**Executive Summary of the Epigenetic Mechanisms in Cancer Think Tank**

The purpose of the Epigenetics Think Tank was to bring together experts
from basic and translational sciences to discuss the potential of
epigenetic research to inform cancer biology, diagnostics and
therapeutics, and to make specific recommendations to NCI for action in
this field. The think tank began with brief presentations by each
participant on the first evening. On the second day, three discussion
sessions addressed cancer epigenetics, basic mechanisms in chromatin
structure, and epigenetic technology and epigenomics. A special
presentation was included on the European Epigenomic Network of
Excellence. On the final morning, the panel summarized the discussions
and prioritized recommendations.

The Think Tank highlighted a clear link between epigenetic alterations
and cancer. Epigenetic changes such as DNA methylation and imprinting
are targets of environmental carcinogens and diet, and probably are
responsible for much of the population variation relevant to cancer
risk. The role of hypomethylation in gene activation, aging and
chromosomal instability is an emerging significant issue. The panel was
particularly excited by the apparent role of epigenetic changes in the
earliest stages of carcinogenesis, because these are potentially
reversible with treatment. This provides an epigenetics-based strategy
for cancer prevention. Epigenetic changes also clearly play an important
role in tumor progression, tumor cell heterogeneity and possibly
metastasis. A key need is to map these early changes through the
analysis of methylation and other epigenetic marks in a well-defined set
of reference human normal and tumor tissues. In addition, it is
important to determine the temporal relationship of epigenetic
alterations, as well as their relationship to underlying DNA sequence
variation in the population.

This is an exciting time in the understanding of the mechanisms of
epigenetic transcriptional regulation and chromatin structure. Protein
complexes that manipulate nucleosomes, organize larger chromatin domains
and set boundaries of chromatin structure have been discovered in recent
years; now research must focus on discovering how they work in such
cellular functions as replication, transcription and differentiation.
Crucial next steps are to understand the role of key histone
modifications, cis-acting elements, and regulatory proteins in setting,
maintaining, and reprogramming epigenetic memory. Other key unknowns are
the determinants of higher-order chromatin structure, the function of
micro RNAs or siRNA in mammalian gene regulation and genome stability,
and the mechanism of transmission of epigenetic marks through cell
replication.

The Think Tank concluded that the highest priority should be development
of a U.S. Human Epigenome Project of analogous scope to the Genome
Project. The immediate establishment of a Working Group to define the
criteria and plan the approach to the Epigenome Project is timely and
highly recommended. There was strong consensus that we are now at the
stage at which we can \"genomicize\" epigenetic information. Most of the
necessary technologies are already available, although some tools for
analysis of epigenetic alterations at the genome-wide level are still
needed. New quantitative and computational methods may be required,
including integrating epigenetic information with existing databases.
Mapping epigenetic information to the human genome will maximize its
usefulness and allow us to determine the relationship between genetic
and epigenetic variation in the population, and its relationship to
cancer. Such a project would be immediately worthwhile for its basic
science insights as well as for enabling the use of epigenetic
information in prevention or therapeutic strategies.

**Introduction**

The think tank began on the first evening with brief presentations of
scientific advances, questions and barriers by each participant. The
following day, three discussion sessions addressed Human Cancer
Epigenetics; Basic Mechanisms in Chromatin Structure and Epigenomics;
and Technology and Translation. There was also a presentation on the
European Epigenome Program. On the final morning, the discussions were
summarized, and the participants created a prioritized list of major and
minor recommendations to NCI which will best advance the science.

In each session, the discussion was organized around a set of major
questions. The panel began with these questions, and proceeded to
discuss the issues and most critical next steps, and concluded each
discussion with the next crucial questions to be answered and needs for
the field to progress.

**Human Cancer Epigenetics**

*Mechanisms of cancer initiation and progression by epigenetic events*

Epigenetic changes are modifications to the genome that are heritable
during cell division but do not involve change in DNA sequence.
Expression of individual genes, of gene regions or a whole
transcriptional program are regulated not by the DNA sequence, which is
the same in every cell, but by the epigenetic marking and packaging that
regulates chromatin structure. DNA methylation, histone variants and
post-translational modifications, nucleosome positioning factors,
boundary setting elements, and chromatin loop and domain organizer
complexes are all elements of the epigenome.

Alterations in methylation, imprinting and chromatin are ubiquitous in
cancer. A clear link between epigenetic changes and cancer, as well as
other diseases, has in many case been established. And beyond these
specific instances, epigenetics has the potential to explain many
aspects of cancer: these marks can be modified by environmental factors;
the age dependance of cancer corresponds to epigenetic changes in aging;
and the quantitative nature of epigenetic changes corresponds to changes
seen in cancer. The frequency of epigenetic alterations is orders of
magnitude greater than that of genetic mutation, but unlike mutation,
they are reversible.

