The estrogen receptor alters large-scale chromatin structure
Anne C. Nye, Ramji R. Rajendran, Benita S. Katzenellenbogen and Andrew S. Belmont
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign
BACKGROUND
Whether higher levels of chromatin folding influence transcriptional regulation remains unclear. A strong transcriptional activator, VP16, was previously found to unfold chromatin at the large-scale level, suggesting the importance of this level of structure. We investigated the estrogen receptor (ER), a member of the nuclear receptor superfamily which activates transcription in response to estrogens. To study the effects of ER on large-scale chromatin structure, we targeted a fusion protein containing ER to a chromosome arm which is labeled with GFP.
As shown in the movie, we have found that the ER is in fact capable of dramatically altering large-scale chromatin structure. In brief, the estrogen receptor causes unfolding of fibers in the absence of hormone (not shown), and this unfolding is reversed upon addition of estradiol (shown in the movie).
It is interesting to note that for many of its target genes, the ER's transcriptional activity peaks within an hour and is then downregulated. This downregulation could correlate with the rapid refolding of chromatin, which we have observed.
MATERIALS & METHODS
In these experiments, we used a system developed recently to enable visualization of changes in large-scale chromatin structure, which accompany the targeting of proteins to specific chromosome regions (Tumbar et al., 1999). Lac repressor protein was fused with estrogen receptor (ER) and GFP, and this fusion protein was expressed in a Chinese hamster ovary (CHO) cell-line containing lac operator DNA sequences that are integrated into the genome in huge blocks of up to 90 mega-base pairs. Thus, both ER and GFP are targeted to a large chromosome arm, which is visible by fluorescence microscopy. Images were collected on an inverted light microscope (IMT-2, Olympus; Success, NY, USA) equipped with a cooled, slow-scan CCD camera (Photometrics; Tucson, AZ, USA) as described previously (Hiraoka et al., 1991). Optical sections of nuclei were collected and deconvolved as previously described (Agard et al., 1989).
REFERENCES & ACKNOWLEDGEMENTS
Agard, D.A. et al. (1989) Methods Cell Biol. 30, 353-377
Hiraoka, Y. et al. (1991) Semin. Cell Biol. 2, 153-165
Tumbar, T. et al. (1999) J. Cell Biol. 145, 1341-1354