G ENES IN ACTION

NEWS ECTION S Searching for the ’s Second Code The genome has more than one code for specifying life. The hunt for the past 20 years. We have to change our way

PECIAL various types of noncoding DNA that control gene expression is heating up of thinking.” S

Molecular biologists may have sequenced time and in the right amount. From genes to regulation the human genome, but it’s going to take Most are tackling the functions of At the Medical Research Council’s Lab- molecular cryptographers to crack its com- regulatory elements one at a time, oratory of Molecular in Cambridge, plex code. Genes, keystones to the devel- although a few are taking more global and U.K., Francis Crick—co-discoverer of opment and functioning of all organisms, bioinformatics approaches (see sidebar on DNA’s structure—Sydney Brenner, and their can’t by themselves explain what makes p. 635). At the Institute of colleagues took the first steps toward cows cows and corn corn: The same genes Technology (Caltech) in Pasadena, one figuring out how work. In 1966, have turned up in organisms as different group is trying to identify all the regulatory they proved that genes are written in a three- as, say, mice and jellyfish. Instead, new interactions in maturing embryos; their unit code, each of which specifies a particular findings from a variety of researchers have goal is to elucidate how genes and regulatory amino acid. By combining these threesomes, made clear that it’s the genome’s exquisite DNA work together to guide development called codons, in different ways, the genome control of each gene’s activity—and not and also how those interactions change encodes instructions for thousands of the genes per se—that matters most. over evolutionary time. proteins. This discovery focused the spot- “The of the genetic diversity All this work is making clear that buried light on genes themselves and the coding of animal forms is really due to differences in DNA sequence is a regulatory code akin regions within them; for decades biologists in gene regulation,” says Michael Levine, an to the “but infinitely more called the intervening DNA “junk.” evolutionary biologist at the University of complicated,” says Michael Eisen, a Consequently, the notion of gene regula- California (UC), Berkeley. Turning on a computational biologist at Lawrence tion languished, even when results pointed gene at a different time, or in a new place, Berkeley National Laboratory in California. to its importance. In the early 1970s, Allan or under new circumstances can cause vari- Researchers can predict the proteins Wilson of UC Berkeley and his student, ations in, say, size, coloration, or behavior. specified by the genetic code, but, he adds, Mary Claire King, demonstrated that If the outcome of that new regulatory pat- “we can’t predict gene expression by humans and chimps are quite similar in tern improves an organism’s mating success simply looking at the sequence.” their genes. The key to what makes the two or ability to cope with harsh conditions, Manolis Dermitzakis of the Wellcome species so different, they suggested, lies in then it sets the stage for long-term changes Trust Sanger Institute in Cambridge, U.K., where and when those genes are active. and, possibly, the evolution of new species. agrees: “The complexity of the genome is But not until 2 years ago did experiments Unfortunately, “people don’t look to reg- much higher than we have defined for the begin to bear out this idea. Wolfgang Enard ulatory elements as the cause of the varia- of the Max Planck Institute for Evolutionary tion they see,” says Stephen Proulx of the Anthropology in Leipzig, Germany, and his University of Oregon, Eugene. These ele- colleagues compared the expression of ments are “a major part of the [evolution] many genes in tissues from chimps and story that’s been overlooked,” Levine says. humans. Certain genes are far more active in That neglect is now being righted. the human brain than in the chimp brain, Although many biologists remain gene- they reported in the 15 April 2002 issue of centric, an increasing Science (p. 340), a find- number are trying to fac- ing that supports Wilson tor in the effects of gene and King’s ideas. regulation. Researchers Enard’s 2002 work are beginning to come up came on the heels of with efficient ways to dozens of other studies SCIENCE locate the different regu- suggesting that gene latory regions scattered changes are not the be-all along the strands of and end-all of evolution- DNA. Others are piecing ary innovation. Instead, together the workings of researchers increasingly transcription factors, pro- attribute innovation to a teins that interact with variety of types of regula- regulatory DNA and with tory DNA, some just re- each other to guide gene cently detected. Certain activity. They are homing genes code for the pro- in on exactly where tran- teins that make up the scription factors operate transcription machinery, along the DNA and what Model organism. Fruit flies have played a critical role in the search for stretches of which binds to promot- they do to ensure that a regulatory DNA called enhancers, which control gene expression by binding to one or ers, the DNA right at the

gene turns on at the right more transcription factors. beginning of a coding se- (TOP): CREDITS RESEARCHERS INC., PHOTO 2002; N. (BOTTOM) KEVITIYAGALA/

