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ScienceWeek
SCIENCEWEEK
ScienceWeek
August 29, 2003
Vol. 7 Number 35A
An Online Digest of Research in the Sciences
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Theories come and theories go. The frog remains.
-- Jean Rostand
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Section 1
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Part A - Symposium: Epigenetics
1. Introduction
2. DNA Methylation in Mammalian Epigenetics
3. RNA-directed DNA methylation in Arabidopsis
4. RNA and Gene Silencing
5. Nuclear Cloning and Epigenetic Reprogramming
6. Transgenerational Inheritance of Epigenetic States
7. Epigenetic Monoallelic Expression in the Immune System
8. Induction of Tumors in Mice by Genomic Hypomethylation
9. Self-Perpetuating Epigenetic Pili Switches in Bacteria
10. Epigenetics and Neuropathologies
Notices and Subscription Information
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Section 2
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1. INTRODUCTION
ON EPIGENETICS
The following points are made by Judith Bender (Current Biology
2002 12:R412):
1) Epigenetic changes in gene expression have fascinated
researchers over several decades. These processes have received
particular attention in plants, where they can result in
beautiful variations in conspicuous phenotypes such as
pigmentation. Epigenetic control is also a key issue in the
development of transgenic plants with appropriate expression from
newly introduced transgene segments.
2) The term "epigenetic" refers to heritable gene expression
patterns determined by how the DNA of a gene is packaged rather
than its primary DNA sequence. Within tightly packed DNA, genes
are not readily available to the transcription machinery and are
poorly expressed. Normally the patterns of DNA packaging are
carefully controlled to give predictable patterns of gene
expression. However, the process can occasionally go awry to
cause altered gene expression.
3) In higher organisms, DNA is packaged into the nucleus of the
cell by association with histone proteins; this DNA–protein
complex is "chromatin". Some regions of the genome are loosely
packaged into "euchromatin", whereas other regions are tightly
packaged into "heterochromatin". One factor that determines
chromatin patterning is modification of histone proteins by
attachment of small chemical groups to particular amino acid side
chains. Specific patterns of histone modification are thought to
recruit specific chromatin remodeling proteins that direct either
heterochromatin or euchromatin formation.
4) In mammalian and plant genomes, chromatin patterning is also
determined by the attachment of methyl groups to cytosine
residues in the DNA by cytosine methyltransferases. When a region
of genomic DNA has cytosine methylation it is typically assembled
into heterochromatin. Methylated DNA appears to recruit methyl-
DNA binding proteins, which in turn recruit histone-modifying
enzymes and chromatin-remodeling factors necessary for
heterochromatin formation. Cytosine methylation is a fundamental
epigenetic mark that can be maintained after each round of DNA
replication because the template strand of DNA will retain the
modification. Although changes in the cytosine methylation mark
often correlate with epigenetic variation, there are also likely
to be cases where chromatin changes occur independently of
methylation.
5) In mammals, DNA methylation marks are reprogrammed during
early embryogenesis and altered methylation patterns are not
usually transmitted to progeny. In plants, however, it seems that
DNA methylation changes can persist throughout development and
can be inherited between generations.
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TERMINOLOGY AND NOTES
The nucleosome is a tertiary structure of chromosomal DNA found
in eukaryotic cells. In chromosomes, about every 200 nucleotides,
the DNA double helix is coiled around a complex of 8 histone
proteins, the entire assembly having the appearance of beads on a
string. The beads of nucleosomes are in turn supercoiled into a
solenoid structure, and the entire complex of the eukaryotic
chromosome is called "chromatin". The small histones proteins are
basic (as opposed to acidic) proteins, and they are essential in
forming nucleosomes.
Genomic imprinting is an epigenetic process that leads to
inactivation of paternal or maternal allele of certain genes
susceptible to epigenetic regulation.
The term "parasitic DNA" (selfish DNA) refers to functionless
segments of DNA that are replicated along with the rest of the
chromosomal regions that serve vital functions. Examples are
pseudogenes and tandemly repeated and dispersed DNA segments that
appear to serve no function.
In this context, the term "promoter" refers to a region on a DNA
molecule to which an RNA polymerase binds and initiates
transcription.
In this context, in general, an "exon" is any DNA sequence
encoding and giving rise to a translated polypeptide sequence. An
"intron" is a portion of DNA that lies between two exons. Introns
are intervening sequences that are not expressed; they are
eliminated during the process that forms messenger RNA.
Neurospora: (pink bread mold) A genus of fungi grown in culture
and widely used in research in genetics and biochemistry. In the
wild (i.e., natural) state, Neurospora crassa will grow on a
nutrient medium containing sugar as the only organic compound
except for a small required concentration of biotin. Induced
mutations (e.g., produced by x-rays) can result in mutants that
require other organic substances, and systematic analysis of the
genetics of these mutants and their new requirements made
possible an understanding of the genetics of a number of
biochemical pathways and of the enzymes that control these
pathways.
