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Volume 272, Number 49, Issue of December 5, 1997 pp. 31118-31129
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleosome Positioning and Transcription-associated Chromatin Alterations on the Human Estrogen-responsive pS2 Promoter

(Received for publication, April 30, 1997, and in revised form, August 21, 1997)

From the Department of Molecular Genetics, Dana Farber Cancer Institute, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

The positioning of nucleosomes on a promoter is a significant determinant in its responsiveness to inducing signals. We have mapped the chromatin structure of the human, estrogen-responsive pS2 promoter at nucleotide level resolution within the context of its normal genomic location in human mammary epithelial cells. In vivo digestion by nucleases followed by ligation-mediated polymerase chain reaction analysis revealed two rotationally phased and translationally positioned nucleosomes within the promoter between nucleotide positions 450 and +7. The estrogen response elements at 400 and TATAA box at 35 are each located at the edge of a nucleosome. The two precisely positioned nucleosomes exist in both transformed and nontransformed human mammary epithelial cells, regardless of estrogen receptor status or transcriptional activity of the gene. However, two structural alterations correlate with the transcriptional potential of the promoter. In MCF-7 cells, in which the pS2 promoter is inducible, the chromatin exhibits an increased sensitivity to DNase I in a region of DNA adjacent to the TATAA box and an additional micrococcal nuclease-hypersensitive site in the linker DNA between the two positioned nucleosomes. We were also able to demonstrate that nucleotides 1100 to +10 of the pS2 promoter are sufficient to determine the positioning of these two nucleosomes. Our results establish the structural features of the chromatin covering the pS2 promoter as well as transcriptionally associated alterations, suggesting how the nucleosomal template influences transcriptional regulation by estrogen receptor.

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