The Scientist

A Biochemist by Nature
by Karen HopkinDanny Reinberg has broken down everything from transcription factors to chromatin. Then he builds them back up, and the discoveries come.Danny Reinberg is a biochemist—a hard-core, purebred, columns-in-the-cold-room kind of biochemist. Born in Santiago, Chile, in 1954, Reinberg took his first biochemistry course as an undergraduate at the Catholic University in nearby Valparaiso. “I liked it so much that even though I got an A-minus, I went back and said, ‘You know, I don’t think I have a complete understanding and grasp of biochemistry. I’d like to take the course again.’” The professor, says Reinberg, “looked at me and said, ‘You’re crazy! Nobody wants to take it again.’”
Although his grade actually went down the second time around, Reinberg says, “I loved it.” That same passion for all things biochemical—and for protein purification in particular—has served Reinberg well. Now a Howard Hughes Medical Institute investigator at the New York University School of Medicine, Reinberg made a name for himself in the field of transcription by isolating the “factors that get together to form the monstrous apparatus that transcribes protein-coding genes,” says Rick Young of the Whitehead Institute. “Because of the sheer number and complexity of these components, purifying them and characterizing their activities was a biochemical tour de force.” Reinberg has since applied that same level of rigor, says Young, “to characterizing the factors that modify chromatin in the vicinity of the transcription apparatus and thereby contribute to gene regulation.” “I think Danny is one of the best people working in the chromatin/transcription field—and it’s a pretty big field with a lot of very good people,” adds Bob Tjian of the University of California, Berkeley. “He’s just a really good protein biochemist who knows how to purify proteins. That was—and is—a big advantage if you want to get down to the mechanistic aspects of complicated reactions and complex molecular machinery.”DannyTheScientist
ENTER THE COLD ROOM
Reinberg learned his way around a fraction collector in the late 1970s working with Jerry Hurwitz, who was then at the Albert Einstein College of Medicine. On the recommendation of Arturo Yudelevich—his undergraduate advisor, who’d earned his PhD with Hurwitz—Reinberg started in the lab the summer after finishing his studies in Valparaiso. “Summer in Chile, you close the lab, the universities close. It’s two months I would have been doing nothing,” says Reinberg. He immediately felt at home. “Einstein was like a very large family. Everyone’s door was always open. I absolutely loved it.” Six months after his summer stint, Reinberg returned to the Hurwitz lab as a graduate student, studying DNA replication in phage Phi X174. “I was fascinated by isolating proteins and then being able to take all these proteins, put them together, and see that you can take one molecule of DNA and make many molecules of DNA. How you could replicate genetic material,” says Reinberg. That same approach characterized Reinberg’s work on purifying transcription factors, which he began as a postdoc in Bob Roeder’s lab at Rockefeller University in 1983. “Deconstructing a system and then reconstructing it, that’s Danny’s specialty,” says Shelley Berger of the Wistar Institute. “He’s been able to do that over the years, first with the basal transcriptional machinery, then with chromatin. It’s not easy to do, but I think he’s exceptionally good at it.”
“This is not simple cloning,” notes Hurwitz. “This is identifying an activity, and then fishing out from these partially purified components that you yank out of a cell the protein that’s doing what you’re interested in. There are not many people who can do that. That’s what he does and he does it very well.” Using this brute-force approach, Reinberg “pieced together the basal transcription apparatus factor by factor,” says Berger. To transcribe a protein-coding gene, RNA polymerase must recognize the promoter and become part of an active initiation complex before it can produce an mRNA. “Danny was instrumental in figuring out how the basal factors associated with the polymerase help it to carry out those steps. It was beautiful biochemistry, really quite a magnificent job.” With Reinberg’s help, says Jim Kadonaga of UCSD, “we went from having a somewhat nebulous mixture of factors that we knew carried out transcription to having a much better idea of the specific factors”—transcriptional accomplices such as TFIIA, TFIIB, TFIID, TFIIE, and TFIIF. “He took us from extracts and crude fractions to factors. And that work holds up to this day.”