Caenorhabditis elegans: This is a small (1 mm) nematode worm. It
is transparent, hermaphroditic, free-living, and found in soil.
It has a relatively small genome (approximately 3000 genes), and
only a few types of cells in its body. It has a 16-hr
embryogenesis that can be achieved in a petri dish, and is thus
highly suitable for the study of developmental and behavioral
genetics.
The term "pronucleus" refers to the nucleus of either the ovum or
the sperm cell following fertilization. Once the ovum is
fertilized, there are two pronuclei, one originating from the
ovum, the other from the sperm cell that produced fertilization.
The two nuclei do not fuse until immediately before the first
cleavage, when each pronucleus loses its membrane to release its
contents.
In this context, the term "transposable elements" refers to a
class of DNA sequences that can move from one chromosomal site to
another. A "retrotransposon" (retroposon) is a mobile genetic
element that transposes via an RNA intermediate. The best
understood retroposons are the retroviruses.
Telomeres are defined ends of chromosomes that contain specific
repeated DNA sequences. They are essential for normal chromosome
replication, and since their length shortens a bit with each
replication, they are believed to be involved in the aging of the
cell.
The term "trisomy", in general, refers to the state of an
individual or cell with an extra chromosome instead of the normal
pair of homologous chromosomes.
The term "CpG island" refers to a region of 1 to 2 kilobases
containing a high density of methylated cytosine residues and
occurring immediately 5' to G residues, i.e., in the sequence
CpG. These islands are frequently found in animal genomes at the
5' end of genes.
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2. DNA METHYLATION IN MAMMALIAN EPIGENETICS
The following points are made by P.A. Jones and D. Takai (Science
2001 293:1063):
1) DNA methylation is essential for the development of mammals
(1,2), but despite 25 years of work, researchers still do not
know exactly why. Recent advances have led to the cloning and
preliminary characterization of the three known active DNA
cytosine methyltransferases (DNMT1, -3a, and -3b) (3,4) and to a
greater understanding of how the methylation signal is
interpreted in mammalian cells.
2) The post-synthetic addition of methyl groups to the 5-position
of cytosines alters the appearance of the major groove of DNA to
which the DNA binding proteins bind. These epigenetic "markers"
on DNA can be copied after DNA synthesis, resulting in heritable
changes in chromatin structure. Methylation of CpG-rich promoters
is used by mammals to prevent transcriptional initiation and to
ensure the silencing of genes on the inactive X chromosome,
imprinted genes, and parasitic DNAs. The potential role of
methylation in tissue-specific gene expression or in the
regulation of CpG-poor promoters is less well established. There
is also tantalizing evidence that normal chromosome structure may
be affected by methylation and that human diseases, including
cancer, are caused and impacted by abnormal methylation.
3) CpG dinucleotides, the sites of almost all methylation in
mammals, are underrepresented in DNA. Clusters of CpGs, called
"CpG islands", are often found in association with genes, most
often in the promoters and first exons but also in regions more
toward the 3' end (5). The exact definition of a CpG island is
evolving. The original suggestion by Gardiner-Garden and Frommer
(1987) of a region greater than 200 base pairs (bp) with a high-
GC content and an observed/expected ratio for the occurrence of
CpG > 0.6, should probably be modified to slightly higher
stringency in terms of length and GC content, thus excluding a
substantial number of small exonic regions and repetitive
parasitic DNAs. The salient property of a CpG island is that it
is unmethylated in the germline (and indeed in most somatic
tissues), thus ensuring its continued existence in the face of
the strong mutagenic pressure of 5-methylcytosine deamination.
4) CpG islands often function as strong promoters and have also
been proposed to function as replication origins. Even though
these islands are generally not methylated, most investigations
on the role of DNA methylation in mammals have focused on CpG
islands rather than on the regions in which the majority of
methylation is found.
5) In summary: Genes constitute only a small proportion of the
total mammalian genome, and the precise control of their
expression in the presence of an overwhelming background of
noncoding DNA presents a substantial problem for their
regulation. Noncoding DNA, containing introns, repetitive
elements, and potentially active transposable elements, requires
effective mechanisms for its long-term silencing. Mammals appear
to have taken advantage of the possibilities afforded by cytosine
methylation to provide a heritable mechanism for altering DNA-
protein interactions to assist in such silencing. Genes can be
transcribed from methylation-free promoters even though adjacent
transcribed and nontranscribed regions are extensively
methylated. Gene promoters can be used and regulated while
keeping noncoding DNA, including transposable elements,
suppressed. Methylation is also used for long-term epigenetic
silencing of X-linked and imprinted genes and can either increase
or decrease the level of transcription, depending on whether the
methylation inactivates a positive or negative regulatory
element.
