A Technology That Fits the Genome Like a Glove
By Jason Socrates
Bardi
People with thick hair know that one of the best ways to
comb it is with their fingers, but could it also be the best
way of combing the gene-thick strings of the human genome?
Combing the genome for genes that underlie biology and disease
is a hot topic now that the human genome has been solved,
with the final draft to be published next month. Now three
scientists from The Scripps Research Institute (TSRI) are
proposing that one of the easiest ways to identify genes and
explore genetic pathways in the genome is to, well, run their
fingers through it.
Research Associates Pilar Blancafort and Laurent Magnenat
and Professor Carlos Barbas III, who holds the Janet and Keith
Kellogg II Chair in Molecular Biology at TSRI, have published
a paper in this month's Nature Biotechnology that describes
a new technique for looking for genes with a combinatorial
library of zinc finger proteins.
Zinc finger proteins contain particular zinc finger domains
that each specifically bind to a particular three-base-pair
sequence of DNA—a codon. Zinc fingers are a common protein
motif in nature because they bind to DNA. They come in various
shapes and sizes, but they all chelate a zinc ion in their
binding domain, and they all have a long alpha helix that
inserts into the major groove of DNA, making contact with
the bases.
Several years ago, Barbas found he could string several
of these zinc finger domains together into a "hand" to create
a highly selective specificity for a longer, more unique sequence
of DNA. Using phage display and oligonucleotide hairpins (short,
single-strand pieces of DNA that twist into tiny helices to
which the zinc fingers can bind), the Barbas laboratory selected
for zinc fingers that bound to sequences of interest and in
cases where selections failed, they designed the desired protein
domain.
A Gene Regulation Library
One of the sequences of interest are those "regulatory"
regions within genes, where proteins known as transcription
factors bind and turn the genes on or shut them off. By fusing
zinc fingers with other, repressor or activator domains, Barbas
and his colleagues found a way to design transcription factors
to specifically down- and up-regulate genes for which they
knew the regulatory regions.
In a series of reports a few years ago, Barbas demonstrated
the efficacy of using polydactyl zinc finger proteins to bind
to two 18-base-pair sequences in the regulatory regions of
the protooncogenes ERBB-2 and ERBB-3. These two genes are
involved in human cancers, particularly breast and ovarian
cancers, and show increased expression in cancerous cells.
Now Blancafort, Magnenat, and Barbas have extended their
technique to generate a library of zinc finger proteins that
can be used to discover genes and to turn on or off virtually
any gene in the genome.
The team used a combinatorial strategy to generate a library
of nearly 100 million zinc finger protein variants from previously
optimized zinc finger domains into multimodular 3- or 6-zinc
finger proteins, and they have the ability to deliver up to
10 million at a time into cells using a retroviral vector.
Many of the sites where these fingers bind do not lead to
regulation of genes, but with many more zinc fingers than
there are genes in the human genome, they have many more chances
to hit any one particular gene. If one zinc finger does bind
within a promoter region of a gene, it can be linked to a
promoter or repressor protein and become a regulator that
activates or suppresses the gene to which that zinc finger
binds.
In their study, the researchers applied the libraries of
zinc fingers to cells and selected cells for the expression
of surface markers that were up or down regulated by the zinc
fingers. In this way, they were able to select for those that
bound to the regulatory region of the target gene.
To read the article, "Scanning the human genome with combinatorial
transcription factor libraries" by Pilar Blancafort, Laurent
Magnenat, and Carlos F. Barbas III, please look at the March
2003 issue of Nature Biotechnology, page 269, or see:
http://www.nature.com/cgi-taf/DynaPage.taf?file=/nbt/journal/v21/n3/abs/nbt794.html
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