In vivo mapping of Polycomb and trithorax group proteins in chromatin of Drosophila melanogaster

Strutt, Helen Lesley (1997). In vivo mapping of Polycomb and trithorax group proteins in chromatin of Drosophila melanogaster. PhD thesis The Open University.

Abstract

The Polycomb group and trithorax group genes of Drosophila are required for maintaining the repressed or active expression state of many developmental regulatory genes, such as the homeotic genes, throughout development. The Polycomb group genes have been suggested to act by regulating higher order chromatin structure.

This work describes an improvement in a formaldehyde cross-linking and chromatin immunoprecipitation technique for analysing in vivo protein-DNA interactions in tissue culture cells. Using this method, Polycomb protein was found to be strongly associated with previously-identified Polycomb group response elements (PREs) in repressed genes of the bithorax complex. Polycomb does not cover entire chromosomal domains, but spreads over a few kilobases of DNA surrounding PREs.

GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus binding sites. This suggests that GAGA factor binds constitutively to PREs in the bithorax complex, which also function as trithorax group response elements.

Finally, Polycomb, Polyhomeotic and Posterior sex combs proteins are shown to participate in a common multimeric complex, and to be associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. These three proteins are however differentially distributed on regulatory sequences of the engrailed-related gene invected. This suggests that there may be multiple different Polycomb group protein complexes which function at different target sites. Furthermore, Polyhomeotic and Posterior Sex Combs are associated with expressed genes, suggesting that the inclusion of Polycomb protein in the complex at PREs is required for stable silencing.

These results give insight into the mechanism by which the Polycomb group proteins mediate silencing of their target genes, and how this silencing is antagonised by the trithorax group genes. In addition, they reveal the structural and functional diversity of Polycomb group protein complexes.

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