References (abridged):
1. E. Li, T. H. Bestor, R. Jaenisch, Cell 69, 915 (1992)
2. M. Okano, D. W. Bell, D. A. Haber, E. Li, Cell 99, 247 (1999)
3. T. Bestor, A. Laudano, R. Mattaliano, V. Ingram, J. Mol. Biol.
203, 971 (1988)
4. M. Okano, S. Xie, E. Li, Nature Genet. 19, 219 (1998)
5. F. Larsen, G. Gundersen, R. Lopez, H. Prydz, Genomics 13, 1095
(1992)
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3. RNA-DIRECTED DNA METHYLATION IN ARABIDOPSIS
The following points are made by W. Aufsatz et al (Proc. Nat.
Acad. Sci. 2002 99:16499):
1) The term "RNA silencing" refers to epigenetic gene silencing
effects that are initiated by double-stranded RNA (dsRNA) (1).
Discovered independently in plants, fungi, and animals, RNA
silencing phenomena are revealing new ways to repress gene
expression and to subdue transposable elements and viruses that
produce dsRNA during their replication cycle (2-5). A fundamental
step in RNA silencing pathways is cleavage of dsRNA into short
RNAs, which are believed to act as guides for enzyme complexes
that either degrade or modify homologous nucleic acids.
2) The most familiar type of RNA silencing occurs primarily in
the cytoplasm and is termed "post-transcriptional gene silencing"
(PTGS) in plants, "quelling" in Neurospora, and "RNA
interference" (RNAi) in animals. PTGS/RNAi involves a dsRNA that
is processed by an RNase III-like enzyme called "Dicer" into
short interfering (si) RNAs 21-22 nucleotides (nt) in length. The
antisense siRNAs associate with a ribonuclease complex and guide
sequence-specific degradation of complementary mRNAs (5).
3) A second form of RNA silencing involves sequence-specific
changes at the genome level. RNA-directed DNA methylation (RdDM),
which has been described so far only in plants, leads to de novo
methylation of almost all cytosine residues within the region of
sequence identity between the triggering RNA and the target DNA.
Similarly to PTGS/RNAi, RdDM requires a dsRNA that is cleaved to
short RNAs 21-24 nt in length. It is not yet certain whether the
short RNAs or dsRNA guide methylation of homologous DNA
sequences, although the length of short RNAs is consistent with
the minimum DNA target size of RdDM (30 bp).
4) In summary: In plants, double-stranded RNA that is processed
to short RNAs 21-24 nt in length can trigger two types of
epigenetic gene silencing. Post-transcriptional gene silencing,
which is related to RNA interference in animals and quelling in
fungi, involves targeted elimination of homologous mRNA in the
cytoplasm. RNA-directed DNA methylation involves de novo
methylation of almost all cytosine residues within a region of
RNA-DNA sequence identity. RNA-directed DNA methylation is
presumed to be responsible for the methylation observed in
protein coding regions of post-transcriptionally silenced genes.
Moreover, a type of transcriptional gene silencing and de novo
methylation of homologous promoters in trans can occur if a
double-stranded RNA contains promoter sequences. Although RNA-
directed DNA methylation has been described so far only in
plants, there is increasing evidence that RNA can also target
genome modifications in other organisms. The authors report that
to understand how RNA directs methylation to identical DNA
sequences and how changes in chromatin configuration contribute
to initiating or maintaining DNA methylation induced by RNA, a
promoter double-stranded RNA-mediated transcriptional gene
silencing system has been established in Arabidopsis. A genetic
analysis of this system is helping to unravel the relationships
among RNA signals, DNA methylation, and chromatin structure.
References (abridged):
1. Matzke, M. A., Matzke, A. J. M. & Kooter, J. (2001) Science
293, 1080-1083
2. Vance, V. B. & Vaucheret, H. (2001) Science 292, 2277-2280
3. Waterhouse, P., Wang, M. B. & Lough, T. (2001) Nature 411,
834-842
4. Voinnet, O. (2001) Trends Genet. 17, 449-459
5. Bernstein, E., Denli, A. M. & Hannon, G. J. (2001) RNA 7,
1509-1521
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4. RNA AND GENE SILENCING
The following points are made by M. Matzke et al (Science 2001
293:1080):
1) RNA silencing is a new field of research that has coalesced
during the last decade from independent studies on various
organisms. Researchers who study plants and fungi have known
since the late 1980s that interactions between homologous DNA
and/or RNA sequences can silence genes and induce DNA methylation
(1). The discovery of RNA interference (RNAi) in the worm
Caenorhabditis elegans in 1998 (2) focused attention on double-
stranded RNA (dsRNA) as an elicitor of gene silencing, and
indeed, many gene-silencing effects in plants are now known to be
mediated by dsRNA (3).
2) RNAi is usually described as a post-transcriptional gene-
silencing phenomenon in which dsRNA triggers degradation of
homologous mRNA in the cytoplasm (4). However, the potential for
nuclear dsRNA to enter a pathway leading to epigenetic
modifications of homologous DNA sequences and silencing at the
transcriptional level should not be discounted. Although the
nuclear aspects of RNA silencing have been studied primarily in
plants, there are hints that similar RNA-directed DNA or
chromatin modifications might occur in other organisms as well.
