Sci. Aging Knowl. Environ., 10 April 2002
Vol. 2002, Issue 14, p. nf6
[DOI: 10.1126/sageke.2002.14.nf6]

NEWS FOCUS

An Untwister's New Twist

R. John Davenport

http://sageke.sciencemag.org/cgi/content/full/sageke;2002/14/nf6

Helicase enzymes seem to put the brakes on aging: Defects in these DNA-unwinding proteins result in a number of diseases that mimic aspects of growing old. But a helicase implicated in one of these conditions--xeroderma pigmentosum (XP)--apparently helps hormones activate genes in addition to straightening DNA, according to new work. DNA-repair defects account for the majority of the problems in XP--but not all of them. The protein's newly discovered role in hormone signaling, however, might underlie those unexplained XP symptoms.

Faulty hormone signaling is "one viable mechanism" for the XP symptoms that aren't readily explained by flaws in DNA repair, says Errol Friedberg, a pathologist at the University of Texas Southwestern Medical Center (UT Southwestern) in Dallas, although he cautions that it's not the only possible cause. The next step, says Friedberg, is for scientists to correlate particular symptoms with specific mutations in XP, which could help nail down a link. Matching physiological effects with protein actions might connect defects in hormone signaling to XP.

The symptoms of diseases caused by defective helicases vary, but they commonly include a dramatically elevated risk of cancer--something every one of us faces with age (see "Dangerous Liaisons"). Understanding these conditions might help explain why. The most common symptoms of XP--sensitivity to light and a propensity for skin cancer--stem from insufficient ability to mend DNA. As part of a protein machine called TFIIH, the XPD helicase untwists DNA so that other proteins can fix bases that have been harmed by ultraviolet light. Defective XPD loses that ability and leaves DNA permanently marred. Some people with XP, however, suffer from additional symptoms, including mental retardation, sterility, and dwarfism, that appear unrelated to crippled DNA-repair mechanisms.

Treatment with a hormone called retinoic acid improves the health of XP patients with mutations in the XPD gene but not those with mutations in one of the six other genes that cause the disease, so Jean-Marc Egly of the French Institute of Health and Medical Research (INSERM) in Strasbourg and colleagues wondered whether the mutations alter hormone signals. Other observations suggested that it might. XPD-containing TFIIH not only repairs DNA damage but also joins a larger gathering of molecules that kick-start transcription, the process by which genes are copied into messenger RNA. And hormones link to transcription because they bind to nuclear receptor proteins, which then attach to genes and turn them on. Previous work in Egly's lab hinted at a possible mechanism by which TFIIH could tweak hormone signaling: In a test tube, TFIIH adds a phosphate group to the retinoic acid receptor (RAR), a modification that frequently switches proteins on or off. But it wasn't clear whether TFIIH similarly alters RAR in cells and, if so, whether such a change influences the receptor's function.

In the new research, Egly and co-workers asked whether defects in XPD disrupt hormone signaling in cells. To find out, the researchers constructed a test molecule with two parts: a DNA sequence to which RAR binds and attached to whose protein product emits light. If hormone activates transcription of the gene, the protein is produced and illuminates the cell. The scientists inserted the test gene into two types of cultured human cells: normal ones and those that carry a mutation in the XPD gene found in XP patients. Then they treated each type of cell with retinoic acid, which normally would turn on the gene. Cells that produced normal XPD shined brightly, whereas cells with aberrant XPD did not (see figure). Adding a normal copy of the XPD gene to the mutant cells restored the glow. The data indicate that the mutation in XPD hinders hormonal activation of the test gene. Additional experiments suggest that the XPD mutations thwart other hormones: Mutant proteins prevent activation by estrogen and androgen, but not by a nonhormonal gene activator.



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Hormone helper. Normal XPD helicase spurs retinoic acid to turn on a test gene inside cells (top); a defective form of XPD fails to perform the same feat. [Credit: A. Keriel et al., Cell]

 
The work also suggests that XPD promotes hormone signaling by holding TFIIH together: TFIIH isolated from cells with defective XPD falls apart more easily than that from normal cells. In particular, the core proteins of TFIIH don't hook as tightly to cdk7, the part of the complex that adds phosphates to other proteins. Loosening cdk7's attachment to TFIIH, the scientists reasoned, might translate into less phosphate on nuclear receptors--and diminished gene activation.

To test that idea, Egly and his co-workers added radioactive phosphate to cells, burst them open, and then used antibodies to pull out RAR. As they had predicted, phosphate tagged half as many receptor molecules in XPD mutant cells as in controls. And in test tubes, TFIIH from normal cells tagged more molecules with phosphate than did TFIIH from mutant cells. In addition, the researchers created an RAR variant that resembles the phosphate-carrying form by engineering a bulky amino acid at the position that likely receives the phosphate group. Then they tested this version of the protein to find out if it could circumvent the mutation in XPD. When they put the altered RAR into cells with the defective XPD, the cells glowed as brightly as controls that carried normal RAR and XPD. The authors conclude that XPD mutations hinder TFIIH from phosphorylating RAR, which limits its gene-spurring capacity.

The work provides fundamental information about how hormones flip on genes, experts say. Usually activators turn on the basic transcription machinery, says biochemist Marc Timmer of the University of Utrecht Medical Center in the Netherlands. But in this case, there seems to be a "retrograde activation," in which the basic machinery (TFIIH) turns on the activator (RAR). Scientists have traditionally thought of the sequence of events that stimulate genes as "a linear process that goes from A to B to C to D," says Timmer. "Now it turns out that you can go back from D to B and strengthen the whole process. It's remarkable."

Although the work advances understanding at the molecular level, it's not clear how much it will tell us much about diseases such as XP. A hormonal defect might explain certain symptoms, such as sterility or slow growth, but researchers aren't convinced that this new idea is correct. Because XPD is part of the basic transcription machinery, alterations in the protein potentially affect numerous genes, not just the ones controlled by hormones, says UT Southwestern's Friedberg. Those changes could be sufficient to cause the unexplained symptoms of XP. Inability to phosphorylate hormone receptors "may or may not be linked to XP," says Friedberg. But unraveling XPD's varied capabilities will likely clarify how its malfunctions cause trouble.

April 10, 2002

R. John Davenport is an associate editor of SAGE KE. Already famous for his pies, he's hoping people will someday discover his still-developing banjo skills.

Suggested ReadingBack to Top

  • A. Keriel, A. Stary, A. Sarasin, C. Rochette-Egly, J.-M. Egly, XPD mutations prevent TFIIH-dependent transactivation by nuclear receptors and phosphorylation of RAR{alpha}. Cell 109, 125-135 (2002). [Abstract] [Full Text]








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