A modified chromatin-immunoprecipitation protocol for silkmoth ovarian follicular cells reveals C/EBP and GATA binding modes on an early chorion gene promoter.
Standard Chromatin immunoprecipitation protocols have been designed to suit studies performed on cell line cultures or yeast cells growing in liquid cultures. In these cases cross-linking/fixation takes place directly in the growing medium of the cells by the addition of a general fixation reagent. When applied on whole isolated silkmoth follicles, this procedure results in poor release of follicular cells from the basal membrane and lower yield of cross-linked chromatin. We present a modification to the standard protocol, where detachment of follicular cells from the basal membrane of the egg and nuclei isolation precedes formaldehyde-mediated cross-linking. We also discuss application of the modified method for the identification of distinct BmC/EBP and BmGATAbeta binding modes on a chorion gene promoter from the Er1.A/B early gene pair.
Papantonis
Argyris
A
Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, 15701 Athens, Greece.
Lecanidou
Rena
R
D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002823:Chorion / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D005260:Female; D050980:GATA Transcription Factors / Q000378:metabolism*; D006080:Ovarian Follicle / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013997:Time Factors
D022762:CCAAT-Enhancer-Binding Proteins; D050980:GATA Transcription Factors