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CODE BREAKERSScientists tease out the secrets

of proteins that DNA wraps aroundBY JOHN TRAVIS

Jamming a week’s worth of clothing into a carry-on suitcase is tough, but consider the challengea human cell faces with its DNA. More than6 feet of this double-stranded molecule, makingup a cell’s 23 pairs of chromosomes, must get

stuffed into the cell’s microscopic nucleus. Just as peo-ple might roll or fold their clothes in special ways to stuffa piece of luggage, cells have devised tricks of their own to cram in all their DNA. One trick is to tightly wind the DNAaround complexes of proteins called histones, much as thread iscoiled around a spool. The histone-DNA combos, in turn, arefolded and refolded to make up individual chromosomes.

When scientists originally discovered this packing system, theywere befuddled. To make new proteins, certain cellular enzymesmust read the sequence of nucleotides that make up a cell’s DNA.But the enzymes can’t do their job, the scientists reasoned, if thegenetic sequences are locked in a tight embrace with histones.

Scientists have learned more recently that cells use various chem-ical modifications of histones to sometimes expose and sometimessequester, thus turning genes off or on. As biologists start to under-stand these alterations, appreciation for the importance of his-tones is growing.

“They’re not just spools on which DNA is organized andpacked into the nucleus. They’re intimately involved in regu-lating access to genes,” says Shelley L. Berger of the Wistar Insti-tute in Philadelphia.

In fact, 4 years ago, Brian D. Stahl and C. David Allis, both thenat the University of Virginia Health Science Center in Char-lottesville, coined the term histone code to represent the idea thatspecific histone modifications can be paired with specific geneticactivity within a cell. For example, one pattern of histone chem-istry turns up when a cell is dividing, while another pattern fore-casts the death of a cell. More-permanent histone modificationsmay maintain a cell’s specific identity, such as brain cell or liver cell.

Further deciphering this histone code and developing ways tomanipulate it could have major medical payoffs, say both Bergerand Allis. Compounds that interfere with how cells modify histoneshave already shown promise in treating tumors and Huntington’sdisease.

“The implications for human health are quite strong,” says Allis,who now leads a histone-biology research team at Rockefeller Uni-versity in New York.

TALE OF THE TAILS When it comes to biology, the genetic codehas earned fame. Check any life science textbook and it will describehow different triplets of DNA nucleotides represent the 20 aminoacids that make up natural proteins. The three-nucleotidesequences generally signal for one or another amino acid, but somesimply tell a cell when to stop building a protein.

A histone code may be much more complex. Peer inside the

nucleus of a human cell and zoom in on a chromosome. Scientistscompare its structure to that of a string of beads, with each beadconsisting of a 146-nucleotide-long DNA strand wrapped almosttwice around a complex of eight histones.

Each bead contains two copies of four histones: H2A, H2B, H3,and H4. Several decades ago, as scientists began to piece togetherthe structure of the beads, they observed that small groups of atomsknown as acetyl or methyl groups frequently adorn the histones.“It began to emerge that the histone proteins were phenomenallydecorated by these chemical flags,” says Allis.

Scientists also noticed general correlations between certain pat-terns of histone decoration and gene activity. In particular, partsof chromosomes in which histones are covered with acetyl groupstend to have active genes, whereas deacetylated histones tend toharbor inactive genes. DNA near methylated histones is generallyshut down.

Slowly, as researchers learned more about the structure of his-tones, it became evident that patterns of acetylation and methy-lation could be quite precise. It turns out that each histone has a

tail, a flexible string of amino acidsjutting out from the DNA-wrappedspool. Acetyl and methyl groups tendto plant themselves on particularamino acids in the tails, scientistsfound.

Histone tails have a lot to teachbiologists, according to Allis. Fromspecies to species, he notes, these tails

are nearly identical, implying that they are important to the cell.“Nature has held these things constant for a reason,” says Allis.

In 1992, Bryan Turner of the University of Birmingham Med-ical School in England and his colleagues discovered that themale fruit fly’s single X chromosome, but not its other chromo-somes or the female’s two X chromosomes, has a specific acety-lated amino acid on the tail of histone H4. Since genes on themale fly’s X chromosome are extra-active to compensate for theirabsence on the smaller Y chromosome, the investigators sug-gested that the acetylation accounts for the increased male-geneactivity.

Another major breakthrough in histone biology occurred in1996. That year, Allis’ group identified the first histone acetylase,an enzyme that places acetyl groups on histone tails. A month afterthat work was published, a research team led by Stuart Schreiberof Harvard University reported the discovery of a histone deacety-lase, an enzyme that strips the tails of such groups. Moreover, theacetylase and deacetylase had already been implicated in turninggenes on and off, respectively. Together, the two reports made itclear that the enzymes regulate genes via histone tails. “All of asudden, a beautiful mechanism emerged,” recalls Berger.

By 2000, when Stahl and Allis proposed that there’s an elab-orate code of histone modifications, scientists had tallied sev-eral histone-tail decorations beyond methylation and acetyla-tion and identified additional enzymes involved in this chemical

“This opens uptherapeuticopportunities. ”— DAVID ALLIS ROCKEFELLER UNIVERSITY

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accessorizing. In some cases, sugars or whole proteins, albeitsmall ones, mark histones. Biologists found, for example, that theprotein ubiquitin, which was originally thought only to markproteins for destruction, attaches to histone tails that remainintact.