632 22 OCTOBER 2004 VOL 306 SCIENCE www.sciencemag.org Published by AAAS G ENES IN ACTION S PECIAL Hot on the trail dozen of what seem to be complex enhancers; To understand the role of recently the count has more than doubled. enhancers in development, And in pinning down these enhancers, the

Levine is studying their archi- researchers uncovered almost 50 genes that S tecture and function in the fruit seem to be controlled by this same set of tran- ECTION fly genome. The first challenge scription factors and thus are likely to work he encountered was simply together to guide early development finding the elusive quarry: So far, the researchers have confirmed Several years ago he encour- that at least some of these newly identified aged his graduate student clusters really are enhancers by testing their Michele Markstein (and her activity in transgenic fruit flies. They add computer-savvy parents) to DNA consisting of the putative enhancer write a computer program that and a marker gene. If the marker gene could begin to do just that. shows up in the same place as the The trio worked first with enhancer’s target gene, then the researchers a , dorsal, know they have got what they want. These which was known to affect a data are showing that when several gene called zen. They already enhancers have a similar binding site knew that the enhancer for composition, they tend to work together and zen contained four binding coordinate the expression of sets of genes. Genome cryptography. The regulatory code is encoded in the sites for dorsal, packed close arrangement of an enhancer’s DNA binding sites (A), in the together. The researchers used Enhancer encryption spacing between binding sites (B), or by the loss or gain of one that signature sequence as With the first enhancers in hand, Levine and or more of these sites (C). a probe for finding other his colleagues were ready to take the next enhancers that also had step: to figure out how enhancers orches- quence. Other genes code for transcription clusters of dorsal binding sites. trate development and effect the changes factors that can be located anywhere in the The method worked. Proof positive came underlying evolution. They began to dissect genome. All affect their target genes by at- when the program pinpointed three previously the architecture of these bits of sequence, taching to regulatory DNA—sometimes identified enhancers that control other genes. determining exactly where the transcription called modules—that’s usually near but not It also turned up a dozen more clusters factor dorsal attached and whether those next to a gene. Protein-laden modules that containing three or four of dorsal’s binding locations had anything to do with the stimulate gene activity are called en- sites. The researchers have since shown that enhancer’s function. They also tracked down hancers; those that dampen activity are at least two are definitely enhancers. Levine transcription factors that interacted with the called silencers. is encouraged: “Sometimes the clustering of same enhancers as dorsal. As a plethora of meetings and research a single factor’s binding sites is sufficient to Through these efforts, Levine and UC reports suggests, enhancers are hot. They find new enhancers,” he says. Indeed, using a Berkeley collaborator Albert Erives have are small genetic command centers, similar strategy, Eisen identified a set of been able to decipher another layer of consisting of stretches of 500 or so bases. enhancers responsible for anterior-posterior “code” scattered in the arrangement of Those clusters in turn are peppered with development in the fruit fly. The groups binding sites within the enhancer. This code transcription factor binding sites, which published their results 2 years ago. can be less than 10 bases long. The target That same year, Eisen, Levine, of a particular enhancer—and its effect— and UC Berkeley’s Benjamin depends on the spacing and order of the Berman teamed up to use this binding sites within it. approach, along with other Sometimes the enhancer simply contains bioinformatics tools, to look for

; (BOX WITHIN) PHOTO RESEARCHERS INC.,;WITHIN) PHOTO (BOX 2002 multiple copies of the same binding site and more complex enhancers. Instead

