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The Platelet-Derived Growth Factor B (PDGF-B) gene product is an important protein during development and adult life, as well as being associated with the development of many tumour types. It has been ascribed roles in many normal and pathological processes including angiogenesis, tissue repair, atherosclerosis and cytotrophoblast proliferation.

The transcriptional regulation of the PDGF-B gene is very complex and multiple levels of regulation have been demonstrated. A full understanding of how levels of this biologically important molecule are controlled may allow therapeutic control of this gene. The control of the expression of PDGF-B in choriocarcinoma cells (JEG-3) was shown to involve a unique promoter-specific enhancer. The PDGF-B promoter was characterised and an element within the promoter was shown to control its specific interaction with the enhancer. The enhancer was subsequently characterised and was shown to be composed of two independent elements. One of these elements was shown to be responsible for the interaction of the enhancer with the promoter, while the other was shown to be responsible for the activation by the enhancer. The activation element failed to independently activate the PDGF-B promoter at a distance without the cooperation of the second element. We propose the possibility that many "proximal-only" elements may be directed to work via promoters in this fashion.

During development and tumourigenesis, there will be times that cells/tissues are deprived of oxygen (hypoxia) and to survive, they need to take measures to overcome this deprivation. PDGF-B has previously been reported to be up-regulated by hypoxia in endothelial cells. In contrast, however, we show a down-regulation of PDGF-B and suggest a link between prehypoxia levels of PDGF-B and the response to hypoxia.

Much emphasis has centred on the effect of histone acetylation on gene transcription. The effect of acetylation on endogenous PDGF-B expression and on the promoter/enhancer system we have described, was examined in multiple cell-types.

Embryonic development consists of a vast number of fine-tuned, complex processesthat organise the body plan and individual organs. To enable developmental decisions,genes pivotal for normal development must be stably activated and silenced, asappropriate. Imprinted genes are exclusively expressed from the paternal or the maternalallele, such that one allele is activated and the opposite allele silenced in the same cell,illustrating an excellent example of epigenetic silencing.

We investigated H19, a putative tumour suppresser gene, which is highly expressedduring embryonic and foetal development, exclusively from the maternal allele. A novelmethod, allele-specific in situ hybridisation (ASISH), was developed to examine allelespecific activation/silencing events during early mouse development and in experimentalsystems. In order to understand the function of the H19 gene, we also investigated post-transcriptional regulation processes, as well as potential roles for the H19 transcript.

We were able to show that the H19 gene can be expressed from the same chromosome as Igf2 in the choroid plexus of the mouse, contrary to previous suggestions.We also found that the silencing of the paternal H19 allele is stepwise, leading to avariegated expression pattern during early mouse development. The manifestation of theimprinting status of H19 involves deacetylation of histones, since the silencing of thepaternal HI9 allele could be prevented by trichostatin A, an inhibitor of histonedeacetylases. Collectively, the results suggest an analogy between genomic imprintingand position-effect variegation in Drosophila.

We have also discovered a novel splice variant of the H19 transcript which isgenotype-specific. Moreover, we document that the genotype and epigenotype synergiseto control the expression of the splice variant. It was also shown that H19 transcript isassociated with polysomes and may control expression of the IGF2 gene in trans.

The first experimental link between genomic imprinting and the fetal alcohol syndrome was also documented, since the ethanol metabolite, acetaldehyde perturbs the manifestation of the H19 impritning status during early mouse development.