Summary:
The DNA sequence information in the human
genome is read and interpreted by proteins. Without interactions between
proteins and DNA, life would not exist. My laboratory studies how proteins
are directed to specific parts of the genome, so that they can do their
specific jobs. Using a tool called the DNA microarray, we can determine the
location of thousands of protein-DNA interactions in a single experiment.
Then, we use this data to reconstruct the rules that the protein used to find
out where it was supposed to go. Our studies may lead to better predictions
of where defective proteins that cause cancer bind to the genome.
Research Description
The Lieb laboratory was established in the
summer of 2002 at the University of North Carolina in Chapel Hill. We aim to
understand a very basic unsolved problem in biology: How do proteins that
interact with DNA find their proper targets in living cells? We study this
problem in yeast, a single-celled fungus, and C. elegans, a very
simple roundworm that lives in the soil. C. elegans is not the kind of
worm you may have in mind: it is transparent, about the size of a piece of
lint, and is barely visible with the naked eye.
Why do
we use these simple creatures to study this problem?
Researchers often use simpler organisms, like
yeast, to figure out the details of processes that are fundamental to all
living things. Then these discoveries are tested in more complicated systems,
where the knowledge can be used as the basis for creating drugs or other
therapies to cure disease. The advantage is that yeast cells grow very
quickly and are easy to manipulate in the lab, allowing discoveries to be
made much more rapidly than they would be if human cells were studied first.
We use yeast and worms to study how
DNA-binding proteins called transcription factors find their proper targets
in living cells. Transcription factors control which genes get turned on and
off, when those genes get turned on and off, and in what tissues they get
turned on and off. Using a new research tool called the DNA microarray (see
below), we are able to map all of the sites of interaction between any given
transcription factor and the entire yeast genome (a "genome" is
simply all of the DNA that makes up the chromosomes that are in a cell). We
have begun to uncover some of the rules that proteins use to find their
targets in yeast. For example, we were able to provide conclusive genome-wide
evidence that DNA sequence information alone is not sufficient to direct a
binding event. Even though many regions of the genome may contain a DNA
sequence a transcription factor likes to bind, only sequences that are in
particular parts of the genome are actually bound. We would also like to test
whether the rules that proteins use in yeast also are used in a much more
complicated system, C. elegans (these are the worms). Worms are simple
compared to humans, but are much more complicated than a yeast cell. For
example, worms have muscle, a nervous system, and a gut, all things yeast
don't have. Therefore, worms are an important stepping stone in determining
whether the way transcription factors behave in yeast is similar to the way
they behave in humans.
Why is understanding how transcription
factors find their targets important?
In humans,
cancers occur when mutations in DNA lead to improper regulation of genes that
control cell growth. It is likely that in all cancers, alterations in the
abundance or binding specificity of at least one transcription factor
contributes directly to uncontrolled cell proliferation. As one example, Burkitt’s lymphoma is caused when a transcription
factor called c-myc is turned on in improperly in immune cells, which in turn
leads to inappropriate gene regulation and cancer. Another example is a
protein called CTCF, which is involved in the occurrence of breast, prostate,
and kidney tumors. When CTCF is mutated, the DNA binding spectrum of CTCF
changes such that it no longer bind to its proper targets involved in
regulating cell proliferation, but instead binds other targets. In both
examples, which serve as general illustrations for numerous other cancers,
alterations in the natural range of a transcription factor’s gene
targets are likely major factors in the development of cancer. We anticipate
that our studies of DNA-binding specificity will lead to better predictions
of the targets of human transcription factors that have the potential to
cause cancer, which may ultimately lead to new therapies based on inhibiting
binding to inappropriate targets. Our work may also lay the groundwork for
experiments in mammalian systems to examine how any cancerous state affects
the distribution of DNA-binding proteins, and how that altered distribution
contributes to progression towards malignancy.
What are DNA microarrays?
Traditionally,
biologists have studied one gene at a time, often focusing on a single gene
for their entire career. DNA microarrays allow a single investigator to study
all of the genes in an organism in parallel, in a single experiment.
Microarrays have therefore greatly accelerated the pace of biological
discovery. Even more important than the increase in throughput is our ability
to for the first time obtain a complete picture of how genes act to together,
in groups, to specify biological function. This was impossible before; the analogy
would be trying to understand how a battle was won by studying the behavior
and fate of an individual soldier. Now, we can zoom out and see the movement
and behavior of entire regiments, while retaining the resolution to focus in
on particular soldiers of interest, who may have performed critical
functions.
DNA microarrays
are simply arrays of thousands of discrete DNA sequences, for example PCR
products or oligonucleotides, representing all
6,200 genes in Saccharomyces cerevisiae
(baker's yeast), printed at high density onto a glass slide. A typical DNA
microarray fabricated in our laboratory contains about 20,000 spots of DNA,
which represent all of the genes and regulatory DNA in Saccharomyces
cerevisiae. The spots are about 100 microns in diameter and are spaced at
a center-to-center distance of 175 microns. A home-made
robotic machine produces the DNA microarrays we use. It is housed in
Fordham hall, and was produced from off-the-shelf electronics, motors, and
motion-control software that are used in the semiconductor industry.
Because of the
property of DNA that allows complementary DNA fragments to hybridize to each
other, we can use these arrays to measure the relative levels of specific
nucleic acids in biological samples. For example, we can separately grow
yeast under two different conditions (for example cold and hot), and then
extract mRNA from both yeast cultures. The RNA from each of the cultures can
be labeled with a different fluorescent dye (for example red and green). The
labeled RNAs are then mixed together and hybridized
to the array. By the ratio of fluorescent signal coming from each spot, we
can monitor the relative levels of every mRNA expressed by an organism under
any given condition. The same can be applied to human cells, for example
comparing gene expression from cancerous and non-cancerous tissue. There are
many other uses for microarrays. My primary use of them is for mapping the
position of protein-DNA interactions in the genome.
