Nucleosomal organisation over the ovine β-lactoglobulin gene

• Main Author: Boa, Simon Andrew
• Published: University of Edinburgh 2000
• Subjects: 591.35
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641744

To date, there have only been limited studies on the nucleosomal organisation of genes in their natural environment. The majority of these studies have concentrated on short regions of positioned nucleosomes spanning either repetitive DNA or the promoter regions of specific genes. However, nucleosome positioning over an entire gene domain may have a significant impact on its regulation and compaction. I have mapped the nucleosomal organisation over 10kb of a tissue specific, temporally regulated gene using the enzymatic probe, micrococcal nuclease and the chemical probe, cuprous phenanthroline. The ovine b-lactoglobulin (BLG) gene studied has a well characterised developmental profile, a minimal transcriptional domain and has been used extensively as an expression cassette in transgenic animals to driver heterologous gene transcription. When the gene is inactive, in the liver, it displays a tightly defined array of positioned nucleosomes that modulate between two specific phases over the gene domain. A similar, less tightly defined array is present when the gene is active, in the mammary gland, except over the promoter and actively transcribing regions. The same arrays are present over the BLG promoter region in transgenic mice in both active and inactive states. To extend this analysis, I used the monomer extension assay to identify nucleosomal positioning <i>in vitro</i>. This procedure identifies sequence dependent nucleosome positioning of single reconstituted nucleosomes. This shows an interesting relationship with known transcription factor binding sites within the BLG promoter and correlates well with the <i>in vivo</i> results. A number of other milk protein genes have a similar pattern of key transcription factor building sites over their promoter regions. If the nucleosome positions were conserved in these genes, with respect to these binding sites, it would support a role for positioned nucleosomes in their regulation. A total of four genes, each in two different organisms, were used to test this hypothesis.