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Epigeneticsand methylation
1. Epigenetics and Methylation
Shubhda Roy
Dennis Lloyd Jr.
December 4, 2006
Epigenetics
In Bioinformatics, we often consider the effect of the genetic code: how do we find
genes in the genome; what proteins do the genes code for; what are the shape and
function of those proteins. However, an important and emerging research area is arising
within genetic and bioinformatics study: epigenetics. Epigenetics is the study of gene
expression and suppression, and how these phenomena affect genetic and other
biological processes. Various epigenetic properties may be passed from one generation
to another and may also develop throughout the lifetime of an individual. The
inheritance of epigenetic properties is often referred to as “cell memory”.1
Epigenetics
also describes the important specialization of cells from a common set of stem cells.
Epigenetic changes can last generations and be affected by environmental factors such
as diet, exposure to toxins and the nurturing care of parents. One study found that the
offspring of rats exposed to a fungicide while pregnant had lower sperm counts. While
this did not surprise the researchers, they were amazed to find that after several
generations, the descendants still had lower sperm counts. The epigenetic effects of
fungicide exposure had carried for several generations. But all these changes are
temporary and reversible since they arise from regulatory mechanism and do not cause
a genetic mutation.2
Epigenetic changes bring about a phenotypic change in the cell
functionality but the genotype does not change since the nucleotides remains the same.
Since the time when scientists laid the initial foundations for heredity through DNA, the
concept of epigenetics has been hotly contested. However, new research has given
significant evidence for many types of epigenetic systems existing within the cell. Each
epigenetic system attempts to describe the biological processes at work on gene
expression and how these factors are inherited through cell division and reproduction.
Epigentics determines the cell functionality of the stem cells and the state of the cell is
called epigenome.
Types of Epigenetic Systems1
Gene silencing and promotion may result from variations in RNA transcripts and their
protein products. For example, an active gene may code for a protein product that
regulates that same gene or other genes. During cell division, RNA transcripts and
proteins are transferred to the daughter cells and they continue to regulate the genes of
the copied chromosomes. Currently, research is being conducted to determine how
RNA may be passed from parent to child through the egg and sperm cells.
1
2. Another type of protein epigenetic system works by passing on what are known as
prions. Prions are proteins that have an abnormal structural conformation such that they
cause the abnormal conformation of other proteins. These dysfunctional distorted
proteins are often present in cells due to infection and might be passed during cell
division.
Structural elements of the cell are also inherited through cell division. For example, the
cellular plasma membrane and various organelles are replicated during cell division.
These structures may have properties that yield certain phenotypes that are inherited by
the daughter cells.
DNA takes on its compact form within the nucleus of a cell because it is wrapped
around structural proteins known as histones. The DNA bonds to the histones such that
gene expression only occurs at points where the DNA is capable of unwinding from the
histone, allowing room for transcription. Enzymes within the cell can alter the chemical
bonding properties of the histones, making it more or less difficult for expression of
genes neighboring the histone. This is known as a chromatin-marking system. Much
research is being devoted to understanding how the marking of histones affects gene
expression. It is believed that chromatin-marking may occur primarily during cell division
because the histones are more exposed to the enzymes within the cell at this time.
One project, located at National Human Genome Research Institute, is collecting
sequences of histone proteins. Histone protein sequences are cross-linked to the NCBI
Entrez database and are aligned through multiple sequence alignment using the
CLUSTAL-W and MUSCLE algorithms. Users of the database may enter sequence
fragments and narrow their searches by specifying species and histone type.
One final form of epigenetic inheritance is known as methylation. This epigenetic
system will be the focus of the remainder of this paper.
Methylation
One of the most researched epigenetic systems is the study of methylation. Methylation
is the process of attaching a methyl group (CH3) to the 5-carbon of the pyrimidine ring
of cytosine. This may eventually silence a gene. There are specific places within a DNA
sequence where methylation is likely to occur. These are places where cytosine is
present next to guanine, abbreviated as CpG and referred to as the CG dinucleotide.