FROM CHALLENGES TO CHROMATIN
It wasn’t all smooth sailing for Reinberg. “I killed myself, working day and night,” he says of his two years at Rockefeller. Though Roeder was extremely supportive, the atmosphere there left Reinberg depressed. “I have never met so many weird people in my life as in that lab,” he says. “They made my life miserable.” One even accused him of faking his data—a claim that Roeder was obligated to check up on by dispatching another senior researcher to a competitor’s lab to see whether Reinberg’s results jibed with theirs. At the time Reinberg was working on purifying TFIIB, which he’d found to be a 33 kD polypeptide. Pierre Chambon and his colleagues in Strasbourg, who were hunting the same protein, had also pulled down a polypeptide with a mass of around 33 kD. That bit of news got Reinberg off the hook. But he was lucky. It turned out the French group had purified the wrong protein—one that by coincidence had the same molecular weight as Reinberg’s TFIIB prep. “Had this person come from Europe and told Bob that what Chambon had was 40 or 50 or 60 kD, I don’t know what he would have done,” says Reinberg. “Probably my career would have been terminated.”Instead, Reinberg was able to focus his attention on reconstituting transcription in vitro, using all the factors that bring eukaryotic RNA polymerase to the promoter—a problem that occupied him well into the 1990s, by which time Reinberg was well established as an independent investigator at the University of Medicine and Dentistry of New Jersey (UMDNJ) in Piscataway, where he set up his lab in 1986. Once he’d helped to define the factors that allow RNA polymerase to dock to a promoter and then shove off, “the next step was regulation,” Reinberg says. And that involved working with chromatin. “Chromatin is the natural state of DNA in the nucleus, so in a sense it didn’t take high level of thinking to come to the conclusion that studying transcription in the context of chromatin might be important,” says Kadonaga. “But from a social point of view, I think people in the field just didn’t like chromatin—like they wouldn’t like some type of music or some type of art. Danny wasn’t one of those people. Danny was open-minded.” “I give Danny a lot of credit for being one of the first people to utilize chromatin templates in in vitro transcription reactions,” says UMDNJ’s Michael Hampsey, a colleague and collaborator. “There were many people working in the transcription field who knew it had to be done. But because they were not chromatin people, they just shied away from developing that system. I don’t think Danny ever doubted he could set it up in a timely manner and make significant strides. And indeed that’s what he did.” After sending one of his postdocs, George Orphanides, to Kadonaga’s lab to learn how to prepare chromatin, Reinberg and his colleagues discovered FACT: a protein that dismantles and then reassembles the core histone proteins, allowing RNA polymerase to transcribe through the nucleosomes that help to package—and regulate the expression of—eukaryotic genes. The first FACT paper appeared in Cell in 1998 and by 2003 FACT had made the cover of Science. “Fast forward to now, this is all the rage: how nucleosomes are removed during the process of transcription,” says Berger. “Danny really led that area.”  “He is very quick to size up a problem and very quick to move in and take advantage of it,” says Hurwitz. “He really knows how to focus, so if he picks up a hint of something he can very quickly bring it to fruition.” That quality makes Reinberg a valued collaborator. “Collaborations with Danny are always productive and fast-paced,” says Hampsey. “You know things will move ahead quickly and you’ll have a manuscript to show for it.”That aggressive curiosity also makes Reinberg a lively addition to any conference. “He’s highly engaged, always sits in the middle of the front row, and he really stimulates honest, straightforward, and often tough discussion,” says Young. “When he’s at a meeting, you know he’s there.” Bob Kingston of the Massachusetts General Hospital agrees. “His passion is sort of legendary. He has strong opinions on a lot of topics and he voices them,” he says. At a recent meeting, for example, Reinberg stood up and criticized his colleagues for using the term “epigenetics” too loosely. Subsequent speakers at that meeting confessed to being scared to use the word at all, but Reinberg’s comments raised an issue that Kingston says needed airing. “The field would be, in my opinion, much poorer without Danny and his brash outspoken statements on any topic you can imagine,” he says. “That combined with his scientific expertise have really made a difference.” Reinberg is now directing some of that expertise into his own exploration of nucleosome modification and epigenetics. In 2002,  he and his team discovered an enzyme called PR-Set7, which methylates lysine 20 on the histone H4 protein and associates with silenced chromosomes in vivo. The researchers have shown that the modification is heritable, because when they inactivate the enzyme and allow the cells to replicate, “we see that the mark gets diluted and eventually disappears,” says Reinberg. That satisfies one of the criteria for being a true epigenetic change. The other would be whether the modification carries information that helps to establish a heritable particular pattern of gene expression, enabling, say, a liver cell to produce progeny that are also liver cells. “Whether the mark is fundamental for cellular identity, time will tell,” says Reinberg. “At this point we have no idea.” Reinberg continues to pursue the question at NYU, where he moved in the fall of 2006 after UMDNJ was paralyzed by financial scandal. “It’s so sad,” says Reinberg. “I was there for 20 years.  People came from all over to make it a great university. Then lousy administrators destroyed all that. It’s going to take another 20 years—and a lot of money—to rebuild that place.” In the meantime, he says, “moving [to NYU] has been the best thing I’ve ever done. I love it.” And being in the city suits him. “Danny may be from Chile, but he’s a New Yorker,” says Hampsey. Kadonaga agrees. “It’s a fast-moving place. There’s a lot going on, a lot of energy. It kind of reflects Danny.This article is reprinted with permission from the January 2008 edition of ©2008 The Scientist, LLC. All rights reserved. Further duplication without permission is prohibited.