The most frequently identified epigenetic alterations in cancer are
changes in DNA methylation, although others have been identified as
well. DNA methylation at CpG sequences (frequently in stretches called
"islands") is a basic marker of the genome that appears to have several
functions. Methylation of gene regulatory regions has long been
associated with transcriptional inactivation, and differences in
methylation pattern in the promoter regions of tumor suppressor genes
are documented in some cancers (e.g., p16/INK4a in pancreatic cancer).
Hypomethylation is thought to either permit gene expression where it
should not occur, or to encourage chromosome instability, especially
when it occurs in the centrosome region. In general, global
hypomethylation with local hypermethylation of CpG sequences is
associated with cancer, although this concept may eventually be revised.

Two well described instances of epigenetic changes involved in cancer
were used to illustrate different epigenetic mechanisms of cancer
initiation throughout the meeting. Both involve loss of imprinting
(LOI), which is DNA methylation that silences specific genes on either
the maternal or paternal allele lineage. In Wilms' tumor, approximately
95% of cases do not follow the two-hit model as it has been understood,
with an inherited mutation in one allele of a tumor suppressor followed
by a mutation in the other allele; instead, the second hit usually is
LOI that activates the IGF2 gene. In colon cancer, IGF2 is also
frequently activated, but another mechanism is involved, in which
genetic and epigenetic mechanisms appear to be complementary. The LOI
seen in these tumors also exists in the surrounding normal tissue and
appears to predate the tumor; the theory is that this "field effect"
renders the mucosa highly susceptible to subsequent insults, such that
the overexpression of IGF2 (or other growth factors) due to LOI renders
mutations such as APC mitogenic rather than apoptotic.

While the link between epigenetic changes and cancer is clear, the
mechanisms behind the abnormal changes -- indeed, the mechanisms by
which normal patterns are established and maintained -- at this time are
largely unknown. The relationship between cause and effect is also
controversial; while histone acetylation and DNA methylation are
permissive for transcription, there is conflicting evidence as to
whether these marks are the signals for gene expression, or if they
follow the passage of the polymerase and serve to reinforce the "active"
gene structure. DNA methylation differences at specific loci exist in
Wilms' tumor and Beckwith-Wiedemann syndrome, but a mosaic of
methylation states is observed in the population of tumor cells. This
raises the question of the degree to which the methylation pattern is a
cause of the malignant growth. Because normal methylation patterns are
not well defined, it is difficult to determine what is abnormal in
cancer. In spite of the widely documented link between methylation
changes and cancer, none of the known methyltransferase genes are known
oncogenes or tumor suppressors. Thus, several crucial questions remain
to be answered for methylation to be optimally used in cancer diagnosis
or treatment.

A fascinating current issue is whether there are sequence variations in
the human genome that create susceptibility differences by predisposing
the site to epigenetic changes. Noncoding DNA such as repetitive
elements and transposons may play a role in genome-wide methylation
patterns. These elements are the main sites of CpG methylation in the
genome. The example discussed by one think tank participant was that of
a transposed element that altered the methylation pattern at a gene
locus in a mouse model in a manner that responded to dietary factors. In
the offspring, the transposon could be differently methylated depending
on the maternal diet, with a higher methylation state leading to an
obesity phenotype, demonstrating a means by which disease states could
have an underlying genetic/epigenetic basis that responds to
environmental factors. In this example, a genetic event led to the
possibility of stochastic epigenetic changes. There is currently no
known human locus where methylation variation leads to trait or
susceptibility variation, or where a transposable element creates a site
of potential expression variation; however, the likelihood was thought
to be high that such examples will be found.

Histone modification, especially acetylation, has long been recognized
to influence transcriptional capacity, but we must now begin to
understand how DNA methylation and histone modifications have
coordinated effects. A self-reinforcing cycle between these two
epigenetic marks appears to exist, such that transcriptionally active or
inactive regions of chromatin (i.e., euchromatin or heterochromatin) are
self perpetuating through replication. The most important next step will
be to understand the degree to which changes in these epigenetic marks
are the causative regulatory factor in chromatin function changes, or
whether other complexes play the regulatory role and these marks play a
mainly reinforcing or maintenance role. This issue was the focus of some
debate, since different results have shown these marks to play both an
active and a passive role. Histone acetylation, for example, has been
shown to cause transcriptional activity and also to result from the
passage of the polymerase complex, thus being self-reinforcing, while
loss of single specific histone acetylations can cause the spread of
heterochromatin over a gene region. The recently proposed histone "code"
of modifications was challenged by some panel members because such
questions of cause and effect cannot yet be answered.

