Jh. Millonig et al., MOLECULAR ANALYSIS OF THE DISTAL ENHANCER OF THE MOUSE ALPHA-FETOPROTEIN GENE, Molecular and cellular biology, 15(7), 1995, pp. 3848-3856
The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels i
n the visceral endoderm of the yolk sac and fetal liver and at much lo
wer rates in the endoderm of the fetal gut. Expression of the gene in
vivo requires the presence of at least one of three enhancers which li
e in its 5' flanking region. In this report, we establish that the mos
t distal AFP enhancer directed consistent expression of a linked AFP m
inigene in all three endodermal tissues in transgenic mice. The enhanc
er is composed of three domains, each of which is essential for full e
nhancer function by transient transfection assays. DNase I footprintin
g identified three regions of the enhancer which are protected by huma
n hepatoma nuclear extracts, one of which corresponded to a consensus
site for HNF-3 binding. Site-directed mutations in this site caused a
10-fold reduction in enhancer function by transient transfection. In t
ransgenic mice, however, the mutation resulted in sporadic expression
of the transgene, dependent on the site of integration. A similar acqu
isition of position-dependent sporadic expression of the transgene was
observed with a mutation in a second protein binding site, despite th
e fact that this mutation had very little effect on enhancer function
as assessed by transient transfection. These studies underscore the va
lue of examining the functions of specific protein binding sites in vi
vo.