3) Although they may differ in detail, RNAi in animals and the
related phenomena of post-transcriptional gene silencing (PTGS)
in plants and quelling in Neurospora crassa result from the same
highly conserved mechanism, indicating an ancient origin (5). The
basic process involves a dsRNA that is processed into shorter
units that guide recognition and targeted cleavage of homologous
mRNA. dsRNAs that trigger PTGS/RNAi can be made in the nucleus or
cytoplasm in a number of ways, including transcription through
inverted DNA repeats, simultaneous synthesis of sense and
antisense RNAs, viral replication, and the activity of cellular
or viral RNA-dependent RNA polymerases (RdRP) on single-stranded
RNA templates. In C. elegans, dsRNAs can be injected or
introduced simply by soaking the worms in a solution containing
dsRNA or feeding them bacteria expressing sense and antisense
RNA.
4) In summary: In diverse organisms, small RNAs derived from
cleavage of double-stranded RNA can trigger epigenetic gene
silencing in the cytoplasm and at the genome level. Small RNAs
can guide post-transcriptional degradation of complementary
messenger RNAs and, in plants, transcriptional gene silencing by
methylation of homologous DNA sequences. RNA silencing is a
potent means to counteract foreign sequences and could play an
important role in plant and animal development.
References (abridged):
1. M. Fagard and H. Vaucheret, Annu. Rev. Plant Physiol. Plant
Mol. Biol. 51, 167 (2000)
2. A. Fire, et al., Nature 391, 806 (1998)
3. M. A. Matzke, A. J. M. Matzke, G. Pruss, V. Vance, Curr. Opin.
Genet. Dev. 11, 221 (2001)
4. S. M. Hammond, A. A. Caudy, G. J. Hannon, Nat. Rev. Genet. 2,
110 (2001)
5. V. B. Vance and H. Vaucheret, Science 292, 2277 (2001)
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5. NUCLEAR CLONING AND EPIGENETIC REPROGRAMMING
The following points are made by W.M. Rideout et al (Science 2001
293:1093):
1) Epigenetic modification of the genome ensures proper gene
activation during development and involves (i) genomic
methylation changes, (ii) the assembly of histones and histone
variants into nucleosomes, and (iii) remodeling of other
chromatin-associated proteins such as linker histones, polycomb
group, nuclear scaffold proteins, and transcription factors (1).
2) The two parental genomes are formatted during gametogenesis to
respond to the oocyte environment and proceed through
development. The zygote biochemically remodels the paternal
genome shortly after fertilization and before embryonic genome
activation (EGA) occurs. To successfully recapitulate these
processes, the somatic nuclei transferred into an oocyte must be
quickly reprogrammed to express genes required for early
development.
3) Epigenetic reprogramming after fertilization and nuclear
transfer has been studied in Xenopus and mammals (1).The
programming of the genome that occurs as primordial germ cells
(PGCs) differentiate into mature gametes establishes the markedly
different chromatin configurations of sperm and oocyte. As
demonstrated by normal preimplantation development of uniparental
embryos, both parental genomes share the ability to independently
direct cleavage (early development to the blastocyst stage)
despite profound differences in their epigenetic organization
(2,3).
4) In spermatogenesis, chromatin is sequentially remodeled,
silenced, and ultimately compacted with protamines (4), processes
crucial for normal fertilization (5). However, completion of
these events is not strictly required for development as normal
pregnancies can result from intracytoplasmic sperm injection with
round spermatids or secondary spermatocytes. In contrast, the
genome of the oocyte is organized in a structure more like that
of a somatic cell, with chromatin whose nucleosomes contain an
oocyte-specific linker histone. In comparison with the male
pronucleus, the female pronucleus is more transcriptionally
repressive, contains relatively deacetylated histone, and is
deficient in generalized transcription factors. This repressive
chromatin structure may protect the oocyte genome against the
extensive epigenetic modifications imposed on the paternal genome
after fertilization.
5) In summary: Cloning of mammals by nuclear transfer (NT)
results in gestational or neonatal failure with at most a few
percent of manipulated embryos resulting in live births. Many of
those that survive to term succumb to a variety of abnormalities
that are likely due to inappropriate epigenetic reprogramming.
Cloned embryos derived from donors, such as embryonic stem cells,
that may require little or no reprogramming of early
developmental genes develop substantially better beyond
implantation than NT clones derived from somatic cells. Although
recent experiments have demonstrated normal reprogramming of
telomere length and X chromosome inactivation, epigenetic
information established during gametogenesis, such as gametic
imprints, cannot be restored after nuclear transfer. Survival of
cloned animals to birth and beyond, despite substantial
transcriptional dysregulation, is consistent with mammalian
development being rather tolerant to epigenetic abnormalities,
with lethality resulting only beyond a threshold of faulty gene
reprogramming encompassing multiple loci.
