| Summary: | Expression of milk proteins including β-lactoglobulin is controlled by prolactin activation of the transcription factor Stat5 via the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) pathway. Stat5 has previously been shown to tetramerise where binding sites are tandemly linked and the proximity of these binding sites appears to be important for these interactions. This work and previous large scale mapping of the β-lactoglobulin promoter shows that the dyad of a strongly positioned nucleosome lies at -184 bp from the transcription start on the promoter of the β-lactoglobulin gene. This brings together two binding sites for Stat5, at the points of entry and exit of DNA from the nucleosome that would otherwise be spaced 185 bp apart, an arrangement that could potentially bring bound Stat5 dimers closer enough to facilitate tetramerisation. The chromatin structure over the active and inactive gene promoter is different; there are two alternative nucleosome positions in the active and only one in the inactive promoter. One of these positioning sites would not allow the tetramerisation interaction to take place. In order to understand better the mechanisms by which the expression of β-lactoglobulin is regulated by Stat5 we set out to investigate the role of these positioned nucleosomes in Stat5 binding <i>in vitro</i>. Stat5A and B binding patterns on both naked DNA and on reconstituted chromatin probes are shown by a series of bandshift experiments using purified recombinant Stat5 produced in a baculovirus expression system. Characterisation of Stat5 reveals the protein to be phosphorylated and able to bind DNA. A mutation, W37A, which removes the ability of Stat5 to form dimer-dimer interactions was employed to further investigate a potential role of tetramerisation influencing Stat5 binding in a chromatin context. This architectural feature could act to control the temporal and tissue specific expression of β-lactoglobulin.
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