PNAS therefore uses the same transcription factor of containing repeated binding throughout its length. Other times, it has sites for the same transcription multiple transcription factors, and slight factor, complex enhancers contain differences among these proteins can cause binding sites for several different one gene to turn on and another to turn off. factors, thereby providing precise Both enhancers and silencers are regulation of gene expression. especially hard to find because they are To find these sequences, very small pieces of sequence and, unlike Berman and his colleagues looked promoters, reside at varying distances from for pieces of DNA with binding ; (BOTTOM MAIN BOX) ANAND & BELTING/ ; BOX) MAIN (BOTTOM the gene they regulate. “We have a lot of sites for five transcription factors. potentially short stretches of DNA where They didn’t have a specific SCIENCE the action is and stretches of DNA that enhancer in mind but sought out don’t matter,” Patrick Phillips, an evolutionary DNA with those binding sites developmental biologist at the University sitting close enough together to of Oregon, Eugene, points out. Theorists suggest they formed an enhancer. predict that humans could have as many as The five transcription factors all One gene, many sizes. Enhancers from different species alter 100,000 enhancers and silencers, but fewer affect embryonic genes. Initially, the extent of a Hox gene’s expression (dark stain), variation

CREDITS: N. (TOP) KEVITIYAGALA/ than 100 are known, says Levine. Berman’s program found several that leads to species-specific numbers of thoracic vertebrae.

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is critical to directing patterns of differen- After adding a reporter gene to each sorts of enhancers, pave the way to the tiation in embryonic tissue, they reported enhancer so they could see where it was different developmental pathways that

ECTION in the 16 March Proceedings of the active, Shashikant’s group inserted the com- make each species distinctive. Five years

S National Academy of Sciences. bination into mouse embryos. Then they ago, he and his colleagues embarked on an Dorsal, whose concentration in the compared the pattern of expression generated ambitious project to map all the genetic embryo is highest on the ventral side and with the zebrafish and puffer fish enhancers interactions necessary to build the embry- decreases toward the dorsal, is key in defin- to that of the mouse enhancer. In all three onic sea urchin’s endomesoderm, cells that PECIAL ing these regions. Gene activity varies along cases, the activity of the gene was restricted are precursors to the skeleton, gut, and S the dorsal-ventral axis, leading to the differ- to the back part of the developing spine. other tissues. They worked gene by gene, entiation of tissue types. High amounts of Shashikant suspects that the subtle differ- determining the expression pattern of each dorsal lead to mesoderm, a precursor to ences among the enhancers in each species and deducing their functions by knocking muscle; low amounts stimulate the develop- changes the physical boundaries of Hoxc8 them out in developing embryos. From ment of precursor nervous system tissue. expression within the embryo, thereby help- these data and more, they pieced together a When the researchers probed more ing explain why chickens wind up with computer model of the 50-gene circuitry closely into the enhancer, they found that a seven thoracic vertebrae and snakes about controlling the embryo’s first 30 hours as binding site for dorsal was always next to the 200, he says. Shashikant is also looking at an initial glob of cells began to differentiate binding site for a transcription factor called the sequences of this enhancer in other into endomesoderm. twist. Levine found that the close proximity species, including whales and