Purification of GAGA factor of Drosophila and its role in nucleosome disruption.

Publisher Summary This chapter describes a protocol to express the GAGA factor protein in Escherichia coli, and to purify it in a form that is active in a nucleosome disruption assay. The GAGA factor is identified as a transcription factor for developmentally regulated genes of Drosophila and as a binding factor at GA CT-rich sequences in the promoters of the Drosophila heat shock and histone genes. In vitro transcription experiments showed that the GAGA factor is capable of counteracting the repression by nonspecific DNA-binding proteins such as histone HI, but is incapable of activating transcription by itself. The GAGA factor was also found to be involved in the disruption of the chromatin structure at Drosophila heat-shock promoters as suggested by the loss of DNase I hypersensitivity in vivo when the GA CT elements are deleted, and by the ability of recombinant GAGA factor to alter nucleosomes and create a DNase I hypersensitive site in vitro . Hence, the GAGA factor may be the prototype of a transcription factor whose primary function is to condition the chromatin template for the subsequent binding of other factors that activate the RNA polymerase.