References (abridged):
1. K. E. Latham, Int. Rev. Cytol. 193, 71 (1999)
2. M. H. Kaufman, S. C. Barton, M. A. Surani, Nature 265, 53
(1977)
3. J. McGrath and D. Solter, Cell 37, 179 (1984)
4. K. Steger, Anat. Embryol. 199, 471 (1999)
5. C. Cho, et al., Nature Genet. 28, 82 (2001)
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6. TRANSGENERATIONAL INHERITANCE OF EPIGENETIC STATES
The following points are made by V.K. Rakyan et al (Proc. Nat.
Acad. Sci.. 2003 100:2538):
1) It is generally assumed that a phenotype is determined by the
interaction of a specific genotype and a specific environment,
but there are a number of examples where variable expressivity
and incomplete penetrance cannot be explained by genetic or
environmental heterogeneity. One of the earliest documented
examples is the axin-fused (AxinFu) allele, first identified in
1937 (1). Axin regulates embryonic axis formation in vertebrates
by inhibiting the Wnt signaling pathway (2). AxinFu is a dominant
gain-of-function allele that has a 5.1-kb intracisternal-A
particle (IAP) retrotransposon (subtype I1) inserted in an
antisense orientation (relative to the axin locus) in intron 6
(3). The characteristic AxinFu phenotype is kinks in the tail
caused by axial duplications during embryogenesis (1,2). The
phenotype is variably expressed among AxinFu individuals, and in
some mice the tails appear completely normal; i.e., the mutant
phenotype is silent.
2) The variable expressivity of AxinFu is reminiscent of that
observed for the Aiapy, Ahvy, and Avy alleles of the agouti
locus, all of which contain IAP (subtype I1) insertions upstream
of the agouti gene (4,5). The coats of isogenic mice carrying
these alleles vary from wild-type agouti to completely yellow,
with a spectrum of intermediate mottled coats (4,5). The variable
expressivity correlates with differential DNA methylation at a
cryptic promoter within the long terminal repeat (LTR) of the
IAP, which can override the endogenous agouti promoters (4,5).
Hypomethylation is associated with ectopic agouti expression and
consequently a completely yellow coat, whereas hypermethylation
correlates with normal agouti expression, resulting in an agouti
coat. Furthermore, at the Avy allele the epigenetic state can be
inherited transgenerationally after maternal transmission,
resulting in the inheritance of phenotype, i.e., the range of
coat colors of the offspring correlates with the coat color of
the dam.
3) In summary: Phenotypic variation that cannot be explained by
genetic or environmental heterogeneity has intrigued geneticists
for decades. The molecular basis of this phenomenon, however, is
largely a mystery. Axin-fused (AxinFu), first identified in 1937,
is a classic example of a mammalian allele displaying extremely
variable expression states. The authors demonstrate that the
presence or absence of its characteristic phenotype, a kinked
tail, correlates with differential DNA methylation at a
retrotransposon within AxinFu. The authors identify mutant
transcripts arising adjacent to the retrotransposon LTR that are
likely to be causative of the phenotype. Furthermore, the
epigenetic state at AxinFu can be inherited transgenerationally
after both maternal and paternal transmission. This is in
contrast to epigenetic inheritance at the murine agouti-viable
yellow (Avy) allele, which occurs through the female only. Unlike
the egg, the sperm contributes very little (if any) cytoplasm to
the zygote, and therefore paternal inheritance at AxinFu argues
against the possibility that the effects are due to cytoplasmic
or metabolic influences. Consistent with the idea of
transgenerational inheritance of epigenetic marks, the authors
find that the methylation state of AxinFu in mature sperm
reflects the methylation state of the allele in the somatic
tissue of the animal, suggesting that it does not undergo
epigenetic reprogramming during gametogenesis. Finally, the
authors demonstrate that epigenetic inheritance is influenced by
strain background.
References (abridged):
1. Reed, S. C. (1937) Genetics 22, 1-13
2. Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J.,
Perry, W. L., Tilghman, S. M. & Costantini, F. (1997) Cell 90,
181-192
3. Vasicek, T. J., Zeng, L. I., Zhang, T., Costantini, F. &
Tilghman, S. M. (1997) Genetics 147, 777-786
4. Argeson, A. C., Nelson, K. K. & Siracusa, L. D. (1996)
Genetics 142, 557-567
5. Michaud, E. J., van Vugt, M. J., Bultman, S. J., Sweet, H. O.,
Davisson, M. T. & Woychik, R. P. (1994) Genes Dev. 8, 1463-1472
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7. EPIGENETIC MONOALLELIC EXPRESSION IN THE IMMUNE SYSTEM
The following points are made by C. Rada and A.C. Ferguson-Smith
(Current Biology 2002 12:R108):
1) Pathogenic agents, such as bacteria and viruses, are
recognized by the B and T cells of the immune system via a vast
repertoire of antigen receptors. The diversity of this repertoire
is created from a relatively small number of V, D and J gene
segments that become shuffled by DNA rearrangements in somatic
cells in a process termed V(D)J-recombination (1). Importantly,
only one of the two alleles of the antigen receptor is expressed
in B cells and T cells, whereas the other one is silenced by a
mechanism known as "allelic exclusion". It is often the case that
the expressed allele is rearranged completely and productively
but the silent one is not. It was therefore believed that
rearrangement occurred randomly with respect to the two alleles,
but that further rearrangement was inhibited when a functional
receptor was expressed on the surface of the cell. But new
evidence (2,3) indicates that both the initial selection of which
allele is to be rearranged, as well as the maintenance of
silencing of one of the alleles, is under the control of
epigenetic mechanisms.