While DNA methylation of specific genes or general pattern changes are
the most widely reported epigenetic signature in cancer, a cancer
connection with chromatin organization is also well documented. SNF5, a
subunit of the SWI/SNF chromatin remodeling complex family, is deleted
in the aggressive pediatric cancer rhabdosarcoma. It acts as a tumor
suppressor, activating expression of p16/INK4a and repressing several
cyclins and CD44, a cell surface glycoprotein implicated in metastasis.
Another example is that of the tissue-specific BUR (base unwinding
region)-binding protein SATB1, which appears to organize chromatin loop
domains and act as a positioning factor that attaches genes to Matrix
Attachment Regions (MARs), giving them the proper nuclear localization
for transcriptional activation. SATB1 specifically binds to the major
break region in the characteristic chromosomal translocation of some
lymphomas, creating the MAR which is thought to be a susceptible site.
It will be interesting to search for similar proteins specific to other
cell types and determine their expression or activity in cancer cells,
since SATB1 has the ability to cause global changes in gene expression
pattern.

Several specific needs were identified to advance our understanding of
the epigenetic mechanisms functioning in cancer. It will be important to
define genetic-epigenetic relationships; e.g., to identify mutations
that lead to epigenetic changes. It will also be necessary to determine
the relationships among such transacting modifiers of the epigenome as
methyltransferases, transcription factors and genome organizers, as well
as identify the factors and/or pathways that are activated in
conjunction with these relationships. Another significant area in need
of investigation is the effects and dynamics of epigenetic modifications
of repetitive elements, transposons, pseudogenes, and novel transcripts.
New insight into the organization and functioning of transcriptional
networks and pathways may be gained from understanding principles of
chromatin organization. New experimental model systems (e.g., to study
RNAi; global CpG methylation and hypomethylation in cancer; pseudogenes
in heterochromatin origins; and histone acetylation/deacetylation) are
needed to study these features. Finally, it will be important to
identify and develop applications for reagents for site-specific
modifications of histones, such as acetylation-site-specific antibodies,
and identify ways to decrease cost and increase availability of these
reagents.

*Interplay of epigenetics and cancer B where does epigenetics fit in the
big picture of initiation and progression?*

Despite the cancer associations highlighted above, the question remains
of how frequently epigenetic gene activation or silencing is a "first
cause" event that initiates dysplasia or creates susceptibility.
Depending on the cancer, the first hit may be a mutation followed by
epigenetic changes leading to a functional second hit, or abnormal or
age-related epigenetic changes may create conditions that enhance
environmental assaults or release suppression of tumor progression. Once
the primary tumor is under way, the many steps by which a motile,
metastatic phenotype emerges are likely to include epigenetic changes in
gene regulation. Tumor cells generally must obtain a motile, spindle
phenotype while traveling to the new site, then change phenotype again
as they become established as a metastatic lesion. Regarded in this
light, the reversibility and changeability of epigenetic states, as
opposed to mutations, is a thought provoking idea. The reexpression of
genes in metastatic cells, and the regulation of some genes involved in
the angiogenic switch, are thought potentially to have an epigenetic
etiology. In all of these questions, the lack of experimental models is
a major barrier. Specifically lacking are models of very early
epigenetic changes or very early stages of disease.

One promising focus for research is the question of epigenetic
methylation changes as a timing event in the path from stem cells to
differentiated cells to tumor cells (i.e., DNA methylation as a
developmental timer). Another area of investigation might involve
examination of epigenetic changes in "normal" cells and cells
predisposed to cancer, to distinguish between normal epigenetic
variation or changes with age, and potentially dysregulating epigenetic
changes leading to disease. The epigenetic status of normal cells
surrounding tumors, including angiogenic, immune, and stromal cells, may
also play a significant role, and is virtually unknown. It will also be
necessary to identify the relationship of epigenetic changes to
chromosome instability and recombination in cancer.

*Comprehensiveness of epigenetic studies of cancer B how much
information do we need?*

It will be important to conduct a genome-scale analysis of domains of
epigenetic change at the local level, in gene groups or clusters, and
globally. A genome-wide screen for DNA methylation changes would avoid
the bias from candidate gene studies; on the other hand, the data set
from a single region would allow us to study all epigenetic features,
and determine how they relate to each other. There were conflicting
opinions on which approach would be most valuable at this time. It will
be necessary to investigate the functional significance of specific
epigenetic marks in order to better understand how to resolve this
question. It is important to determine the relationship between
hypomethylation and gene activation, as there are several examples of
loss of methylation linked to new gene expression and cancer. Before
demethylating agents become widely used to treat cancers, this question
must be addressed. The relationship of epigenetic changes to histone
modifications is also important, and may involve the results of clinical
applications, such as the effects of drugs on gene expression.