In the Nov. 11, 2003 Proceedings of the National Academy ofSciences, Yuzuru Shiio and Robert N. Eisenman of the FredHutchinson Cancer Research Center in Seattle report that genesare turned off when members of a family of ubiquitin-like pro-teins are added to the tails of histone H4.

“There are all kinds of sites [on histone tails] that can be mod-ified,” says Berger. She adds, “The possibilities for a code are reallyquite enormous. It’s not going to be a simple code.”

How does acetylation or any other histone-tail modificationinfluence gene activ-ity? The original theory was thatchemically modify-ing histones wouldmake their electricalcharge less positive.As a result, theywouldn’t hold on astightly to their DNA,which is negativelycharged.

Today, biologistsare more inclined toargue that modifiedhistone tails act aslanding pads forother proteins thatinfluence the acces-sibility of DNA forgene activity. Turner put forth this idea a decade ago, but it proveda challenge to identify proteins that recognize specific histone-tailconfigurations.

In 2001, however, two research teams reported that heterchro-matin protein 1, a molecule known to mediate the silencing ofgenes, binds to the amino acid lysine on the tail of histone H3 onlyif methyl groups adorn the lysine. Other proteins that bind specif-ically to modified histones have subsequently turned up.

“Now, we’re finding these docking molecules,” says Allis.

LIFE AND DEATH Allis notes that some investigators may quib-ble with the notion of a histone code. “Code suffers a bit from beinga buzzword,” he says.

Turner agrees. “I think we have to agree on what we mean bythe histone code and what we expect from it,” he cautions. “Ithink if the code is going to be worth anything, it has to havepredictive value. It has to be passed on from one cell generationto the next.”

Histone methylation, for example, appears stable, says Turner.Scientists haven’t yet found an enzyme that strips methyl groupsoff a histone, and the patterns of this chemical tag appear to betransferred into both sister cells when a cell divides.

As a cell specializes, it may use histone methylation to perma-nently turn off unneeded genes and activate those that are essen-tial to the cell’s function. Histone methylation “looks like it’s lesstransient and like it’s more involved in the long-term setting ofthe genome,” says Berger.

In contrast, a cell’s pattern of histone acetylation may not qual-ify as a code, says Turner. Acetyl groups frequently hop on and offof histone tails, making it difficult to argue that they provide a cellwith a discernible identity.

Arguing that there is a histone code, Allis cites other additionalinstances in which he can make predictions about a cell by read-ing its histones. He and his colleagues have found that if a cell has

a phosphorus-containing chemical group tacked onto several aminoacids on the tail of H3, the cell is dividing. On the other hand, if aparticular serine on the tail of H2B has a similar phosphate group,the cell is about to commit suicide, the researchers reported in theMay 16, 2003 Cell.

Allis refers to these two distinct histone markings as life codesand death codes. “It’s cool to think that there may be a uniqueproperty of the H2B tail that encodes death” for a cell, he says.

If so, perhaps researchers can use the death code to kill tumorcells. Or, they could thwart the death code and thereby stop cellsfrom dying in a variety of human illnesses, such as degenerativebrain disorders. “This opens up therapeutic opportunities,” says Allis.

Drugs that disrupt the putative histone code are already beingwielded against lymphomas, leukemia, and other cancers. After

cell and animal stud-ies suggested thatinhibiting histone-deacetylase activitycould kill cancer cells,physicians cautiouslybegan to testinhibitors of theenzyme on people.The initial concernwas that such drugswould have danger-ous side effectsbecause they wouldalso affect deacetyla-tion in normal cells.That hasn’t been amajor problem so far.

“These drugs don’tseem overly toxic,”

says Allis. “People are tolerating them reasonably well. More impor-tantly, their tumors are disappearing.”

Animal studies have also indicated that histone-deacetylaseinhibitors might thwart the brain-cell loss characteristic of Hunt-ington’s disease (SN: 2/15/03, p. 102). Mutant proteins generatedin the illness seem to gum up the workings of histone acetyl-transferases, so blocking deacetylase function may create a morenormal histone state within cells.

DESIGNER HISTONES Even as researchers attempt to exploittheir new understanding of histones for medical purposes, manyquestions remain about these DNA-wrapped entities. Considerthe mystery of how a dividing cell produces two cells with seem-ingly identical methylation of the histones’ tails. “How [cells]inherit the histone code is something we have to scratch our headsabout,” says Allis.

Scientists are coming up with new tools to probe histone biol-ogy. In the Oct. 14, 2003 Proceedings of the National Academy ofSciences, a research team described a strategy to synthesize largequantities of a histone with a chosen modification, such as acety-lation of a particular amino acid in the histone’s tail.

In essence, the investigators manufacture just the tail, with thechosen modification, and chemically glue it to a tailless histone cre-ated separately. The researchers can then wrap these designer his-tones with DNA and examine how combinations of histone-tailmodifications influence a gene’s activity.

“We’re looking at creating these different types of molecules enmasse. We’re now making entire libraries of all possible modifi-cations,” says study coauthor Dewey McCafferty of University ofPennsylvania School of Medicine in Philadelphia.

With such designer histones, it seems that researchers are ontheir way to having in their hands all the words of the histone code.But, it may still be a stiff challenge to figure out what those wordsmean. ■

TAGGING TAILS — DNA wraps around complexes of proteins called histones. On a his-tone in one of the complexes in this diagram, an enzyme attaches a phosphate chemicalgroup to a tail-like structure (dotted circle). Such tail modifications regulate gene activity.

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