2) The RAG recombinase, which is responsible for V(D)J-
recombination, recognizes a short consensus sequence known as a
recombination signal sequence or RSS that is present in the
flanks of the segments to be rearranged. However, since all
antigen receptor gene segments are flanked by this signal, it
occurs many times in the genome and therefore the accessibility
of these sequences to RAG has to be carefully regulated. In
vitro, the relative positioning of the RSSs with respect to
nucleosomes can affect the initiation of V(D)J-recombination by
RAG (4). In vivo, the accessibility of a locus is usually
indicated by active transcription, by features affecting the
chromatin structure including histone acetylation, and by the
replication time of the DNA during S phase of the cell cycle --
transcriptionally silent heterochromatic regions replicate later
in S phase than actively transcribed euchromatic loci.
3) Replication asynchrony is therefore considered to be a
suitable marker for allelic differences in the epigenetic state
of the locus. Chromatin immunoprecipitation experiments have
shown that histone acetylation correlates strikingly with V(D)J-
recombination in the T-cell receptor loci (5), and the histones
in the immunoglobulin heavy chain locus are hyperacetylated in a
stepwise manner, domain by domain, so that the DJ regions are
made accessible first while the distal V segments remain
hypoacetylated.
4) In summary: Epigenetic modifications to DNA and chromatin
program important genome functions including gene expression,
chromosomal architecture and stability, and the maintenance of
developmental states. Recent findings further implicate
epigenetic modifications in the control of allelic choice in the
immune system.
References (abridged):
1. Hesslein, D.G. and Schatz, D.G. (2001). Factors and forces
controlling V(D)J recombination. Adv. Immunol. 78, 169-232
2. Mostoslavsky, R., Singh, N., Tenzen, T., Goldmit, M., Gabay,
C., Elizur, S., Qi, P., Reubinoff, B.E., Chess, A., and Cedar, H.
et al. (2001). Asynchronous replication and allelic exclusion in
the immune system. Nature 414, 221-225
3. Skok, J.A., Brown, K.E., Azuara, V., Caparros, M.L., Baxter,
J., Takacs, K., Dillon, N., Gray, D., Perry, R.P., and Merken
schlager, M. et al. (2001). Nonequivalent nuclear location of
immunoglobulin alleles in B lymphocytes. Nat. Immunol. 2, 848-854
4. Kwon, J., Imbalzano, A.N., Matthews, A., and Oettinger, M.A.
(1998). Accessibility of nucleosomal DNA to V(D)J cleavage is
modulated by RSS positioning and HMG1. Mol. Cell 2, 829-839
5. McBlane, F. and Boyes, J. (2000). Stimulation of V(D)J
recombination by histone acetylation. Curr. Biol. 10, 483-486
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8. INDUCTION OF TUMORS IN MICE BY GENOMIC HYPOMETHYLATION
The following points are made by F. Gaudet et al (Science 2003
300:489):
1) Human cancer cells often display abnormal patterns of DNA
methylation. The role of aberrant hypermethylation in the
silencing of tumor suppressor genes is now well documented (1).
In contrast, the role of aberrant hypomethylation -- which is
observed in a wide variety of tumors (2–5), often together with
regional hypermethylation -- has remained unclear.
2) To investigate whether DNA hypomethylation has a causal role
in tumor formation, the authors generated mice with highly
reduced levels of Dnmt1, the enzyme that maintains DNA
methylation patterns in somatic cells. Because mice homozygous
for a Dnmt1 null allele (Dnmt1c/c) die during gestation, the
authors combined a hypomorphic allele [Dnmt1chip (9)] with a null
allele to generate Dnmt1chip/c (referred to here as Dnmt1chip/–)
compound heterozygotes with a substantially reduced level of
genome-wide DNA methylation. Dnmt1chip/– embryonic stem (ES)
cells expressed 10% of wild-type levels (Fig. 1A). To test
whether the reduced Dnmt1 expression affected DNA methylation in
vivo, the authors generated mice carrying the different Dnmt1
alleles and determined their global methylation levels with the
use of a probe for endogenous retroviral A type particles (IAPs)
and centromeric repeats.
3) In summary: Genome-wide DNA hypomethylation occurs in many
human cancers, but whether this epigenetic change is a cause or
consequence of tumorigenesis has been unclear. To explore this
phenomenon, the authors generated mice carrying a hypomorphic DNA
methyltransferase 1 (Dnmt1) allele, which reduces Dnmt1
expression to 10% of wild-type levels and results in substantial
genome-wide hypomethylation in all tissues. The mutant mice were
runted at birth, and at 4 to 8 months of age they developed
aggressive T cell lymphomas that displayed a high frequency of
chromosome 15 trisomy. The authors suggest these results indicate
that DNA hypomethylation plays a causal role in tumor formation,
possibly by promoting chromosomal instability.