It will be highly informative to add epigenetic information to existing
databases of genetic information. Gene regions could be layered with
information on methylation and other marks that relate to
transcriptional activity. However, as discussed in the final section on
Epigenomics, a judicious approach to selecting experimental material and
identifying the regions of chromatin to analyze must be used. To date,
drugs that target DNA methylation and histone acetylation have been used
with some degree of success in the clinic, albeit the more that is
learned about the function of these epigenetic marks the less clear it
becomes how the drugs work.

**Basic Mechanisms in Chromatin Structure**

*Key Players in Chromatin Organization*

A number of molecules with critical roles in chromatin organization,
remodeling and epigenetic modification have been identified in recent
years. However, our understanding of which ones are the key regulators
and how they accomplish their roles in development, in the identity of
different tissues, or during cellular functions such as replication is
far from complete. Further basic research to better understand the
mechanisms of action and determine the regulatory pathways of the known
enzymes and complexes is crucial and timely. At the same time, it is
important to try to broaden the spectrum of known chromatin modifying
enzymes, if we wish to form a comprehensive picture. To understand how
chromatin structure marked by specific epigenetic modification is
established, it is necessary to identify additional players at the
levels of DNA, RNA and proteins, and then go on to identify the dynamic
relationships between them. The role of non-coding RNAs is addressed in
a later section.

There is evidence that DNA methylation/demethylation affects gene
transcription by signaling chromatin structure changes, rather than
simply "blocking" access by polymerases or transcription factors. Thus,
since DNA methylation status is the most widely and readily measured
epigenetic mark, it will be important to determine its role in chromatin
organization. At the same time, it is important to search for long-term
epigenetic marks other than DNA methylation and histone modification.

A major challenge in the near future will be to identify key
determinants of locus-specific chromatin structures; that is, key
histone modification sites and key cis-acting DNA elements (e.g., BURs,
which are recognized by global gene regulators) which have dominant
roles in the formation of specific chromatin structure, as well as the
protein complexes which interact with these sites. To understand how
epigenetic states are established, it will be necessary to identify new
interacting proteins that are involved in chromatin targeting and
remodeling. These partner proteins currently include PCNA, RNA
polymerase, nucleosome assembly factors, CTCF, and loop organizers.
Other factors must be presumed to exist which have yet to be identified;
however, it is not too soon to begin to ask which part of the genome is
regulated by which of the known factors.

The known molecules that regulate accessibility of the DNA to regulatory
factors include DNA methyltransferases, histone variants, and enzymes
that covalently modify histones through acetylation, phosphorylation,
methylation and other chemical moieties. Another class of
chromatin-modifying complex includes those that move nucleosome
positions to reveal or occlude DNA sequence. Three families of these
complexes have been identified -- SWI/SNF, ISW1, and NuRD -- all of
which consist of a core subunit with an ATPase domain, plus associated
subunits presumed to be regulatory. Other chromatin modifiers include
the Methyl-CpG-Binding Domain (MBD) protein family, Position-effect
variegation (PEV)-related factors, and chromatin loop organizers.
Although presumed to exist based on the observed dynamics, neither DNA
demethylases nor histone demethylases have yet been identified.
Discovery of such crucial enzymes will have high impact on understanding
of chromatin structure maintenance and remodeling. Additionally, any of
these enzymes, loop organizers or boundary element-binding proteins may
be subject to post-translational modifications that regulate their
functions, although very little is known at all about such regulation of
these molecules. Many of these proteins can be studied in yeast; for
example, yeast studies have demonstrated that Sir2 histone deacetylase
induces the spread of heterochromatin, while Sas2 acetyltransferase
disrupts heterochromatin, and together, they appear to establish
chromatin boundaries. Efforts are now being directed at understanding
the roles of known higher eukaryote analogs of these enzymes.

*Mechanisms for higher-order folding of chromatin*

To understand chromatin structure and function, it is essential to
expand our knowledge of how chromatin is packaged beyond the nucleosome
structure. Which proteins are responsible for higher-order packaging
(e.g., loop domain organization) and the biological significance of such
chromatin folding are fundamental questions that have only recently been
formally addressed. It will be an important step to identify more loop
organizers in addition to SATB1, in different tissues and at different
developmental stages, and to distinguish between general and specific
proteins. This will require improvement in the highly variable loop
capture assay, and the development of new technologies to analyze
higher-order chromatin structures (e.g., in mega-base domains).