This is different from the bond formed by cytosine and guanine in the DNA double-helix.
Methylation at a CpG site is abbreviated as 5mCpG. The ratio of the number of 5mCpG
sites to CpG sites in a sequence is the percent of methylation.
2
3. 3
CpG sites may be methylated or de-methylated, causing genes to be suppressed or
expressed. A change in DNA methylation often effects the growths of certain cancers.
For example, de-methylation of certain genes may cause cells to reproduce
uncontrollably, while methylation of tumor suppressing genes may cause increase in
growth of tumors leading to cancer.1,4
The link between methylation and parental nurturing was studied by researchers
observing methylation of certain genes affecting the hippocampus in the brains of rats.
The study conducted by Michael Meaney and Ian Weaver showed that parental
nurturing by mother rats through licking and grooming their young had significant
impacts on the amount of methylation of a gene that contributes to the growth of the
hippocampus. They were able to measure significant differences in the amount of gene
expression between highly methylated samples taken from nurtured rats and low
methylated samples taken from rats that did not receive adequate nurturing.
Specifically, they were studying methylation on a promoter region for the glucocorticoid
receptor gene which is known to have an effect on reactions to stress. Behavioral
differences are observable in the rats; those that where were nurtured by their mothers
were less skittish due to the secretion of serotonin the happy happy harmone.5
Contrarily rats not nurtured were more nervous if left in a new environment due to
secretion of stress hormones.
DNA Methylation Database
In recent years there has been a growing interest in DNA methylation leading to a
growing amount of data on this topic. To make it easily available for research, a public
database has been established known as DNA Methylation Database or MethDB6
. At
present, this is the only dedicated database which is consistently growing and holds
methylation patterns and profiles. Currently MethDB contains data for 46 species, 160
tissues and 72 phenotypes, resulting from 6667 experiments as of September 4, 2002.7
The submission of data has increased in the last few years providing the genetic
community with a resource to standardize DNA methylation data. This database has
gained popularity and acceptance over the years.
MethDB publishes the method used for each experiment, preventing repetition of an
existing experiment. Earlier methyl patterns were only represented numerically. Now
3
4. pictorial representation in array like formats is available, making it easier to compare
experiments. In the picture below, each row represents an individual test subject from
which samples are taken. Each block represents a sample taken from that individual.
They are color coded to represent healthy and unhealthy tissue. The shading of the
circles within the block represents the percent methylation of the CpG sites within the
sample.
There is a direct correlation between tissue and the phenotypic stage in the case of
DNA methylation as epigenetic markers. Therefore the tool contains short descriptions
for tissues and disease types that are referenced in the database. MethDB allows for
textual and pictorial annotation as shown below:
It also contains an online submission tool to make new additions to the database easier.
Methylation Tools
For new studies, it is important to determine what level of methylation has occurred at
given genomic sites and if there is any correlation between level of methylation and
4
5. disease states. To aid in this analysis, a package of methylation tools is available for
download or use over the Internet (results are automatically e-mailed to the user)8
.
As input, the tools take a series of aligned sequences which may have been aligned
using a tool such as CLUSTAL-W. The sequences used will include one reference
sequence and other sequences that have been obtained using a technique called
bisulfite genomic sequencing. Bisulfite genomic sequencing is a process by which
converts un-methylated cytosine to uracil, leaving methylated cytosine intact. This way,
the sequencing tracks where methylation has occurred. Having appropriately aligned
sequences is critical for getting meaningful results from these tools.
Included in the output are two graphs useful for analyzing methylation at the genomic
site of interest. The first graph, shown below, depicts the alignment of the sequences.
Unmethylated CpG sites are shown in blue whereas methylated sites are shown in red.
If sequences are known to represent phenotypes, this graph can help determine if
methylation is a factor and indicate which methylated sites are important.
Another graph of interest
shows the density distribution of
methylation within the aligned
sequences. The height of this graph
represents the percent methylation
while the color represents the
number of sequences with
that percentage.