References (abridged):
1. P. A. Jones, S. B. Baylin, Nature Rev. Genet. 3, 415 (2002)
2. A. P. Feinberg, B. Vogelstein, Nature 301, 89 (1983)
3. M. Ehrlich, Oncogene 21, 5400 (2002)
4. M. A. Gama-Sosa, Nucleic Acids Res. 11, 6883 (1983)
5. J. N. Lapeyre, F. F. Becker, Biochem. Biophys. Res. Commun.
87, 698 (1979)
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9. SELF-PERPETUATING EPIGENETIC PILI SWITCHES IN BACTERIA
Depending on the species, bacteria have various types of
extensions protruding through the external capsule and into the
ambient medium. One or more "flagella" may be present, long
filamentous and flexible structures whose mechanical motions
provide the bacterium with motility. Two types of rigid
extensions are common, "pili" (Latin for "hairs") and "fimbriae"
(Latin for "fringes") (singular: pilus, fimbrium), both much
shorter than flagella, and fimbriae much shorter than pili. These
structures are 7 nanometers in diameter, and much thinner than
flagellae (which are 25 nanometers in diameter). Both pili and
fimbriae are believed to be involved in the adhesion of
pathogenic bacteria to the surfaces of host cells.
The following points are made by A. Hernday et al (Proc. Nat.
Acad. Sci. 2002 99:16470):
1) Bacteria have developed phase variation mechanisms to control
cell surface pili-adhesin complexes between expression (phase ON)
and nonexpression (phase OFF) states. Pili phase variation can
occur by site specific (1,2) and homologous recombination (3)
mechanisms. In addition, a large group of pili operons including
pyelonephritis-associated pili (pap) are regulated by an
epigenetic switch directly controlled by DNA methylation pattern
formation (4,5).
2) The expression of pap is positively controlled by the local
regulators PapI (8 kDa) and PapB (12 kDa) in concert with the
global regulators leucine-responsive regulatory protein (Lrp) and
catabolite gene activator protein (CAP). DNA adenine methylase
(Dam) is also required for pap transcription. Knockout mutations
in each of the genes encoding these regulatory proteins inhibit
the pap phase OFF to phase ON switch. In addition, histone-like
nucleoid structuring protein modulates pap phase switching
because hns mutants show a decreased OFF to ON switch rate.
3) In summary: Bacteria have developed an epigenetic phase
variation mechanism to control cell surface pili-adhesin
complexes between heritable expression (phase ON) and
nonexpression (phase OFF) states. In the pyelonephritis-
associated pili (pap) system, global regulators [catabolite gene
activator protein (CAP), leucine-responsive regulatory protein
(Lrp), DNA adenine methylase (Dam)] and local regulators (PapI
and PapB) control phase switching. Lrp binds cooperatively to
three pap DNA binding sites, sites 1-3, proximal to the papBA
pilin promoter in phase OFF cells, whereas Lrp is bound to sites
4-6 distal to papBA in phase ON cells. Two Dam methylation
targets, GATCprox and GATCdist, are located in Lrp binding sites
2 and 5, respectively. In phase OFF cells, binding of Lrp at
sites 1-3 inhibits methylation of GATCprox, forming the phase OFF
DNA methylation pattern (GATCdist methylated, GATCprox
nonmethylated). Binding of Lrp at sites 1-3 blocks pap pili
transcription and reduces the affinity of Lrp for sites 4-6.
Together with methylation of GATCdist, which inhibits Lrp binding
at sites 4-6, the phase OFF state is maintained. The authors
hypothesize that transition to the phase ON state requires DNA
replication to dissociate Lrp and generate a hemimethyated
GATCdist site. PapI and methylation of GATCprox act together to
increase the affinity of Lrp for sites 4-6. Binding of Lrp at the
distal sites protects GATCdist from methylation, forming the
phase ON methylation pattern (GATCdist nonmethyated, GATCprox
methylated). Lrp binding at sites 4-6 together with cAMP-CAP
binding 215.5 bp upstream of the papBA transcription start, is
required for activation of pilin transcription. The first gene
product of the papBA transcript, PapB, helps maintain the switch
in the ON state by activating papI transcription, which in turn
maintains Lrp binding at sites 4-6.
References (abridged):
1. Fulks, K. A., Marrs, C. F., Stevens, S. P. & Green, M. R.
(1990) J. Bacteriol. 172, 310-316
2. Blomfield, I. C., Kulasekara, D. H. & Eisenstein, B. I. (1997)
Mol. Microbiol. 23, 705-717
3. Mehr, I. J. & Seifert, H. S. (1998) Mol. Microbiol. 30, 697-
710
4. Krabbe, M., Weyand, N. & Low, D. (2000) in Bacterial Stress
Responses, eds. Storz, G. & Hengge-Aronis, R. (Am. Soc.