It is necessary to determine the role of loop structures in delineating
functional chromatin domains that are marked by specific epigenetic
modifications. Such an approach, which introduces the third dimension
into epigenetic analysis, will reveal the contribution of higher-order
folding to epigenetic modification and overall genome organization. It
will be important to identify the mechanisms that regulate functions of
loop organizers such as SATB1, and to discover additional analogous
protein complexes. Finally, it will also be important to analyze the
roles of loop structure and loop organizers in cancer and explore their
diagnostic utility. Some observations suggest that the organizer protein
BORIS competes with the related protein CTCF to disrupt boundary
function, and may alter transcription pattern on a large scale when
abnormally expressed in cancer. Additionally, the global organizer SATB1
is aberrantly expressed in some cancers. It will be significant to
explore whether these proteins could be effectively targeted in cancer
therapy.

*The role of RNA in gene regulation and genomic organization*

Non-coding RNA appears to play several roles in chromatin regulation.
Emerging evidence suggests that micro RNAs (miRNA) or siRNAs can
regulate over very large chromatin territories. These small RNAs,
produced by defined processing pathways, are known to silence genes at
the translational and perhaps also the transcriptional level; however, a
role in maintaining heterochromatic regions is currently only
postulated. It will be important to determine the generality of RNAi
control in the genome beyond repetitive elements, and the function of
RNAi in genome organization. Currently it is unknown how many miRNAs are
present in our genome. It is still to be determined how they can be
identified, and how their targets can be validated. Then, moving beyond
quantification of miRNAs, we will have to determine the role of miRNA in
gene expression, where it acts, and what link if any it has to cancer.
Another intriguing question is whether viruses and other pathogens
modify the epigenetic profile of the genome in ways that involve siRNAs
and/or miRNA, leading to dysregulation and potentially diseases such as
cancer.

Intergenic transcription is a novel mechanism by which heterochromatin
may be excluded from transcriptionally active regions. It will be
necessary to determine the link between intergenic transcription and
RNAi. An important question is whether siRNA-based regulatory networks
exist, and what the role and mechanisms of large noncoding RNA
transcripts (e.g., Xist) are in chromatin function.

*Epigenetic inheritance in somatic cells*

This area of chromatin epigenetics research in particular is still in
its early stages and warrants further, extensive investigation. DNA
methylation is initiated de novo by DNA methyltransferases and
maintained by Dnmt1, and this provides a mechanism of epigenetic
inheritance. However, there are epigenetic phenomena in organisms such
as *Drosophila* that lack any significant DNA methylation. Epigenetic
states may be transmitted through specific chromatin structures, such as
histone modifications, but how this is achieved is largely an enigma.
Lysine methylation on histones 3 and 4 provides relatively stable marks
on histones. But, again, how such marks are maintained through the cell
cycle or through development is not known, given that there are
mechanisms for active loss of histone methylation, in addition to
passive loss by dilution at each round of replication. Replication
timing may be a crucial mechanism in setting or preserving the
epigenetic marks, since euchromatin replicates early in the cell cycle,
while heterochromatin replication is delayed. The histone modification
Acode@ has not yet been demonstrated to be heritable. The true function
of histone modification patterns remains to be determined, and there was
disagreement as to whether an active role in determining chromatin
functions has been convincingly demonstrated, as opposed to a more
passive role as a result of replication or transcription.

The question of what epigenetic information is preserved through
replication, as well as the related question of what modifications are
causative of chromatin organization as opposed to simply the more
passive result of replication, were the topics of extensive discussion.
The roles of DNA methylation and histone modifications were discussed in
this light.

A critical next step is to determine the mechanisms of the replication
of specific chromatin structure, identifying the DNA sequences and the
targeting of chromatin remodeling factors involved in setting up
specific chromatin structures during or after DNA replication. Part of
the same effort will be to test the role of histone modifications in
epigenetic inheritance through the cell cycle, and to determine the
dynamics of histone modifications during DNA replication and the cell
cycle. We must deepen our understanding of how DNA methylation relates
to gene expression and chromatin structure.

*Epigenetic remodeling and genome stability*

It will be important to determine the mechanisms of regulating or
reversing the more stable epigenetic marks. For example, DNA methylation
can be either an active or a passive process, as discussed above, and
once methylated DNA demethylation may be via an active or passive
mechanism. Also, when histones are replaced with variants that have
different functions or modification sites, are these mechanisms active
regulatory mechanisms or secondary results of other functions? In any
case, it remains unknown if they have relevance to cancer.

