Transcription activation mediated by the bZIP factor SPA on the endosperm box is modulated by ESBF-1 in vitro.

A modified in vitro transcription system has been used to study the function of the cloned bZIP transcription factor SPA and the binding activity ESBF I in activating transcription from the bifactorial endosperm box region of the wheat prolamin LMWG-1D1 gene. Recombinant SPA expressed in Escherichia coli activated transcription from the endosperm box motif, and this was dependent upon the binding of the nuclear protein ESBF I. ESBF I did not activate transcription independently, but potentiated SPA-mediated transcriptional activation. ESBF I is likely to be the equivalent of, or contain the recently characterised DOF class of, Zn-finger protein called WPBF. These data provide new information about the interplay of members of the bZIP and DOF transcription factor families in regulating expression from bifactorial sites found in a variety of plant promoters.

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

The conserved bifactorial endosperm box found in the promoter of wheat storage protein genes comprises two different cis elements that are thought to be involved in regulating endosperm-specific gene expression. Endosperm nuclear extracts contain binding activities. One is called ESBF-I, which binds to the endosperm motif (EM), and the other is called ESBF-II, which binds to the GCN4-like motif(GLM). Here, we present a functional analysis of the endosperm box of a low-molecular-weight glutenin gene found on the 1D1 chromosome of hexaploid wheat (LMWG-1D1) in transgenic tobacco plants. Our analysis demonstrates the necessity of the EM and GLM for endosperm-specific gene expression and suggests the presence in tobacco of functional counterparts of wheat ESBF-I and ESBF-II. Furthermore, we describe the isolation and characterization of cDNA clones encoding SPA, a seed-specific basic leucine zipper protein from wheat that can activate transcription from the GLMs of the -326-bp LMWG-1D1 promoter in both maize and tobacco leaf protoplasts. This activation is also partially dependent on the presence of functional EMs, suggesting interactions between SPA with ESBF-I-like activities.

The maize transcription factor Opaque-2 activates a wheat glutenin promoter in plant and yeast cells.

The promoter of the wheat low-molecular-weight glutenin (LMWG1D1) gene contains a cis element called the GCN4 like motif (GLM) which has low homology to one class of binding site for the maize endosperm-specific b-ZIP transcription factor Opaque-2 (O2). Previous work has shown that the GLM element interacts with the nuclear factor ESBFII during wheat endosperm development at the time of maximum transcription of the LMWG1D1 gene. In this paper we demonstrate that O2 binds to the GLM element and can activate high levels of transcription from the wheat GLM in transient assays in plant protoplasts and in yeast cells. Lower levels of O2 activation through the GLM element in yeast containing a defective GCN4 gene showed that GCN4 was necessary for high levels of O2 transcriptional activation, indicating that O2 may need to heterodimerise with GCN4 to activate transcription in yeast. These observations provide evidence that the GLM represents a new type of O2 DNA-binding site, and support a postulate that an O2 homologue may activate endosperm-specific expression of wheat storage protein genes.

Transcriptional control of plant storage protein genes.

The accumulation of plant storage proteins is controlled primarily by the transcriptional activation of their genes. Two classes of storage proteins, the zygotic or seed-specific, and the somatic, such as tuber proteins, have been studied. Gene expression analysis in transgenic plants has defined small regions of the promoters of such genes that are able to confer the appropriate patterns of expression. Protein-DNA interactions, both in vivo and in vitro, have revealed proteins that bind to regions implicated in expression, and these may be transcription factors. Promoter deletion analysis has determined the role of some of these DNA-binding proteins, such as in determining tissue-specificity or levels of expression. A common theme linking the expression of both classes of storage proteins is the involvement of metabolite levels in directly controlling gene expression.

The human insulin gene-linked polymorphic region adopts a G-quartet structure in chromatin assembled in vitro.

The insulin gene-linked polymorphic region (ILPR), located 363 bp upstream of the human insulin gene, is composed of tandem repeats of the consensus sequence ACAGGGGT(G/C)(T/C)GGGG. It has previously been shown that an insulin gene fragment containing the ILPR adopts an altered DNA structure in vitro. Furthermore, oligonucleotides containing the consensus repeat sequence exhibit multiple quadriplex DNA structures. The present study was undertaken to determine whether such altered DNA structures existed within the ILPR when the insulin gene was assembled into chromatin in vitro. Chromatin assembly was achieved using histones and an extract from unfertilized eggs from Xenopus laevis. The presence of altered DNA conformations within the 5' region of the human insulin gene was investigated using the structural probe nuclease P1. Nuclease P1 recognized multiple distinct sites in the 5' flanking region of the human insulin gene in naked DNA. Most of these sites disappeared when the recombinant plasmid DNA was treated with histones and unfertilized egg extract. In the assembled DNA, the ILPR appeared as the major site of nuclease P1 hypersensitivity. Fine-mapping of the multiple reactive sites within the ILPR showed a pattern characteristic of G-quartet foldback structures similar to those that have been observed for telomeric DNA.

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.

Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo.

An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.

A consensus repeat sequence from the human insulin gene linked polymorphic region adopts multiple quadriplex DNA structures in vitro.

A hypervariable region consisting of repeats of a 14 base pair (bp) consensus sequence ACAGGGGT(G/C)(T/C)GGGG is located 363 bp upstream of the human insulin gene. Different repeat numbers of this oligonucleotide give rise to a polymorphism, and so this region is commonly known as the insulin gene linked polymorphic region (ILPR). Here we present evidence, based on the mobility in non-denaturing polyacrylamide gels of two dissimilarly sized oligonucleotides containing the ILPR consensus sequence, that this sequence can adopt a number of quadriplex DNA structures in vitro.

The human insulin gene linked polymorphic region exhibits an altered DNA structure.

Regulation of transcription of the human insulin gene appears to involve a series of DNA sequences in the 5' region. Hypersensitivity to DNA structural probes has previously been demonstrated in regulatory regions of cloned genomic DNA fragments, and been correlated with gene activity. To investigate the structure of the DNA in the human insulin gene, bromoacetaldehyde and S1 nuclease were reacted with a supercoiled plasmid containing a 5kb genomic insulin fragment. Both probes revealed the human insulin gene linked polymorphic region (ILPR), a region (-363) upstream of the transcriptional start site which contains multiple repeats of a 14-15mer oligonucleotide with the consensus sequence ACAGGGGT(G/C)(T/C)GGGG, as the major hypersensitive site. Fine mapping and electron microscopic analysis both show a very different behaviour of the two DNA strands in the region of the ILPR and suggest the G-rich strand may be adopting a highly structured conformation with the complementary strand remaining largely single stranded.