Until recently, the chemical marks littering the DNA inside our cells
like trees dotting a landscape could only be studied one gene at a time. But
new high-throughput DNA sequencing technology has enabled researchers at the
Salk Institute for Biological Studies to map the precise position of these
individual DNA modifications throughout the genome of the plant Arabidopsis
thaliana, and chart its effect on the activity of any of Arabidopsis?
roughly 26,000 genes.
?For a long time the prevailing view held that individual
modifications are not critical,? says Joseph Ecker, Ph.D., a professor in
the Plant Biology laboratory and director of the Salk Institute Genomic
Analysis Laboratory. ?The genomes of higher eukaryotes are peppered with
modifications but unless you can take a detailed look at a large scale there
is no way of knowing whether a particular mark is critical or not.?

The Salk study, which appears today in the online issue of Cell,
paints a detailed picture of a dynamic and ever-changing, yet highly
controlled, epigenome, the layer of genetic control beyond the regulation
inherent in the sequence of the genes themselves.

Being able to study the epigenome in great detail and in its entirety
will provide researchers with a better understanding of plant productivity
and stress resistance, the dynamics of the human genome, stem cells?
capacity to self-renew and how epigenetic factors contribute to the
development of tumors and disease.

Discoveries in recent years made it increasingly clear that there is
far more to genetics than the sequence of building blocks that make up our
genes. Adding molecules such as methyl groups to the backbone of DNA without
altering the letters of the DNA alphabet can change how genes interact with
the cell?s transcribing machinery and hand cells an additional tool to
fine-tune gene expression.

?The goal of our study was to integrate multiple levels of epigenetic
information since we still have a very poor understanding of the genome-wide
regulation of methylation and its effect on the transcriptome,? explains
postdoctoral researcher and co-first author Ryan Lister, Ph.D.

The transcriptome encompasses all RNA copies or transcripts made from
DNA. The bulk of transcripts consists of messenger RNAs, or mRNAs, that
serve as templates for the manufacture of proteins but also includes
regulatory small RNAs, or smRNAs. The latter wield their power over gene
expression by literally cutting short the lives of mRNAs or tagging specific
sequences in the genome for methylation.

But before Lister could start to unravel the multiple layers of
epigenetic regulation that control gene expression, he had to pioneer new
technologies that allowed him to look at genome-wide methylation at
single-base resolution and to sequence the complete transcriptome within a
reasonable timeframe.

Collaborating scientists at the ARC Centre of Excellence in Plant
Energy Biology at the University of Western Australia in Perth developed a
powerful, web-based genome browser, which played a crucial role in unlocking
the information hidden in the massive datasets.

Cells employ a whole army of enzymes that add methyl groups at
specific sites, maintain established patterns or remove undesirable methyl
groups. When Lister and his colleagues compared normal cells with cells
lacking different combination of enzymes they discovered that cells put a
lot of effort in keeping certain areas of the genome methylation-free.

On the flipside, the Salk researchers found that when they knocked out
a whole class of methylases, a different type of methylase would step into
the breach for the missing ones. This finding is relevant for a new class of
cancer drugs that work by changing the methylation pattern in tumor cells.

?You might succeed in removing one type of methylation but end up with
increasing a different type,? says Ecker. ?But very soon we will be able to
look and see what kind of compensatory changes are happening and avoid
unintended consequences.?

Previous studies had found that a subset of smRNAs could direct
methylation enzymes to the region of genomic DNA to which they aligned.
Overlaying genome-wide methylome and smRNA datasets confirmed increased
methylation precisely within the stretch of DNA that matched the sequence of
the smRNA. Conversely, heavily methylated smRNA loci tended to spawn more
smRNAs.

?We looked at a plant genome but our method can be applied to any
system, including humans,? says Lister. Although the human genome is about
20 times bigger than the genome of Arabidopsis ? plant biologists? favorite
model system not least because of its compact genome ? Ecker predicts that
within a year or so, sequencing technology will have advanced far enough to
put the 3 billion base pairs of the human genome and their methyl buddies
within reach.

?This really is just the beginning of unmasking the role of these
powerful epigenetic regulatory mechanisms in eukaryotes,? says Ecker.

This work was supported by grants from the National Science
Foundation, the Department of Energy, the National Institutes of Health and
the Mary K. Chapman Foundation.

Scientists who also contributed to the study include postdoctoral
researcher and co-first author Ronan C. O?Malley, Ph.D., and postdoctoral
researcher Brian D. Gregory, Ph.D., in Ecker?s lab, graduate student and
co-first author Julian Tonti-Filippini and Professor A. Harvey Millar,
Ph.D., both at ARC Centre of Excellence in Plant Energy Biology at the
University of Western Australia, Perth, and professor Charles C. Berry,
Ph.D., in the Department of Family/Preventive Medicine at the University of
California, San Diego.

The Salk Institute for Biological Studies in La Jolla, California, is
an independent nonprofit organization dedicated to fundamental discoveries
in the life sciences, the improvement of human health and the training of
future generations of researchers. Jonas Salk, M.D., whose polio vaccine all
but eradicated the crippling disease poliomyelitis in 1955, opened the
Institute in 1965 with a gift of land from the City of San Diego and the
financial support of the March of Dimes.


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