Chromosomal rearrangements are a common feature of a wide variety of
neoplastic lesions and are thought to have a causal role in
tumorigenesis. Genome instability, leading to carcinogenesis, may occur
due to the genome-wide DNA hypomethylation observed in aging. Global
hypomethylation is frequently observed in tumor cells, and has been
associated with abnormal chromosomal structures, as observed in cells
from patients with ICF (Immunodeficiency, Centromeric instability and
Facial abnormalities) syndrome. However, the mechanistic link between
genomic instability and hypomethylated genomic DNA remains to be
discovered.

It will be very important to determine the mechanism for the creation of
neocentromeres. Centromeres are key players in genome integrity, and
defects in their function results in mis-segregation of chromosomes and
aneuploidy. Neocentromeres are ectopic centromeres that originate
occasionally despite the complete absence of normal centromeric
alpha-satellite DNA in the regions. No alteration of DNA sequence
occurs, and it remains unknown how they are created, but they are still
able to form a primary constriction and assemble a functional
kinetochore. Neocentromeres have been detected in certain cancers, and
are thought to be generally detrimental. However, no systematic
screening for these structures has been conducted.

Finally, it will be important to investigate the role of chromosome
territories, and factors that organize these territories, in
recombination and genome instability. Chromatin must be viewed in the
context of whole chromosome territories, organized into specific nuclear
zones. These territories occupy non-random positions in the interphase
nucleus. The mechanisms confining each chromosome in a defined territory
remains unknown, however, as well as the question of whether changes in
the position of the territories occur during differentiation or
carcinogenesis. The specific location of genes within nuclei is
important for proper gene expression, and nuclear architecture
contributes to the localization of genes. Thus, improper positioning of
genes may contribute to their improper regulation, potentially leading
to cancer. Cancer is associated with disruption of gene expression
patterns and global disorganization of chromosome organization within
the nucleus. The roles of nuclear architecture, including chromosome
territories, in genome stability and proper gene regulation must be
studied in depth.

**Epigenomics, Technology, and Translational Opportunities**

We are presently approaching the stage where we can begin to
"genomicize" epigenetic information. At the think tank, much of the
discussion of needed technology and translational opportunities took
place in the context of developing a Human Epigenome Project, analogous
to the Genome Project. The science is clearly developed to a point that
it is timely to plan a strategy for such an effort. The key question is
what information the efforts should focus on obtaining.

There are two decisions that need to be made before launching into a
large-scale effort. First, what epigenetic information should be
gathered and incorporated into the Epigenome? DNA methylation is the
first consideration, and most of the investigators want to make
genome-wide DNA methylation profiling using microarrays the top
priority. After that, other important elements that should and could be
incorporated include unequal allelic gene expression (including
imprinting); histone variants and modifications; chromatin organizer
proteins; and *cis*-acting marks for epigenetic signals. It must be
remembered that different types of epigenetic information present
different challenges. For example, the two fundamental epigenetic marks,
histone modifications and DNA methylation, differ in stability; certain
histone modifications can change quickly (acetylation reversed by
deacetylases, methylation reversed by histone replacement, etc.), while
CpG methylation of DNA is more stable.

Second, what tissue samples should be used? Unlike DNA sequence,
epigenetic marks and states certainly differ between tissues and change
with the age of the donor. A discussion is needed about what we know and
what we need to know, to permit these choices to be made. It would be
useful to ask what relationship exists between diversity in the
population and diversity in epigenetic marks, and what new epigenetic
population-based and family-based algorithms need to be developed.

*Key Issues and Potential Pitfalls*

New technologies are needed to study some questions of higher order
chromatin organization and function, and broader training is needed in
current state-of-the-art procedures such as the loop capture assay or
the ChIP-on-chip assay. Nonetheless, current methodology is sufficient
for conducting genome-wide screens of much of the epigenetic
information. Integration of epigenetic information into existing genome
databases would be optimal. A computer screen that incorporates all
levels of epigenetic information for each stretch of DNA sequence in the
genome was envisioned by some of the panel. In principle, data from
genome-wide or chromosome-wide bisulfite conversion/sequencing, histone
modifications, and mRNA expression profiling, can be overlaid on the
existing human genome sequence, to provide an integrated picture of the
genome and epigenome. This information, with associated annotations,
could be displayed at a resolution ranging from the whole chromosome to
the individual nucleotide in a clickable format, simply by adding a
series of new Ainformation fields@ to available, widely used genomic
databases. With this coordination of data, the project would begin to
provide answers about the potential for epigenetic therapy and how the
epigenome can be targeted for chemotherapy or chemoprevention.