coelacanths, The circuit is a hairball of proteins such and specific order of the binding sites were as transcription factors and signaling conserved in an equivalent enhancer in molecules, and their genes, all mosquito and a different fruit fly: “They connected by means of regulatory were not randomly arranged,” Levine says. DNA into multiple feedback loops. These results suggested that the effect Multiple transcription factors partner of an enhancer on a gene is determined with an enhancer to control the activity not just by the combination of transcription of other transcription factors. Thus, factors but also by the spacing between even though the circuit involves just a the binding sites. Levine thinks that dorsal few cell types, “it’s a very complex and twist have to be quite close together network,” says Davidson’s Caltech for the enhancer to work dorsally, where colleague Veronica Hinman. concentrations of dorsal are low; when far Hinman and Davidson have now apart, much more dorsal is needed. Thus it taken the next step: elucidating the seems that the genome can use the same role of gene regulation in helping to subset of transcription factors to regulate define developmental differences in different genes simply by changing the two echinoderms. Hinman has been order or spacing of those proteins, or working out the same genetic circuitry where they bind along the enhancer. With in a starfish. Whereas the starfish and this work, “Levine has gotten inside the the sea urchin shared a common mind of enhancers,” says Eisen. ancestor about 500 million years ago and still have similar embryos, the Enhancers and evolution two species have long since Alterations in the order and spacing of gone their separate ways. binding sites can also affect how the same Adult sea urchins look gene works across several species, new like pincushions, with research is finding, suggest- rounded exoskeletons; ing that it might take only Enhancing differences. Sea urchins and starfish are flat, a relatively simple re- starfish share much of their embryonic with arms protruding arrangement to change an genetic circuitry, but their enhancers can from a central cavity. enhancer and affect evolu- vary, altering developmental pathways. Hinman has focused tion. At a meeting on devel- on the earliest moments opmental diversity held in April in Cold and again he has found of the starfish’s life. Spring Harbor, New York, developmental changes that probably Despite the differences in biologist Cooduvalli Shashikant of Penn- help define each organism’s the adults, much of the sylvania State University, University Park, shape. Sometimes they are sim- embryonic genetic circuitry described his survey of enhancer effects on a ple sequence changes. Other times, studied so far is almost identical in gene called Hoxc8. This gene, found in missing or additional DNA alters the mix of both species, she reported in 2003. many organisms, helps define the number transcription factors involved. Through Yet subtle variations have had a big and shape of thoracic vertebrae. Shashikant evolution, “a lot of tinkering has gone on at impact. For example, there’s a five-gene suspected that the enhancer, rather than the the enhancer level,” Shashikant says. circuit both species share. A key gene in gene alone, plays a pivotal role in delineating this pathway is otx, and it sets off the different vertebrae configurations among Evolving embryonic differences circuit in the sea urchin and the starfish. species. To find out, he and his colleagues Similarly, Eric Davidson, a developmental Hinman has found a tiny change in this analyzed the sequence of the 200-base-pair gene regulation biologist at Caltech, has enhancer: Between the two species, this enhancer that lays just upstream of Hoxc8 in found that a small change in one enhancer’s enhancer varies by just one binding site,

