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Don’t Throw Out the Packing Materials

Most illustrations of DNA depict a kind of ladder spiraling off into the distance, the ladder being the famous DNA double helix consisting of paired nucleotide bases. Although it has long been known that mammalian DNA is packed very tightly and systematically with specialized proteins into material called chromatin, researchers are only now beginning to appreciate the importance of chromatin structure in gene regulation. Gordon Hager, Ph.D., Chief of the Laboratory of Receptor Biology and Gene Expression, has built his considerable scientific achievements on the study of nuclear hormone receptors, using the glucocorticoid receptor (GR) as a prototype. Not without controversy, his research has brought him inexorably closer to the pivotal role and complex dynamics of chromatin structure in the control of gene regulation.

Gordon Hager, Ph.D.
Photo shows Gordon Hager, Ph.D. (Photo: R. Baer)

“In the 1980s,” recalled Hager, “chromatin was a bit of a dirty word.” A wave of experiments done by several different laboratories that were designed to transcribe genes from chromatin fractions had ended in the purgatory of experimental artifacts a few years earlier. Wary scientists shied away from studying chromatin as anything more than DNA packing material. “And there were some groups, including mine, Carl Wu’s, Gary Felsenfeld’s, and Bob Simpson’s that did a lot of the early chromatin work because it simply couldn’t get funded extramurally,” explained Hager, referring to his current and former NIH Intramural Research Program colleagues.

Hager’s laboratory was focused on nuclear hormone receptors—receptors that bind hormones like glucocorticoids, which allow them to interact with particular response elements in the DNA to regulate gene transcription. For reasons that are still only partially understood, the murine mammary tumor virus (MMTV) contains a regulatory element that binds GR when MMTV is integrated into cellular DNA. The team discovered that when MMTV integrated into the mammalian genome, chromatin structural elements called nucleosomes were invariably positioned over the GR binding sites.

This discovery was soon followed by studies showing that GR binds directly to a nucleosome and that, as a result, the nucleosome undergoes a structural transition. To measure the change in chromatin structure, the team used an assay known as DNAse hypersensitivity. DNAse or deoxyribonuclease is an enzyme that will chew up DNA entirely if incubated long enough. However, when incubated only very briefly, DNA is fragmented at easily accessible sites in the chromatin structure that are termed hypersensitive. GR binding sites corresponded to sites of DNA hypersensitivity.

“We proposed that the glucocorticoid receptor was binding to DNA and causing chromatin reorganization at a specifically positioned nucleosome,” said Hager.

A Few Years in the Doghouse

“In 1987, I proposed at a Keystone Meeting in Park City, Utah, that somehow this chromatin reorganization was part of the mechanism by which the glucocorticoid receptor regulated gene expression,” said Hager. Hager was presented with a “Renegade Award” at the meeting, which was not meant as an accolade. “We were in the doghouse,” said Hager.

Within a few years, however, the field had shifted its perspective. Evidence began to accumulate suggesting that the structure of chromatin was playing more than just a passive role in gene regulation. A Postdoctoral Fellow in Hager’s lab, Trevor Archer, Ph.D., now Chief of the Laboratory of Molecular Carcinogenesis at the National Institute of Environmental Health Sciences, published an influential experiment in 1992 that presented the first direct evidence that the structure of chromatin could prevent a transcription factor from binding to its promoter element. A few years later, C. David Allis, Ph.D., now Tri-Institutional Professor at The Rockefeller University, and colleagues identified a known transcriptional regulator in yeast as an enzyme that modified histone proteins in chromatin.

The structure of chromatin was playing more than just a passive role in gene regulation.

“And bingo, chromatin was not such a dirty word anymore,” said Hager.

It is now clear that remodeling of chromatin is a key event in transcriptional regulation. Many chromatin remodeling factors have been identified and an entire field of epigenetics has emerged to investigate the influence of chromatin modifications
on functional gene expression.

There and Back Again

“Most of molecular biology until about the mid-1990s was dead-cell biochemistry,” said Hager. “No matter what you do—a DNA footprint or a chip experiment—the first thing you do is kill the cell and then, often, you do ‘terrible’ things like crosslink the proteins everywhere.”

Hager and his colleagues wanted to study gene regulation in living cells. With the advent of green fluorescent protein (GFP), which could be introduced genetically to label proteins of interest, a Postdoctoral Fellow in the lab created GRs tagged with GFP and showed that their fluorescent signature could be visualized under a microscope in living cells.

“And then we remembered cell line 3134,” said Hager.

The idea that DNA binding proteins were operating on such a fleeting timescale contradicted many accepted experimental paradigms.

Back in the laboratory’s early days, Hager and his team had used the MMTV promoter element as an experimental tool to study glucocorticoid receptor function. “Accidentally, we had a cell line where this structure had amplified itself into a 200-copy tandem array sitting in one place on chromosome 4. That’s two million base pairs of DNA with about 1000 GR binding sites in this one place in the chromosome.” A more perfectly optimized system for visualizing GR binding could not be readily imagined.

Under a fluorescence microscope, Hager and his colleagues were able to see GRs accumulating on this massive stretch of binding elements. They could also study its kinetics through a technique known as photobleaching. When fluorescent molecules are subjected to light of a particular wavelength, they lose their activity and are no longer visible. By shining a laser on the chromosomal segment, Hager’s team could discover whether and when the GRs were replaced by new unbleached molecules.

“We found that they were almost instantaneously replaced,” said Hager. “And we were back in the doghouse.”

Not only was the result surprising, the idea that DNA binding proteins were operating on such a fleeting timescale contradicted many accepted experimental paradigms for which protein-DNA binding was essentially considered fixed. However, other groups began to do the same kind of experiments with different proteins and the dynamic nature of these interactions gradually came to be accepted.

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