Tissue-specific expression of the human α₁-antitrypsin gene is controlled by multiple cis-regulatory elements

Abstract

Human alpha 1-antitrypsin (AAT) is expressed in the liver, and a 318 bp fragment immediately flanking the CAP site of the gene was found to be sufficient to drive the expression of a reporter gene (CAT) specifically in hepatoma cells. The enhancing activity however, was orientation-dependent. The DNA fragment was separated into a distal region and a proximal region. A "core enhancer" sequence GTGGTTTC is present within the distal region and is capable of activity enhancement in both orientations when complemented by the proximal region in the sense orientation. The results strongly suggest that there are multiple cis-acting elements in the human AAT gene that confer cell specificity for its expression. Nuclear proteins prepared from the hepatoma cells bound specifically to the proximal region in a band-shifting assay that was resistant to competition by the globin promoter DNA. Foot-printing analysis showed a protected domain within the proximal region that contains a nearly perfect palindromic sequence TGGTTAATATTCACCA, which may be important in the regulation of AAT expression in the liver.

Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene.

“…The cis-acting elements required for expression of the human AAT gene in the liver have previously been defined by gene transfer experiments (11,14,66), and nuclear factors that interact specifically with the cis-acting elements have been detected in hepatoma and liver cells (14,66). The crucial questions regarding the nature and mechanism of action of the nuclear factors that must operate through these sequences remain to be answered.…”

“…DNase I footprinting assays (21) were performed as described previously by Shen et al (66). A 20-,ul reaction mixture containing 18 mM HEPES (pH 7.9), 90 mM KCI, 4.5 mM MgCl2, 0.2 mM EDTA, 1.8 mM DDT, 18% glycerol, 1 ,ug of poly(dI-dC), and 1 to 3 ng of 32P-end-labeled HpaII-Hinfl (-150 to +46) fragment was incubated with rat liver or HeLa nuclear extract at room temperature for 10 min.…”

“…We performed DNase I footprinting (21) analysis on AAT upstream sequence with liver and HeLa nuclear extracts. The fragment from -150 to +46 of the AAT gene was chosen as the probe in this study because previous studies had suggested that this region contains cis-acting elements for liver-specific expression (14,66). Figure 4A illustrates an example of the results from one set of titration experiments to footprint the noncoding strand of AAT promoter DNA.…”

“…When introduced into the germ line of mice, the human AAT gene was also found to be expressed in a tissue-specific manner (37,59,67). By transfection experiments, we and others (11,14,66) reported that the 5'-flanking sequence of the human AAT gene was able to direct expression of a heterologous reporter gene specifically in hepatoma cells. We observed that this DNA segment was also capable of directing the expression of the reporter gene in the liver in transgenic mice (Shen et al., submitted for publication).…”

HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF.

“…In subsequent studies, HNF-1 was purified and found to be 88 kD , and the purified protein was shown to bind to functional sequences within the promoters of albumin at a site that had previously been termed the albumin B box (Lichtsteiner et al 1987) or the albumin proximal region (Cereghini et al 1987). In addition, purified HNF-1 was found to bind to the al-antitrypsin, a fetoprotein, transthyretin, and the pre-S1 promoter of the hepatitis B virus at sites that had been implicated in the tissue-specific expression of these genes Costa et al 1986Costa et al , 1989Godbout et al 1986;Shen et al 1987;Burk et al 1988;Hardon et al 1988;Li et al 1988;Monaci et al 1988). Furthermore, most of the tissue specificity of the albumin promoter could be attributed to a site for the C/EBP protein (Johnson et al 1987;Landschulz et al 1988) and to a region termed the albumin B box or the albumin proximal sequence (Gorski et al 1986;Cereghini et al 1988;Lichtsteiner and Schibler 1989).…”