Microbiol., Washington, DC), pp. 305-321
5. van der Woude, M., Braaten, B. & Low, D. (1996) Trends
Microbiol. 4, 5-9
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10. EPIGENETICS AND NEUROPATHOLOGIES
The following points are made by L. Tremolizzo et al (Proc. Nat.
Acad. Sci. 2002 99:17095):
1) Studies of heterozygous reeler mice (HRM) have provided
preliminary evidence of a relationship between reelin haplo-
insufficiency, the decrease of dendritic spine expression density
in frontal cortex pyramidal neurons and associated neuropil
hypoplasticity, the down-regulation of glutamic acid
decarboxylase (GAD)67 expression, and the decrease in gamma-
aminobutyric acid (GABA) turnover (1-3). Similar neurochemical
and structural abnormalities were detected in the frontal cortex
of schizophrenia postmortem brains (4,5). Hence, HRM may be a
model to evaluate the efficacy of novel treatments for
schizophrenia by monitoring drug actions on (i) reelin and GAD67
mRNA expression, (ii) GABA turnover, and (iii) cortical neuropil
plasticity including dendritic spine expression.
2) The HRM model several aspects of the molecular neuropathology
expressed in schizophrenia, although the mechanisms operative in
these pathologies may be different. In fact, demographic studies
of schizophrenia inheritance in identical twins show a
concordance of 50%, which supports an epigenetic model but not
gene haplo-insufficiency of Mendelian origin. Investigation of a
putative epigenetic mechanism attending schizophrenia
vulnerability may therefore be in order. Along this line of
thinking, the authors have hypothesized that a protracted
treatment with L-methionine results in an epigenetic
hypermethylation of the CpG islands located in a number of gene-
promoter regions probably including reelin and GAD67. The
selection of reelin as the target for an epigenetic promoter
hypermethylation model of schizophrenia vulnerability received
support from the finding that hypermethylation of the reelin
promoter CpG island down-regulates reelin expression and by
evidence that reelin plays an important role in (i) embryonic
corticogenesis and (ii) the regulation of adult rodent brain
dendritic spine expression density (2,3).
3) In summary: Reelin and glutamic acid decarboxylase (GAD)67
expressed by cortical gamma-aminobutyric acid-ergic interneurons
are down-regulated in schizophrenia. Because epidemiological
studies of schizophrenia fail to support candidate gene haplo-
insufficiency of Mendelian origin, the authors hypothesize that
epigenetic mechanisms (i.e., cytosine hypermethylation of CpG
islands present in the promoter of these genes) may be
responsible for this down-regulation. Protracted L-methionine
(6.6 mmol/kg for 15 days, twice a day) treatment in mice elicited
in brain an increase of S-adenosyl-homocysteine, the processing
product of the methyl donor S-adenosyl-methionine, and a marked
decrease of reelin and GAD67 mRNAs in both WT and heterozygous
reeler mice. This effect of L-methionine was associated with an
increase in the number of methylated cytosines in the CpG island
of the reelin promoter region. This effect was not observed for
GAD65 or neuronal-specific enolase and was not replicated by
glycine doses 2-fold greater than those of L-methionine. Prepulse
inhibition of startle declined at a faster rate as the
prepulse/startle interval increased in mice receiving L-
methionine. Valproic acid (2 mmol/kg for 15 days, twice a day)
reverted L-methionine-induced down-regulation of reelin and GAD67
in both WT and heterozygous reeler mice, suggesting an epigenetic
action through the inhibition of histone deacetylases. The same
dose of valproate increased acetylation of histone H3 in mouse
brain nearly 4-fold. The authors suggest this epigenetic mouse
model may be useful in evaluating drug efficacy on schizophrenia
vulnerability. Hence the inhibition of histone deacetylases could
represent a pharmacological intervention mitigating
epigenetically induced vulnerability to schizophrenia in
individuals at risk.
References (abridged):
1. Liu, W. S., Pesold, C., Rodriguez, M. A., Carboni, G., Auta,
J., Lacor, P., Larson, J., Condie, B. G., Guidotti, A. & Costa,
E. (2001) Proc. Natl. Acad. Sci. USA 98, 3477-3482
2. Costa, E., Davis, J., Grayson, D. R., Guidotti, A., Pappas, G.
D. & Pesold, C. (2001) Neurobiol. Dis. 8, 723-742
3. Costa, E., Davis, J., Pesold, C., Tueting, P. & Guidotti, A.
(2002) Curr. Opin. Pharmacol. 2, 56-62
4. Akbarian, S., Kim, J. J., Potkin, S. G., Hagman, J. O.,
Tafazzoli, A., Bunney, W. E., Jr. & Jones, E. G. (1995) Arch.
Gen. Psychiatry 52, 258-266
5. Selemon, L. D. & Goldman-Rakic, P. S. (1999) Biol. Psychiatry
45, 17-25
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