Discussion of precisely what epigenetic marks and what tissues would be
included first did not produce a consensus at this meeting. All agreed
that future organizational meetings would be required to plan an
Epigenome Project. This project should take an approach somewhat like
the Human Genome Project, with a sequencing effort that provided
baseline information on which further genetic variation could be
identified. A comprehensive analysis of DNA methylation is needed,
including quantitative variation in the density and breadth (i.e.
numbers of modified residues) of methylation marks. In addition, novel
bioinformatic approaches must be developed.

In contrast to the DNA sequence, the epigenome varies among different
normal cell types and, perhaps more dramatically, between normal and
cancer cells. So, selection of the Areference cells@ and Areference
tissues@ for analysis in the Epigenome must be done with extreme care.
To deal with the problem of cell heterogeneity, microdissection will be
essential in preparing the samples. An even more fundamental issue
concerns the identification of the closest normal counterpart for any
give human cancer type, i.e. intelligent and meaningful selection of the
Acontrol@ samples for comparing to the cancer samples. Significant
progress is being made in this area, largely through gene expression
profiling and immunophenotyping, and this information needs to be taken
into account in making this critical decision on sample selection. It
would be a disastrous waste of resources to analyze the epigenome in
tissue samples that subsequently prove to be poorly defined and
heterogeneous.

*Feasibility of a Human Epigenome Project*

Despite these issues to be worked out, it was the consensus of the panel
that beginning an Epigenome Project is feasible now. Methods for
determining the pattern of CpG methylation of DNA and corresponding
patterns of histone modifications across the human genome are well
established. Chromatin immunoprecipitation/ChIP-on-chip assays can
rapidly score histone modification, potentially tiled across an entire
genome, and bisulfite conversion/DNA sequencing, as well as several
complementary approaches including microarray-based methods and genome
fractionation using McrBC endonuclease, can achieve the same objective
for DNA methylation. Furthermore, such a Project will be highly
leveraged by the completed Human Genome Project.

The most useful next steps towards beginning a Human Epigenome Project
were discussed. It will be crucial to identify epigenetic markers other
than methylation, and develop high-throughput tools to target and
characterize these markers fully. Also, it will be necessary to identify
new compounds and reagents, and compare these agents against
standardized reference sets. The tissue bank needed for the project must
be designed carefully. It must contain adequate amounts of material
across appropriate populations, and have samples varying in age, tissue
type, and disease state. A significant consideration is how banked
tissue samples should be processed and stored.

A hypothetical organization of a Human Epigenome Project would include
several Core Facilities, as well as a number of Pilot Projects that
would be expected to leverage the epigenetic data in specific research
areas:

[Core Facilities]{.underline}:

DNA Methylation Core

Histone Modification Core

Gene Expression Core

Bioinformatics Core (interfaced with the CaBIG consortium
[http://cabig.nci.nih.gov/caBIG/]{.underline} )

Tissue/Pathology Core

[Pilot Projects including but not restricted to]{.underline}:

Genome Imprinting and Allelic Asymmetry

Interindividual Variation in Epigenetic Marks

Mechanisms of Epigenetic Marking

Environmental Effects on the Epigenome

Cancer Detection using Epigenomic Targets

Epigenomic Drug Targets in Cancer Treatment

Bioinformatic Approaches to Epigenome Analysis

*Benefits of a Human Epigenome Project*

Epigenetic marks, defined as modifications of the genome that can be
faithfully propagated through cell divisions, and that affect central
biological processes like gene expression and genome stability, have
been amply shown to influence both cancer predisposition and tumor
progression. But in contrast to the DNA sequence, our understanding of
these epigenetic marks remains fragmentary, with most if not all of the
available data coming from highly selected regions of the genome,
examined by non-exhaustive methods. It is likely that a comprehensive
analysis of the epigenome, carried out in carefully chosen and well
defined normal and cancerous cells/tissues, will produce some truly
surprising and paradigm-shifting results, and may well yield fundamental
insights into cancer pathogenesis, detection and treatment. This
integrated epigenomic information will allow us to begin answering the
question of what is the basis for epigenetic therapy and how can the
epigenome be targeted for chemotherapy or chemoprevention.

Following some discussion of the European Epigenomic Network of
Excellence (see below), Think Tank participants discussed pursuing a
similar effort in the United States that would comprise a significant,
dedicated investment whose management and direction would be formulated
by an established network or committee. The justification for pursuing
such a project included:

\$ The series of discoveries in the last 10 years showing that
epigenetics is central to understanding of the development and genetics
of cancer.

\$ Moving forward with an Epigenome Project is timely in light of the
completion and findings of the Human Genome Project.

