Nucleosome positioning on the chromatin strand plays a critical role in regulating accessibility of DNA to transcription factors and chromatin modifying enzymes. Hence, detailed information on nucleosome depletion or movement atcis-acting regulatory elements has the potential to identify predicted binding sites fortrans-acting factors. Using a novel method based on enrichment of mononucleosomal DNA by bacterial artificial chromosome hybridization, we mapped nucleosome positions by deep sequencing across 250 kb, encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene.CFTRshows tight tissue-specific regulation of expression, which is largely determined bycis-regulatory elements that lie outside the gene promoter. Although multiple elements are known, the repertoire of transcription factors that interact with these sites to activate or repressCFTRexpression remains incomplete. Here, we show that specific nucleosome depletion corresponds to well-characterized binding sites for knowntrans-acting factors, including hepatocyte nuclear factor 1, Forkhead box A1 and CCCTC-binding factor. Moreover, the cell-type selective nucleosome positioning is effective in predicting binding sites for novel interacting factors, such as BAF155. Finally, we identify transcription factor binding sites that are overrepresented in regions where nucleosomes are depleted in a cell-specific manner. This approach recognizes the glucocorticoid receptor as a noveltrans-acting factor that regulatesCFTRexpressionin vivo.