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Beyond genomics, biologists and engineers decode the next frontier
Posted November 18, 2009; 09:00 a.m.
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team of Princeton biologists and engineers has dramatically improved
the speed and accuracy of measuring an enigmatic set of proteins that
influences almost every aspect of how cells and tissues function. The
new method offers a long-sought tool for studying stem cells, cancer
and other problems of fundamental importance to biology and medicine.
The research allows scientists an unprecedented look at a special class
of proteins called histones, which are at the core of every chromosome
and control the way instructions in DNA are carried out. Despite rapid
progress in understanding the information encoded in DNA and genes,
scientists have achieved much less insight into the so-called "histone
code," which determines why a gene in one cell functions differently
than the same gene in another cell.
"We take a cutting-edge approach to a field that has been using more or less the same techniques for the past 15 years," said Benjamin Garcia, assistant professor of molecular biology, who supervised the experimental aspects of the study.
The technique reduces by a factor of 100 the time it takes to analyze
histones, while requiring far less sample material and achieving much
more nuanced results than existing methods, said Christodoulos Floudas, the Stephen C. Macaleer '63 Professor in Engineering and Applied Science, who oversaw computational aspects of the research.
The researchers published their results in the October issue of
Molecular & Cellular Proteomics. Their paper was selected as a
"must-read" article in Faculty of 1000 Biology, an online journal that
selects the most interesting papers in all biology based on peer
opinions. A second paper detailing the computational part of the
research appeared in Molecular & Cellular Proteomics this month.
Collaborators
on the papers also include postdoctoral researcher Nicolas Young and
graduate student Mariana Plazas-Mayorca of Garcia's group and graduate
students Peter DiMaggio and Richard Baliban of Floudas' group in chemical engineering.
Despite carrying identical DNA, all cells in a body aren't identical --
a cell in the kidney looks and functions very differently from one in
the brain. What makes this specialization possible is a set of
instructions stored outside of genes or DNA -- "epigenetic" information
-- that helps each cell adapt to its context. Key players in this
process are histones, tiny protein spindles that the 6-foot-long DNA
molecule wraps itself around in forming a chromosome.
Scientists have long known that histones acquire a variety of small
chemical decorations -- small molecules attached here and there along
the length of the histone. The type and location of these add-ons can
regulate nearby genes. Single modifications are known to turn genes on
or off, but what happens when multiple modifications occur in
combinations -- the "histone code" -- remains a mystery.
"The ability to understand this phenomenon and control it with great precision would be revolutionary to medicine," said Young.
Distinguishing between various modified forms of a histone has been
challenging because several combinations of different modifications can
have nearly the same mass. Indeed, under conventional tests two
histones with very different functions could appear identical if they
have the same set of modifications but at different locations on the
molecule. Before now, efforts to distinguish such subtle differences
were extremely difficult and time consuming. "We have now developed the
first practical means to do this," said Young.
The Princeton team combined physical, chemical and mathematical
techniques for separating one histone variation from another. First
they passed a mix of various histones through a very thin, long tube
containing a specially designed material that causes different histone
forms to emerge from the tube at different times over a two- to
three-hour period. They then bombarded selected molecules with ions to
break them apart and send a flood of data into a computer program for
high-throughput analysis.
"We may get several thousand sets of such measurements over the course of a single experiment," said DiMaggio.
When seemingly similar histones are broken into small fragments,
differences in the locations of modifications become more apparent. The
computer algorithm -- based on an area of math called integer linear
optimization -- repeatedly compares all the fragments until it produces
a highly accurate list of modifications and their locations.
"To see separation of nearly identical species and identify and
quantify them with high confidence is very exciting," said Young. He
also noted that of the many millions of combinations of modifications
possible only a few hundred actually appear in real human cells. This
observation implies that combinations of these relatively few
modifications form a code that can now be deciphered.
The next step will be to link specific patterns of modifications with
observable changes in cells. For example, when normal cells transform
themselves into cancerous cells, scientists could track the
corresponding changes in the histones. Similarly, scientists could
identify particular histone codes that are required for stem cells to
change into specific tissue types, such as nerve cells or
insulin-producing cells. Understanding and potentially reprogramming
these processes could have important implications for regenerative
medicine, cancer and other diseases.
As a start, the researchers are collaborating with biologists from the
University of California- Los Angeles to identify histone codes
relevant for stem cell behavior. "We've shown we can measure modified
histone forms, but there's so much to do now," said Garcia. "This is
really the beginning of some true biological breakthroughs."
The work was supported by the American Society for Mass Spectrometry,
the National Science Foundation, the National Institutes of Health and
the Environmental Protection Agency. The National Science Foundation
recently awarded a grant of $1.3 million to Floudas, Garcia and Joshua Rabinowitz, associate professor in the Department of Chemistry and the Lewis-Sigler Institute for Integrative Genomics, to develop the techniques into a generalized platform for the analysis of proteins and other biomolecules.
Biologist Benjamin Garcia (left) and chemical engineer Christodoulos Floudas collaborated to develop a fast and sensitive way to analyze proteins called histones, which play a key role in how genes function. Here, they load samples into a mass spectrometer, which separates and sequences hundreds of modified forms of histones. The work ultimately may aid the development of technology for reprogramming cells to fight cancer or to regenerate damaged tissue. (Photo: Frank Wojciechowski)