\$ To gain a better understanding of human disease, including cancer,
investigators need to take a genome-wide perspective of epigenetics.

\$ It will facilitate the standardization and dissemination of
information across the research community.

\$ It will increase visibility of the research community as a
coordinated group.

\$ Improved cost effectiveness, decreased redundancy, and better
comparisons are expected outcomes with standardization across research
teams, labs, and groups.

\$ Keeping the project in the public domain would help not only to
maintain but to encourage openness of communication, development, and
discoveries, including technological developments and advances.

\$ This strategy would provide an infrastructure for reviewers to
improve genome study sections through increased awareness, expertise,
and innovation.

\$ It will provide a much-needed, coordinated opportunity for
quantitative/statistical epigenetics and the development of new tests,
tools, and measures.

\$ It will provide a missing component of integrative biology.

**Special Presentation: European Union 6^th^ Framework ProgrammeCThe
Epigenome**

> Dr. Patrick Varga-Weisz summarized the EU=s Epigenomic Research
> Program, which provides a platform and structure to facilitate the
> coordination of epigenetics research beyond local efforts. The program
> takes advantage of established research groups and facilitates the
> development of new research talent. A Acore consortium@ of 25 leading
> scientists in epigenetic research receives funding through a Joint
> Program of Activities (JPA) for the first phase of the Epigenomic
> Network of Excellence (NoE), which will run from 2004 through 2009
> with an annual budget of approximately 12.5 million Euros.
>
> The purpose of the Epigenome Program is to integrate European
> epigenetics and epigenome research; to support new researchers; and to
> facilitate dialogue among investigators. More specifically, the three
> key goals are to coordinate and support:
>
> ● European epigenetic research: To unravel epigenetic mechanisms in
> the post-genomic era, the NoE will prioritize research into molecular
> mechanisms of epigenetic control. To achieve this goal, the NoE will
> mount an internationally competitive research program through a Joint
> Program of Activities (JPA).
>
> ● The next generation of European scientists: A funding program for 22
> independent investigators will distribute monies in two rounds over 5
> years to integrate the most promising newly established teams (NET
> program) in Europe.
>
> ● An interactive Website: Scientists and the public will be provided
> with up-to-date knowledge of the field through a Web site maintained
> by the NoE. This Web site \[for the preliminary Web site, go to
> http://www.epigenome.imp.ac.at/index.html\] will develop into a major
> resource for the scientific community, and it will seek to establish a
> dialogue with the public by providing first hand knowledge in an
> appropriate form for a non-specialist audience.
>
> The NOE research program is organized into eight distinct subprograms
> focusing on different aspects of epigenetic control:
>
> ● Chromatin modification
>
> ● Nucleosome dynamics
>
> ● Non-coding RNA and gene silencing
>
> ● Xi and imprinting
>
> ● Transcriptional memory
>
> ● Assembly and nuclear organization
>
> ● Cell fate and disease
>
> ● Epigenomic maps

**Specific Recommendations for the NCI:**

*Specific major recommendations*

1\. Establish a Human Epigenome Project of analogous scope to the Genome
Project. This was the highest priority recommendation. The Project could
include:

> \$ Developing a baseline of epigenetic information and profiles that
> includes reference samples, and expanding exploration of integrative,
> comprehensive epigenetic profiles of normal and tumor tissues.
>
> \$ Developing detection tools to genomicize epigenetics; i.e., to lead
> to robust, cost-effective, high-throughput, genome-level analysis.
>
> \$ Developing novel bioinformatic approaches to epigenetics, including
> computational and statistical tools and comparative epigenomics. A
> computer screen which incorporates all levels of genetic/epigenetic
> and chromatin knowledge was envisioned.

2\. Organize a planning meeting, and establish a Working Group, to
define the needs for a Human Epigenome Project. This was unanimously
agreed upon. The questions to be determined include: tissues to be used;
how many individuals; relationship to Genome Map; what will be measured
B DNA methylation, histone modifications, other chromatin proteins

*General recommendations*

3\. Support technology development for epigenetic profiling as well as
for studies of basic chromatin organization is needed. This may be
addressed as part of Epigenome Project; or may relate to Recommendation
4 below; or may require an R21 initiative to develop technology.

4\. Encourage and foster R01-based epigenetic research, particularly for
basic research in mechanisms of chromatin structure and gene regulation.

5\. Encourage and support translational research with a focus on early
epigenetic variations and translational consequences, e.g., how do these
variations alter adult susceptibility to disease?

6\. Expand training opportunities, including cross-platform training for
post-docs, junior and senior faculty, and institute U.S.-European
collaborations or cross-training agreements.