Exploiting Polytene Chromosomes to Identify Transcription Cofactors and Complexes Recruited to an Estrogen Responsive Promoter In Vivo

Abstract : The purpose of this work was to develop a novel system to study recruitment of factors, especially those involved in chromatin modification and remodeling, to an estrogen-responsive promoter. Transgenic fruitflies were created that conditionally express estrogen receptors and that contain a reporter whose expression is dependent on the estrogen receptor. Six doubly transgenic lines were developed. These transgenic flies express the estrogen receptor in response to heat shock and the reporter (green fluorescent protein) in response to estrogens. For five of six lines, estrogen-dependent expression of the reporter in salivary glands has been confirmed. Whole mounts of glands from control and estrogen-fed larvae were done and estrogen receptor visualized by immunofluorescence. Results suggest that much of the estrogen receptor m both control and hormone-treated larvae, while nuclear, is not bound to DNA. Chromosomal squashes of polytene chromosomes reveal that the chromatin-bound estrogen receptor is bound to euchromatic DNA and excluded from heterochromatic DNA. Surprisingly, estrogen made no difference in the apparent binding of receptor to DNA. These results suggest that estrogen receptors are bound to active regions of chromatin even in the absence of hormone. This may facilitate the search for high affinity binding sites when hormone is present.