Abstract
A 12.5-kilobase pair (kb) segment upstream of the human albumin gene was analyzed for transcription enhancing activity using transient transfection analysis, gel mobility shift assays, DNase I footprinting, and site-specific mutagenesis. Two enhancer regions were identified, one 1.7 kb upstream of the transcription initiation site (E1.7) and the other 6 kb upstream (E6). In E1.7, a nuclear protein from HuH-7 hepatoma cells binds to an AT-rich sequence, GTTACTAATTGAC. Competition gel mobility shift assays suggested that this protein is HNF-1, which regulates the promoter of the albumin gene and several other liver-specific genes. A 60-base pair E1.7 fragment carrying the AT-rich sequence stimulates a heterologous (alpha-fetoprotein) promoter in a dose-dependent manner. In E6, a HuH-7 nuclear protein binds to a GT-rich sequence, TGTTTGGC.A 27-base pair E6 fragment carrying this sequence is able to stimulate the SV40 promoter in an orientation-independent manner. An alteration of this sequence by site-specific mutagenesis resulted in the loss of transcriptional activity as well as binding to the HuH-7 nuclear protein. Competition gel mobility shift assays showed that homologous elements exist in the albumin promoter. These results show that the promoter and enhancer of the human albumin gene are regulated by two common transcription factors through two shared cis-acting elements, one AT-rich and the other GT-rich.
Highlights
Competition gel mobility shift assays suggested that this protein is HNF-1, which regulates the promoter the albumin geneand several other liver-specific gion of the human albumin gene shows a 90% sequence identify and is likely to be regulated by the same
AT- contrast, the human albumin enhancer identified so far has rich sequence stimulates a heterologous (a-fetoproteinb)een mapped adjacent to the promoter (-486 to -221 bp) promoter in a dose-dependent mannerI.n Ea,a HUH-7 (Frain et al, 1990)
A 27-basepair Ee fragment carryingthis at odds with the striking similarityof their promoters. In this sequence is able to stimulate thSeV40 promoter in an study, we examined whether additional enhancers exist furorientation-independent mannerA. n alteration ofthis ther upstream of the human albumin gene
Summary
Yoshitake Hayashi$, Jeannie Chan, Hidekazu Nakabayashi, Tomoko HashimotoQ, and Taiki Tamaokill. E6was treated with the Klenow fragment of DNA polymerase I to form blunt ends, Delimitation of Enhancer Activity Present between -5.1 and -1.1 kb-To determine whether the 5’-half of this region and EglII linkers were attached and inserted into the EglII site of contains the enhancer activity, we compared CAT activities pSV1’-CAT in normal and reverse orientations. The sequence 5’TTGTTACTAATTGACAA-3’containedintheprotection region is similar to the HNF-1-binding site that is present in competitor DNAwas preincubated with nuclear extracts for 5 min the albumin promoter (Table I) Delimitation of Enhancer Activity Present between -10.8 whether additional enhancer activities are associated with and -5.1 kb-To localize theenhancer activityassociated further upstream regions, transfection assays using the CAT with the5.7-kb region from -10.8 to -5.1 kb, we digested this reporter gene were conducted in HUH-7cells.
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