Ad4BP/SF-1 is a nuclear receptor whose expression is restricted to tissues involved in steroid hormone synthesis such as the adrenal cortex and gonads. Recent sequence data analysis has shown that the Ad4BPlSF-1 gene is located only 13 kb downstream of the last exon of the neighboring GCNF gene that is expressed in some neurons and gonadal germ cells. Despite the close proximity of the two genes, regulatory elements from one do not interfere with the transcription of the neighboring gene, resulting in distinct expression patterns of Ad4BPlSF-1 and GCNF. This observation has led to the prediction that an insulator element must exist between the two loci to establish independent transcription units. We performed DNase I hypersensitivity assays on the adrenal cortex cell line., Y-1, to test for the existence of an insulator. Three hypersensitive sites were identified in the region spanning 2.1 kb between the last exon of GCNF and the first exon of Ad4BPlSF-1. The most upstream site contains a binding site for CTCF, a known insulator protein, while the other sites are predicted to associate with the nuclear matrix. Chromatin immunoprecipitation analysis using anti-acetylated historic H3 and H4 antibodies showed a discontinuous Pattern Of historic H3 and H4 acetylation upstream of these sites. Our data suggest that the chromatin architecture specialized by CTCF and the unclear matrix contribute to the distinct pattern of 9 transcriptional regulation of these genes. (c) 2005 Elsevier Inc. All rights reserved.