In the early 1950s, when the world still possessed a measure of anthropological mystery, a young doctor visited a cluster of tiny fishing villages around Venezuela’s Lake Maracaibo and was greeted by a remarkable sight. The streets were dotted with a strange breed of contortionist—people whose limbs would flail at odd moments, causing them to lurch and lose their balance, and whose faces were gripped by their own kind of convulsive motion. According to local legend, the villagers were possessed by a dancing mania, “el mal de San Vito,” but the physician, Americo Negrette, soon realized that they suffered from a rare and fatal disease, Huntington’s chorea. He would spend the next two decades studying and documenting the disease, largely in obscurity. In 1972, one of his films would be shown to a congress of researchers studying the disease.
“People were blown away because this is a rare disorder and here’s this whole town,” said Jang-Ho Cha, HMS associate professor of neurology at Massachusetts General Hospital. The film would galvanize the once quiet field of Huntington’s disease research, turning it and the Lake Maracaibo villages into scientific hot spots. Researchers began flocking there and, in 1983, using data provided by the villagers, James Gusella and colleagues at HMS and elsewhere would link the genetic defect to chromosome four. The Huntington gene was cloned 10 years later. Cha was in one of the Venezuelan villages when the discovery was announced. Over the past 15 years, he and other researchers have been struggling to crack an even harder scientific nut: how does a defect in a single protein, huntingtin, produce such bizarre and idiosyncratic effects?
Cha, Caroline Benn, and colleagues have recently hit upon a surprising, almost ironic, answer. They argue that mutant huntingtin produces the hallmark symptoms of Huntington’s disease—the springlike, kinetic, twisted movements—by a kind of relaxing of the DNA.
In an intricate series of experiments, Benn, until recently a research fellow in neurology at MGH, Cha, and colleagues found that mutant huntingtin binds directly to the genetic material and that this interaction produces an unwinding of DNA’s helical structure. This unraveling makes the genetic material more accessible to transcription factors, the researchers report in the Oct. 15 Journal of Neuroscience. “So you have aberrant transcription of normally silent genes,” said Cha.
The idea that mutant huntingtin carries out its disease-causing activities by unwinding the genetic material would have seemed not just ironic but improbable until quite recently. Most huntingtin is found outside the nucleus, and researchers have tended to focus on how the mutant version disrupts cytoplasmic activities, for example, the functioning of the proteasome. But the notion, while controversial, opens up the possibility that by stabilizing the DNA, Huntington’s disease, which currently affects 30,000 Americans, might be arrested.
“There are DNA-stabilizing drugs out there—certain antibiotics and lots of chemotherapeutic agents stabilize DNA conformation,” said Cha. “But the question is, if you stabilize certain conformations, does that prevent gene expression changes from happening?”
Cha first began to suspect that transcriptional regulation might be awry in Huntington’s disease over 10 years ago after he was sent brains of Huntington’s mice to look at by the British researcher Gillian Bates. One of the first things researchers noticed about the newly discovered huntingtin mutant gene was that it contains an unusually long polyglutamine sequence in exon 1. Bates had produced Huntington’s symptoms in mice by inserting that single exon. Cha and colleagues found that the mouse brains—along with those of Huntington’s patients—exhibited lower levels of certain neurotransmitter receptor proteins and their associated mRNAs. “That was the first real hint that there was a transcriptional problem,” he said.
Meanwhile, researchers had uncovered evidence of huntingtin in the nucleus of neurons and were finding they could kill or preserve cells simply by driving mutant huntingtin in or out of the nucleus.
Benn, Cha, and colleagues wondered whether the mutant huntingtin might be damaging cells by interfering with the way transcription factors bound promoters. Using two common techniques—the gel-shift assay, which reveals protein–DNA interactions in vitro, and chromatin immunoprecipitation (ChIP), which cross-links and pulls out specific protein–DNA complexes in living cells—they found that wild-type and mutant huntingtin were present at select promoters. “So huntingtin was sticking to DNA,” said Cha.
To be sure it was binding directly, they purified the DNA and the huntingtin protein in vitro, a method known as DNA immunoprecipitation (DIP). Normal huntingtin bound the DNA, but the mutant bound more.
Nor did the mutant and wild-type huntingtin always bind the same promoters. Using ChIP-on-chip—a method that combines ChIP with microarray technology to show where in the genome a protein binds—they found that though lots of genes were bound by both, many were bound exclusively by one or the other, suggesting they had distinct genomic targets.
“That still left us with the transcription factor story,” said Cha. It was here they would encounter their biggest surprise. Thinking that the difference in genomic targets would result in the alteration of a few key transcription factors, they decided to do a transcription factor array. “We saw something completely different—we saw lots of transcription factors had altered activity. We thought that was really weird,” said Cha.
Initially they were at a loss to explain the findings, but then came an insight—maybe the huntingtin protein was tinkering with the transcription factors not in a one-to-one manner but by changing the conformation of the DNA, essentially unwrapping it, opening it up to more interaction. Using dyes that bind DNA in a conformationally dependent manner, they tested their hypothesis. “Wild-type huntingtin unwound DNA, but mutant huntingtin unwound it more,” said Cha.
There was still a problem. In his early investigations of Bates’s mouse brains, Cha had found lower levels of certain neurotransmitter receptors and their associated mRNAs, but if transcription factors had greater access, the brains should exhibit higher levels of gene expression. The researchers went back and performed microarray analyses of mutant and wild-type cell lines taken from the striatum, the region of the brain most affected in Huntington’s disease. They compared the number of genes expressed exclusively in one or the other and found that the number was greater in the mutant cells. Intriguingly, genes expressed in both cell lines were more likely to be downregulated in the presence of mutant huntingtin.
How might huntingtin’s nuclear activities be reconciled with its obvious presence in the cytoplasm? “I like the idea that huntingtin maybe is listening to what is going on, integrating a lot of signals in the cytoplasm and upon certain regulatory events, becomes a nuclear signal to alter gene transcription,” said Cha. “Of course, there is not a lot to disprove me.”
For Students: Contact Jang-Ho Cha at cha@helix.mgh.harvard.edu for more information on this and other lab projects.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: Huntington’s Disease Society of America Coalition for the Cure Program, Glendorn Foundation, the National Institutes of Health, and the Massachusetts General Hospital Fund for Medical Discovery
