Presented at the 40th Annual Meeting of the American Society for Cell Biology, December 10, 2000, at San Francisco, California, and published in: Molecular Biology of the Cell, vol. 11, Abstract Supplement, page 8a, (December, 2000):

"The Dynamic Interaction of Transcription Factors with Chromatin: Studies with Living Cells and Reassembled Templates".

Gordon L. Hager 1, James G. McNally 2, Waltraud G. Muller 2, Nianqing Xiao 2, Christopher T. Baumann 2, and Terace M. Fletcher 2,

1 Lab of Receptor Biology and & Gene Expression, National Cancer Institute, Bldg 41, Room B602, Bethesda, Maryland, 20892-5055,
2 National Cancer Institute.

E-mail: hagerg@exchange.nih.gov


Abstract:

Activation of the mouse mammary tumor virus (MMTV) promoter by glucocorticoid receptor (GR) is associated with a specific chromatin structural transition in the B/C nucleosome region of of the viral LTR. We have reconstituted this nucleoprotein transition with chromatin assembled on MMTV LTR DNA, purified GR, and HeLa nuclear extract. Chromatin remodeling in vitro is ATP-dependent, and maps to a region identical to that found in vivo. We demonstrtate specific, GRE-dependent, binding of purified GR to a large multi-nucleosome  MMTV chromatin array, and show that GR-dependent chromatin remodeling is a multistep process. In the absence of ATP, GR binds to multiple sites on the chromatin array and inhibits nuclease access to GR recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzyme sites within the transition region. We conclude [Fletcher et al, MCB (2000) in press] that GR occludes nuclease access by steric hindrance, and is lost from chromatin in an ATP-dependent fashion. The GR interaction with chromatin has also been characterized in living cells. Using a tandem array of (MMTV)-reporter elements and a GR chimera labeled with the green fluorescent protein (GFP-GR), we have observed direct targeting of the receptor to response elements in live mouse cells [Science 287: 1262 (2000)]. While the ligand-activated receptor is associated with the MMTV promoter for observable periods of time, photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. Both the in vitro and in vivo results are consistent with a dynamic model ("hit and run") in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template.

 References:

1.McNally JG, Muller WG, Walker D, and Hager GL, "The Glucocorticoid Receptor: Rapid Exchange with Regulatory Sites in Living Cells", Science 287: 1262-1264 (February 18, 2000).

2. Fletcher TM, Ryu B-W, Baumann CT, Warren BS, Fragoso G, John S, and Hager GL, "Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition", Mol. Cell. Biol. vol. 20, no. 17, pp. 6466-6475, (September, 2000).


Additional References:

1. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature 206: 680-683 (May 15, 1965).

2. Frenster JH,  "Mechanisms of  Repression and De-Repression within Interphase Chromatin", In-Situ, vol. 1, pp.78-101 (1965).

3. Frenster JH, Oncogenes as Molecular Targets within Active Chromatin", Clin. Cancer Res. vol. 5, suppl. 3855a (624), (November, 1999).


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