Abstract
The albumin-alpha-fetoprotein locus epitomizes the main features of transcriptional regulation of fetal and adult hepatocyte-specific genes: developmentally regulated promoters and strong distant enhancers. Full enhancer activity required only a proximal albumin-promoter region containing the TATA box, hepatic nuclear factor 1 (HNF1), and nuclear factor Y (NF-Y) sites. Deletion of the HNF1 site abrogated enhancer and promoter activity, whereas methylation of the site reduced all activity by about 3-fold. Deletion of the NF-Y site attenuated activity by about half, but much of the activity could be replaced by juxtaposition of an upstream region (designated distal element IV). Gel shift and competition analysis demonstrated that binding of architectural factors overlapped NF-Y binding. Moreover, a mutation that eliminated NF-Y binding but only minimally perturbed the surrounding region did not affect enhancer function. In plasmids with a second promoter, the enhancers simultaneously stimulated both albumin and alpha-fetoprotein promoters with minimal competition, but surprisingly some mutations in the albumin promoter attenuated expression from both promoters, whereas another uncoupled their expression. With single promoters, the function of the proximal promoter region was controlled by three parameters in the following hierarchy: HNF1 binding > local architecture > NF-Y binding, but integrated two-promoter function had a much greater dependence on NF-Y.
Highlights
Serum albumin is probably the most characteristic protein synthesized by the mature liver, accounting for more than 10% of total protein synthesis [1]
Albumin Gene Promoter Models—Previous studies have demonstrated that the adjacent ␣-fetoprotein (AFP) gene enhancers are strongly active when combined with the albumin gene promoter [10, 11, 22]
Comparison of additional plasmid constructs with the same enhancer-promoter distances demonstrated that enhancers combined with promoter segments extending to Ϫ394, Ϫ308, or Ϫ175 had essentially the same activity, whereas the combination with a Ϫ84 promoter segment was significantly attenuated. This is consistent with the results of Herbomel et al [29], who reported that the rat albumin promoter consisted of “six positive regulatory elements concentrated within 130 base pairs”
Summary
Serum albumin is probably the most characteristic protein synthesized by the mature liver, accounting for more than 10% of total protein synthesis [1]. Three strong enhancers lie between the two genes [10, 11] These intergenic enhancers are potentially active even when the AFP gene has been developmentally silenced, as demonstrated by studies that combined the enhancers with the albumin promoter. An additional enhancer that lies 10 kbp upstream of the albumin gene promoter [12,13,14] regulates the albumin gene This latter enhancer, in combination with the albumin promoter, shows liver specific expression in transgenic mice [13] and highly differentiated cell lines derived from SV40-transformed hepatocytes [15] but is not active in other cell lines that have strong albumin expression [16, 17]. Such behavior is not observed with plasmid models of the -globin locus, where promoters compete
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