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Nucleic Acids Research, 1989, Vol. 17, No. 6 2333-2350
© 1989

MOLECULAR BIOLOGY

In vivo protein binding sites and nuclease hypersensitivity in the promoter region of a cell cycle regulated human H3 histone gene

Urs Pauli+, Susan Chrysogelos, Harry Nick, Gary Stein* and Janet Stein*

University of Florida, College of Medicine Gainesville, FL 32610, USA

*To whom correspondence should be addressed at: Department of Cell Biology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655, USA

Received December 23, 1987. Revised February 15, 1989. Accepted February 15, 1989.

The chromatin structure and protein-DNA interactions of a cell cycle regulated human H3 histone gene have been examined at different levels of resolution. Using traitional Southern blot analysis we have investigated the accessibility of the H3 coding region and its flanking sequences to DNase I, S1 nuclease and restriction endonuclease digestion. Using the native gencimc blotting method recently developed in our laboratory, two sites of protein-DNA interaction in the proximal 240 bp of the promoter region of this H3 gena were established. Further in vivo analysis of protein-DNA binding sites in intact cells by genomic sequencing revealed, with single nucleotide resolution, the guanine contacts and footprints of the proteins bound to the promoter. The relative locations of protein-DNA interactions in this H3 gene are similar to those identified in vivo ard in vitro in a cell cycle dependent hunman H4 histone gene. The proteins complexed with the H3 histone gene promoter can be dissociated between 0.16 and 0.28 M NaCl. Ihe protein-DNA contacts persist throughout the cell cycle and thus may have a functional relationship with the basal level of transcription of this H3 gene that occurs during and outside of S phase.

+Present address: University of Bern, Institute of Veterinary Virology, Laenggass-Strasse 122, CH-3012 Bern, Switzerland


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