Upstream elements present in the 3'-untranslated region of collagen genes influence the processing efficiency of overlapping polyadenylation signals

3'-Untranslated regions (UTRs) of genes often contain key regulatory elements involved in gene expression control. A high degree of evolutionary conservation in regions of the 3'-UTR suggests important, conserved elements. In particular, we are interested in those elements involved in regulation of 3' end formation. In addition to canonical sequence elements, auxiliary sequences likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We identified highly conserved sequence elements upstream of the AAUAAA in three human collagen genes, COL1A1, COL1A2, and COL2A1, and demonstrate that these upstream sequence elements (USEs) influence polyadenylation efficiency. Mutation of the USEs decreases polyadenylation efficiency both in vitro and in vivo, and inclusion of competitor oligoribonucleotides representing the USEs specifically inhibit polyadenylation. We have also shown that insertion of a USE into a weak polyadenylation signal can enhance 3' end formation. Close inspection of the COL1A2 3'-UTR reveals an unusual feature of two closely spaced, competing polyadenylation signals. Taken together, these data demonstrate that USEs are important auxiliary polyadenylation elements in mammalian genes.     

Efficient identification of regulatory sequences in the chicken genome by a powerful combination of embryo electroporation and genome comparison

Recently expanded knowledge of gene regulation clearly indicates that the regulatory sequences of a gene, usually identified as enhancers, are widely distributed in the gene locus, revising the classical view that they are clustered in the vicinity of genes. To identify regulatory sequences for Sox2 expression governing early neurogenesis, we scanned the 50-kb region of the chicken Sox2 locus for enhancer activity utilizing embryo electroporation, resulting in identification of a number of enhancers scattered throughout the analyzed genomic span. The 'pan-neural' Sox2 expression in early embryos is actually brought about by the composite activities of five separate enhancers with distinct spatio-temporal specificities. These and other functionally defined enhancers exactly correspond to extragenic sequence blocks that are conspicuously conserved between the chicken and mammalian genomes and that are embedded in sequences with a wide range of sequence conservation between humans and mice. The sequences conserved between amniotes and teleosts correspond to subregions of the enhancer subsets which presumably represent core motifs of the enhancers, and the limited conservation partly reflects divergent expression patterns of the gene. The phylogenic distance between the chicken and mammals appears optimal for identifying a battery of genetic regulatory elements as conserved sequence blocks, and chicken embryo electroporation facilitates functional characterization of these elements.     

Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR.     

Analysis of gene regulation in growing pollen tubes of angiosperm and gymnosperm species using microprojectile bombardment

A microprojectile based transient expression assay was used to investigate the functional conservation of gene regulatory mechanisms in the male gametophytes of an angiosperm ( Nicotiana tabacum ) and two gymnospermous ( Picea abies and Pinus pinaster ) species. The activities of two angiosperm gene promoters, which have previously shown to be either preferentially expressed in the male gametophyte ( lat52 ) or highly expressed in both the sporophyte and male gametophyte ( Act I), were analysed. The results showed that in P. abies and P. pinaster , activity of the Act 1 promoter was significantly higher than the activity of the lat52 promoter, while the converse was observed in N. tabacum . Detailed analysis of lat52 5'promoter deletions demonstrated that although the minimal -67 bp lat52 core promoter was active at low levels in all three species, upstream regulatory elements conserved among several pollen-expressed genes, including the PBI element, were not functional in P. abies and P. pinaster . These results suggest that both taxa-specific and conserved regulatory mechanisms operate to control gene expression during pollen germination and tube growth.     

Unique patterns of evolutionary conservation of imprinted genes

During mammalian evolution, complex systems of epigenetic gene regulation have been established: Epigenetic mechanisms control tissue-specific gene expression, X chromosome inactivation in females and genomic imprinting. Studying DNA sequence conservation in imprinted genes, it becomes evident that evolution of gene function and evolution of epigenetic gene regulation are tightly connected. Furthermore, comparative studies allow the identification of DNA sequence features that distinguish imprinted genes from biallelically expressed genes. Among these features are CpG islands, tandem repeats and retrotransposed elements that are known to play major roles in epigenetic gene regulation. Currently, more and more genetic and epigenetic data sets become available. In future, such data sets will provide the basis for more complex investigations on epigenetic variation in human populations. Therein, an exciting topic will be the genetic and epigenetic variability of imprinted genes and its input on human disease.