Conclusion
Epigenetic information helps in the interpretation of DNA expression and suppression in
each cell. Alongside genetic mutation, it is extremely important in deciphering disease
pathways. In the past, epigenetic theory was not taken seriously due to lack of tools to
make the theory a reality. Today with the tight integration of bioinformatics tools,
genomic databases and millions of experiments, research has increased tremendously.
With modern tools available it is easy to organize, analyze, compare and interpret
massive amounts of data patterns. Epigenetic research is helpful in pharmaceutical and
agricultural industry and for society in general. The concept that environment and
nutrition can alter the development of a fetus and that sometimes these changes last
through generation is breaking new ground on understanding biological processes,
disease and potential treatments. We are beginning to realize the responsibility we have
for taking care of ourselves throughout life as this may affect future generations. In the
future, it may be possible that one could take tailored ‘methyl diets’ to stay healthy for
life. Perhaps this can be a reality once the mystery behind epigenetics and a plethora of
diseases, such cancer, is discovered.
References
5
6. 1
Epigenetics at Wikipedia. Accessed online Nov 26, 2006 at:
http://en.wikipedia.org/wiki/Epigenetics
2
Watters, Ethan. DNA is Not Destiny. Discover. Nov, 2006. pg 33-37; 75.
3
Department of Biological Sciences – Graduate School of Science and School of Science. Osaka
University, Japan. http://www.sci.osaka-u.ac.jp/introduction/eng/biology.html
4
The University of Texas. M. D. Anderson Cancer Center. DNA Methylation in Cancer.
http://www.mdanderson.org/departments/methylation/
5
Weaver, Ian, et al. Epigenetic programming by maternal behavior. Accessed online Nov 26, 2006
at http://www.mindfully.org/Health/2004/Maternal-Epigenetic-Programming1aug04.htm
6
Grunau, Christoph. MethDB DNA Methylation Database. Accessed online Nov 26, 2006 at
http://www.methdb.de/
7
Amoreira, Celine; Hindermann, Winfried, and Grunau, Christoph. An Improved Version of the
DNA Methylation Database (MethDB). Nucleic Acid Research. Accessed online Nov 26,2006 at
http://nar.oxfordjournals.org/cgi/content/full/31/1/75
8
Grunau, Christoph; Schattevoy, Ruben; Mache, Niels; Rosenthal, André. MethTools: a toolbox to
visualize and analyze DNA methylation data. Accessed online Nov 27, 2006 at
http://methdb.igh.cnrs.fr/methtools/
7. 1
Epigenetics at Wikipedia. Accessed online Nov 26, 2006 at:
http://en.wikipedia.org/wiki/Epigenetics
2
Watters, Ethan. DNA is Not Destiny. Discover. Nov, 2006. pg 33-37; 75.
3
Department of Biological Sciences – Graduate School of Science and School of Science. Osaka
University, Japan. http://www.sci.osaka-u.ac.jp/introduction/eng/biology.html
4
The University of Texas. M. D. Anderson Cancer Center. DNA Methylation in Cancer.
http://www.mdanderson.org/departments/methylation/
5
Weaver, Ian, et al. Epigenetic programming by maternal behavior. Accessed online Nov 26, 2006
at http://www.mindfully.org/Health/2004/Maternal-Epigenetic-Programming1aug04.htm
6
Grunau, Christoph. MethDB DNA Methylation Database. Accessed online Nov 26, 2006 at
http://www.methdb.de/
7
Amoreira, Celine; Hindermann, Winfried, and Grunau, Christoph. An Improved Version of the
DNA Methylation Database (MethDB). Nucleic Acid Research. Accessed online Nov 26,2006 at
http://nar.oxfordjournals.org/cgi/content/full/31/1/75
8
Grunau, Christoph; Schattevoy, Ruben; Mache, Niels; Rosenthal, André. MethTools: a toolbox to
visualize and analyze DNA methylation data. Accessed online Nov 27, 2006 at
http://methdb.igh.cnrs.fr/methtools/