zebrafish, puffer fish, and mice. structure, and likely many alterations in all for a transcription factor called t-brain. CREDITS: PHILLIP COLLA, (TOP) OCEANLIGHT.COM; JOSEPH DOUGHERTY/ECOLOGY.ORG (BOTTOM)

634 22 OCTOBER 2004 VOL 306 SCIENCE www.sciencemag.org Published by AAAS G ENES IN ACTION S PECIAL A Fast and Furious Hunt for Marcelo Nobrega of Lawrence Berkeley National Laboratory (LBNL) in California. If the animals are too closely related, their genomes will Gene Regulators share many noncoding sequences that have no connection to regula-

tion. If the organisms are too distant, then even functional regions, S Genes may be essential, but researchers increasingly recognize the including regulatory regions, will be too different to detect. ECTION pivotal role that another element of the genome—regulatory DNA— Nobrega’s LBNL colleague Len Pennacchio thinks he has the plays in human disease, speciation, and evolution. In many labs, the answer. When his team compared the human genome to that of a search to find where these regions are buried is intensifying (see main puffer fish, they came up with a whopping 2500 potential text).While some researchers are tackling these regions one at a time, enhancers. To test the effectiveness of this method, the LBNL team others are experimenting with high- has been inserting 100 of the putative speed methods to detect regulatory enhancers, identified on human regions, such as enhancers, en masse chromosome 16, into transgenic mouse and determine what each one does. embryos, which they then analyze for At the Whitehead Institute for signs of regulatory activity. At a meeting Biomedical Research in Cambridge, in Cold Spring Harbor, New York, in May, Massachusetts, Richard Young and Pennacchio reported that 48 of the 60 Ernest Fraenkel are using microarrays to enhancers tested to date were real. analyze yeast in an effort to turn up all But Ewan Birney and his colleagues its promoters, the sites at the beginning at the European Bioinformatics Institute of a gene that bind to activating (EBI) in Hinxton, U.K., and the European proteins. The team starts by treating a Molecular Biology Laboratory in cell and one of its transcription factors Heidelberg, Germany, worry that so that the factor permanently sticks to comparisons alone will yield spurious its DNA-binding sites, thereby tagging matches as well as valid ones. “If you all the promoters that use this factor. Testing, testing. High-throughput screens for enhancers look at conservation itself, it doesn’t tell Then they use that transcription factor can turn up false positives, which can be excluded by you a great deal,” says Birney. to isolate these sequences from the rest looking for enhancer function in mouse embryos. To refine the comparative approach, of the genome. After filling a microarray his team has written a program that with pieces of yeast DNA whose positions on the genome are known, considers only short sequences that are conserved between human they add the tagged DNA, which then links to its matching sequence and mouse—and only when they are present in higher than usual in the microarray, revealing the approximate location of each piece. densities in front of a gene. EBI’s computers divide human and mouse The computer program takes over from there, says Fraenkel, genomes into small pieces, say, six or eight bases, then compare them. refining the location of the binding sites using similar DNA from In the initial experiments, the researchers asked their computers other organisms as a guide. In this way the team has been able to to pick out a well-known piece of regulatory DNA called the TATA pinpoint the promoter-binding sites for each individual transcription box, which is required for the activation of many genes.The program factor in yeast simultaneously. Now, Young and Fraenkel are using did just fine, says Birney, which gives him hope that it will also be this technique to find enhancers and regulatory DNA in organisms able to pin down other regulatory regions elsewhere in the genome. that have more complex genomes than yeast’s. Already they have Birney eventually hopes to tie these data in with forthcoming cornered enhancers on both human liver and pancreatic cells. results from other labs suggesting that gene regulation is controlled Others are also using comparative genomics techniques to fish by other aspects of the genome as well—such as how chromosomes out regulatory regions. The idea is straightforward enough: Compare are twisted around various proteins. And that, he hopes, will enable two or more sequenced genomes to identify those places where DNA him to begin to address “how all this is put together as a code” that outside genes is highly similar and presumably functional. The chal- researchers can use to decipher the true workings of the genome. lenge lies in deciding which genomes to compare, explains postdoc –E.P.

The starfish has this binding site; the sea points out. “The evolution of body plans hap- elements, including enhancers and silencers, urchin does not. In the sea urchin, t-brain pens by changes in the network architecture.” are as important, if not more important, than works in concert with other regulatory gene mutations in introducing genetic inno- genes and sets off the embryo’s skeleton- Enhancing genome studies vations that may set the stage for speciation. forming circuitry, a genetic pathway absent Through these kinds of studies, researchers Changes in one type of regulatory element, in the starfish embryo. But because the otx are beginning to decode the regulatory the transcription factors, can be quite detri- enhancer is missing t-brain, the sea urchin genome. If this code can be made clear, they mental, as each influences many genes. By must also rely on a different transcription should be able to piece together how organ- contrast, enhancer mutations work locally, factor to get the otx gene in the five-gene isms diversify, says Nipam Patel, a biologist affecting just their target genes, and so are circuitry to kick in. at UC Berkeley. For example, through less likely to have deleterious effects on the Meanwhile the t-brain binding site on Davidson and his colleagues’ thorough rest of the genome. the starfish’s otx enhancer keeps otx descriptions of the gene pathways guiding Yet Ludwig and others are the first to focused on genes for the incipient gut. development, they can pin down where admit that they have not cracked this reg- Davidson thinks that ancestral echino- enhancer modules have been added or lost. ulatory code. “We need to learn what it is derms had a t-brain site on the enhancer That understanding, in turn, is changing how and how this information is written in these for otx, one that disappeared from that some researchers make sense of evolution, sequences,” says Eisen. “At this moment, enhancer in the sea urchin. “This looks adds Michael Ludwig of the University of we still do not have that ability.”

CREDIT:LABORATORY BERKELEY NATIONAL SHOUKRY/LAWRENCE MALAK like species-specific jury-rigging,” he Chicago. It’s a vision in which regulatory –ELIZABETH PENNISI

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