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Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.
Weber H
,
Strandmann EP
,
Holewa B
,
Bartkowski S
,
Zapp D
,
Zoidl C
,
Ryffel GU
.
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We review the data available on the structure, developmental appearance and embryonic regulation of the tissue-specific transcription factor HNF1 alpha (LFB1) in Xenopus. The expression of the HNF1 alpha gene starts early in embryogenesis shortly after mid-blastula transition and the protein accumulates in the region of the embryo where liver, pronephros and gut--tissues that contain HNF1 alpha in the adult--are developing. The cofactor DCoH, known to stabilize dimer formation of HNF1 alpha, is present as a maternal factor in the egg and has a partially distinct tissue distribution compared to HNF1 alpha. This implies that DCoH does not only modulate HNF1 alpha dimerization but may also cooperate with other transcription factors. By injecting HNF1 alpha promoter CAT constructs into fertilized Xenopus eggs we obtained activation of the injected gene restricted to the region of the developing larvae expressing endogenous HNF1 alpha. Deletion analysis allowed to define the OZ-element that is essential for embryonic activation. This element also occurs in other promoters activated at mid-blastula transition in the embryo and interacts with the maternal factor OZ-1. As the HNF1 alpha promoter also contains functional binding sites for HNF4 and HNF1, we postulate that all of these transcription factors contribute to the cascade leading to proper embryonic activation of the HNF1 alpha gene.
Fig. 1, Structure of the HNF1o;
gene with its extended homeodomain.
The protein coding region
of HNFla is drawn with the various
protein domains. The position of the
iotrons is given by arrows and the
exons are numbered (Zapp et al.,
1993bJ. The amino acid identity
between the Xenopus XHNF1aa protein
and the rat protein in specific
domains is given. Sfightly different
values are obtained using XHNF1ab
in the comparison (Bartkowski et al..
1993). The composition of the homeodomain
(homeo) of HNF1a is compared
to the classical structure in the
Antennapedia (Antp) protein as
determined by nuclear magnetiC resonance (Leiting et al.. 1993) and by high resofution X-ray (Ceska et al.. 1993). The helixes af, all and (1/11between
the two homeodomains are aligned. The 21 amino acid loop specific for the HNFla homeodomain is located partla/{y in helix all (shaded area).
Comparing the exon/inrron structure of the Xenopus HNF1a with the structure found in the rat genome (Bach et al.. 1992), it is clear that exon 5 of
Xenopus is encoded in 2 exons in the rat (exon 5 and 6) whereas the Xenopus exons 8 and 9 correspond to the exon 9 of the rat. Furthermore it is
notable that the chicken HNFla gene contains ten exons by combining the introns specific for rat and Xenopus (Hor/ein et al.. 1993).
Fig. 2. The interaction of HNF1a with the HPIelement. The tetramer
consisting of HNF1a and DCoH dimer is shown as bound to the regulatory
erement HP 1 upstream of the TATA-box and the transcriprion starr
site (arrow). The consensus shown for HPI is taken from Tronche and
Yaniv (1992) The size of the letter reflects the importance of each
residue.
Fig. 3. XHNF1ua and XHNF1r1b form heterodimers. A gel retardation
assay using the labeled HPI oligonucleotide was performed with the in
vitro translation products XHNFl ab and XHNFl aa made in reticulocyte
Iysates with mRNA encoding a truncated version of XHNFlab (f2.i1 1-410;
and the full-length XHNFl aa, respectively (Bartkowski et a/., 1993). The
addition of the monoclonal antibody XADl is indicated. The complexes
containing aa and bb hamadimers as well as the ab heterodlmer are
marked. The free HPl oligonucleotide has left the bottom of the gel.
Fig. 4. Interaction of XDCoH and HNF1a. Using the labeled HPI
oligonucleotide, gel retardation assays were made with 0.2 or 1 J-li
HNF1a (XHNFlaa) and 5 J-llXenopus DCoH translated in a reticulocyte
lysate. The addition of the monoclonal antibody XAOI specific for HNF1a
(Bartkowski et aI., 1993) and the polyclonal rabbit antiserum against
DCoH (Pogge v. Strandmann and Ryffel, 1995) is given. Camp/exes containing
HNFlo: eno DCoH and the corresponding complexes with the
antibody XAD1 (+XAD1) or the DCoH antiserum (+antl-OCoH) are
marked with arrows.
Fig. 5. Immunostaining of liver and gut sections of adult Xenopus to locate HNF1a. Cryostat sections of the liver (a) and the gut (b) were immunostained with the HNF1a specific monoclonal antibody XAD5 (Bartkowski et al., 1993) as first antibody and the Cy3-conjugated rat anti-mouse IgG as second antibody. The corresponding phase contrast pictures are in panel c and d. The magnification in all panels is the same; bar. 100 um.
Fig. 6. Developmental appearance of HNF1cr: mRNA, HNF1cr: protein, DCoH protein and albumin mRNA. The amount of HNFla mRNA and
HNFla protein at various stages of development are given as determined in Bartkowski et al. (1993). The amount of DCoH is takenfrom Pogge v.
Strandmann and Ryffel (1995) and the albumin mRNA was quantified by RNase protection analysIs (unpublished data) + refers co low amounts and
+ + indicates an approximately 1O-fold higher amount.
Fig. 7. Correct temporal and spatial expression of HNF1a promoter
constructs injected into fertilized Xenopus eggs. (A! Activation of the
endogenous HNFla gene: A Xenopus larvae at stage 38 was dissected
into a head, middle and tail section and the presence of HNF1 a in protein
extracts derived from these pieces was determined in gel retardation
assays using labeled HPI. The HNFla specific antibody XADl was
added as indicated. At the right a liver extract was used as a standard.
(B) Activity of the HNF7a promoter construct in larvae derived from
injected eggs. The relative CAT aerlv/rv in the head, middle and tal/section
is given. The promoter construct used contains the sequence from
~886 to -46 upstream of the translation start codon. The data are taken
from Figure 5A in Zapp et aI., 1993a
Fig. 8. Regulatory elements and factors involved in embryonic activation
of the HNF1a promoter. Schematic drawing of the HNF1a promoter
with the binding sites for OZ1, HNF1 a and HNF4 For details see
text.
Fig. 9. The OZ element is a common regulatory element in genes
activated early in Xenopus embryogenesis. A gel retardation assay
was made with the labeled OZ element from the HNF1a promoter.
Various unlabeled oligonucleotides were added in the amounts (ng) as
indicated.
hnf1a (HNF1 homeobox A) gene expression in sections of Xenopus laevis gut, assayed via immunohistochemistry, NF stage 66.
