abstract

Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.The a,-fetoprotein (AFP) gene, a member of the albumin gene family, is expressed by fetal or malignant hepatocytes and repressed in normal mature hepatocytes. This forms the basis of a long-exploited model system to study cell differentiation and its impairment in neoplasia (1). Molecular genetics have brought the AFP model to molecular levels of cell differentiation, toward finely discerning how genes respond to or escape from developmental and growth signals (2,40,41). As yet, little is known about how differential regulation is exerted on the tandemly organized AFPalbumin locus and whether the two genes operate under shared elements of control. However, at least one wellcharacterized transcription factor, the glucocorticoid receptor, is known to selectively shut off the AFP gene in the developing liver (4, 21). This hormonal effect reaches into the mechanisms of neoplastic resistance to differentiation, because the AFP gene in malignant cells is generally refractory to glucocorticoids (2).Analyses of rat AFP gene and chromatin structures have identified domains in the 5' region of the locus potentially involved in its liver-specific, developmental stage-dependent, and glucocorticoid-regulated expression. A promoter domain spanning '230 nucleotides comprises a chromatin DNase I-hypersensitive (DH) site (32, 42, 44) that is selectively suppressed by dexamethasone (44), glucocorticoid receptor recognition sequences, and octamer motifs similar to those present in other genes under developmental and growth control (8). A distal domain (-2 to -4 kilobases [kb] relative to the AFP trans...

The Fetoprotein Transcription Factor (FTF) Gene Is Essential to Embryogenesis and Cholesterol Homeostasis and Is Regulated by a DR4 Element

“…We started with stable transfection in the well differentiated rat hepatoma 7777.6 subline (4). This stringent system, in which transfected genes are integrated in the genome, has shown much higher sensitivity to mutations affecting basal AFP gene activity, compared with transient expression systems, in which reporter genes function as episomes (4,5). The activity of 4F-CAT stably transfected in 7777.6 cells was reduced by about 75% upon mutation of the FTF DR4 motif (Fig.…”

“…Cells were harvested 48 h after transfection, and CAT activity was measured by thin layer chromatography and phosphorimaging. Wild-type and mutant 4F-CAT constructs were also assayed as stable transfectants in Morris McA-RH7777 rat hepatoma cells (variant 7777.6) (4), by cotransfection with CMV-neo, 10-day selection in G418, and measurement of CAT activity on pools of 200 -300 clones per construct, as described before (4,5).…”

“…One albumin-related gene, the ␣ 1 -fetoprotein (AFP) 1 gene, is activated at the onset of liver differentiation and operates tightly coupled with liver growth (2,3). In 1988, our group circumscribed a proximal AFP promoter element essential to AFP gene activity in hepatocytes, and distinct from promoter components regulating the other albumin loci (4). The AFP-specific activator was then identified as orphan receptor fetoprotein transcription factor (5-7), so named for its first identified target locus (genome data base nomenclature, 2 NR5A2 in the nuclear receptor nomenclature, Ref.…”

Distant Enhancers Stimulate the Albumin Promoter through Complex Proximal Binding Sites

“…About 80% of enhancer stimulation was eliminated by removal of the PCE, but this stimulation also depended on HNF1, as in the albumin promoter. Inactivation of either AFP-promoter HNF1 site reduced activity by about half, whereas inactivation of both totally inactivated transcription (53)(54)(55). The requirement for HNF1 is virtually identical with the heterologous SV40 enhancer.…”

The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation.

“…Distal element II (DEII), at nucleotide -120, binds NF1/CTF, DEI at -90 binds C/EBP20 (1, 5, 27), a CCAAT box at -83 binds ACFINFY (33), and a proximal element (PE) at -60 binds the APF factor (4), also designated HNF-1, LF-B1, or HP1 in other contexts. Present only in hepatocytes and differentiated hepatoma cells, this last factor also participates in the transcriptional control of several other liver-specific genes, including al-antitrypsin (28), pyruvate kinase (S. Vaulont, personal communication), AFP (14,17), and transthyretin (8) genes.The strict tissue distribution of the APF/HNF1 factor, combined with the presence of its target sequence in a 4759 …”