7/27/2019 Epigenetics - The Cancer Code of Silence
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Epigenetics
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SPRING
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FEATURING
Epigenetics: The Cancer Code of Silenceby Jean-Pierre Issa, M.D., Feature Article
PLUS!X Chromosome Inactivation and Chromatin IPHighlight Sections
Chemicon Methylation Products Upstate Products to Histone Modifications
Diagram Inside
Epigenetic Silencing of aTumour Suppressor Gene
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EPIGENETICS NORMAL CELLS
Epigenetics refers to stable changes in gene expression thatare not due to mutations or DNA base changes. Silencing is a
subset of epigenetics, whereby gene expression and function
is permanently lost. We now recognise three related mechanisms
of such silencing DNA methylation, histone code changes and
RNA interference. RNA interference is not well understood in
mammalian cells yet, thus this review will focus on the first
two components of epigenetics.
DNA Methylation
Methylation refers to the biochemical addition of a methyl group
(CH3) to a biological molecule. There are distinct enzymaticsystems to methylate DNA, RNA and proteins. Protein methylation
has been intensively studied recently and is an essential post-
translational modification that affects gene function. RNA
methylation is less understood but probably plays a role in
message stability. DNA methylation is the only normally occurring
modification of DNA and is present from bacteria to man, though
it plays a different role in eukaryotes than in prokaryotes.
In mammals, normal methylation affects only the cytosine
base incorporated into DNA, primarily when it is followed by a
guanosine (hence, we speak of Cytosine-phospho-Guanosine
or CpG methylation). CpG sites are unevenly distributed in thegenome. They are rare (5-10 fold less than statistically expected)
in 99% of the human genome, and most of these CpG sites are
modified by methylation. By contrast, about 1% of the genome
consists of CpG rich areas that are typically 500-2000 bp long,
and are referred to as CpG islands. About half of all CpG islands
correspond to transcription start sites and promoters of expressed
genes, and about half of all genes have CpG islands in their
promoters. Most promoter-associated CpG islands are free of
methylation, regardless of the expression state of the associatedgene. Non-promoter associated CpG islands are less well understood
and can be methylated in normal tissues. Genes that do not
have CpG islands in their promoters show different patterns of
methylation; those rare CpG sites in their transcription start areas
are typically methylated when the gene is inactive and unmethylated
when the gene is active. This non-CpG island methylation does
not prevent gene expression, can be reversed quickly upon gene
activation and may serve primarily to regulate the degree of
acute gene activation by transcription factors.
DNA methylation in promoter-associated CpG islands is a
hot topic these days. A switch from unmethylated to methylated
CpG islands was first demonstrated on the inactive X-chromosome
in women and is now seen as an important mechanism of
epigenetic silencing in mammals. CpG island methylation is now
recognised as an essential contributor to gene silencing in the
rare instances when a cell needs mono-allelic expression for normal
function, primarily the inactive X in women, and about 100 genes
that are imprinted (mono-allelically expressed based on parental
origin). The DNA-methyltransferase enzymes (DNMT1, DNMT3a
and DNMT3b) are essential for establishing and maintaining
normal patterns of DNA methylation, and are helped in this byother proteins, such as DNMT3L.
Histone Code
In the eukaryotic nucleus, DNA is wrapped around an octamer of
core histone molecules to form the nucleosome, the fundamental
subunit of chromatin. Many residues in the histone proteins are
subject to reversible post-translational modifications. These marks
are emerging as important epigenetic mediators of gene expression
EpigeneticsThe Cancer Code of Silence
by Jean-Pierre Issa, M.D., Department of Leukemia, The University of Texas
histone modifications are emerging as important
epigenetic mediators of gene expression
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3
changes. There are numerous possible modifications of histone
tails, and work on regulation of gene expression has focused on
acetylation, methylation, ubiquitylation and phosphorylation. Increases
in histone acetylation generally correlate with gene activation, and
results from the dynamic interplay between histone acetyltransferases
(HATs) and histone deacetylases (HDACs). Histone methylation
can have either positive effects on gene expression (e.g. H3
lysine 4 methylation, mediated by histone methyltransferases
HMTs, such as MLL) or negative effects on gene expression
(e.g. H3 lysine 9 methylation mediated by HMTs, such as Suv39h1
or G9A and H3 lysine 27 methylation mediated by HMTs, such asEZH2). Histone modifications play an important role in chromosome
structure, and silencing marks are enriched at silenced loci, such as
retrotransposons, X-inactive genes and imprinted genes, suggesting
that they play a role there as well. The ultimate mediators of
histone methylation associated gene silencing appear to be proteins
that bind specific histone modifications and recruit effector protein
complexes. H3 Lys9 methylation triggers binding by HP1 family
members, while H3 Lys27 methylation triggers binding by PCG
group proteins. These two complexes are each capable of chromatin
remodelling and transcriptional suppression. H3 Lys4 methylation
recruits the CHD1 protein and the SAGA complex, linking
methylation of lysine 4 to histone acetylation.
Methyl-Binding Domain Proteins (MBDs)
A link between DNA methylation and the histone code is provided
by methylated-DNA binding proteins, commonly referred to as MBDs.
Dense CpG island methylation attracts MBDs in a non-sequence
specific but DNA methylation specific way. These include MeCP2,
MBD1-4 and KAISO. These proteins likely play different roles,
and one of them, MBD4, is involved in DNA repair rather than
gene silencing. MBDs may have some intrinsic transcription
repression properties, but it is thought that their effect on gene
expression is achieved primarily through targeting protein complexes
to specific gene promoters and subsequent local histone
modifications that culminate in gene silencing.
Model for the Epigenetic Silencing of a Tumour Suppressor Gene
Legend A. An actively transcribed gene (yellow arrow, green light) is depicted in a domain of open chromatin, with the hallmarks of open chromatin-histone acetylation (yellow A) and
H3 lysine 4 methylation (not shown for sake of clarity). B. Silencing is initiated by abnormal CpG methylation mediated by a DNA methyltransferases (DNMT). Transcription is beginning to
shut off (small X in yellow arrow, yellow caution light). C. CpG methylation recruits a complex of proteins that includes a methylated-DNA binding protein (MBD) and a histone deacetylase
(HDAC), which removes acetyl groups. D. Histone deacetylation allows histone methyltransferases specific for H3 lysine 9 (K9 HMT), resulting in the the recruitment of Heterochromatin
Protein-1 (HP1). This leads to a compaction of chromatin and total silencing of gene expression (large red X, red stoplight). A.To return the gene to an active, expressed state involves the
recruitment of a putative lysine 9 histone demethylase (HDM) and inhibition of DNA methyltransferase activity. Recruitment of a histone H3 lysine 4-specific HMT occurs during this
process, as well (not shown).
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EPIGENETICS AND CANCER WHY THE FUSS?
DNA methylation is abnormal in cancer cells when compared
to normal cells of the same tissues. This was first recognised
in the late 1970s when total 5-methylcytosine content was shown
to be lower in transformed cells. Subsequent studies have
carefully quantitated this effect, and it appears that, on average,
human cancers lose 10% of all methylated cytosines. Despite
three decades of research, the causes and functional consequencesof this degree of hypomethylation remain mysterious.
In parallel to hypomethylation, cancer cells paradoxically
acquire aberrant locus-specific hypermethylation in normally
unmethylated CpG islands. This phenomenon, first described
almost 20 years ago, is now seen as a universal feature of
neoplasia and affects on average 4-6% of promoter associated
CpG islands. The causes of this increased methylation are also
poorly defined. In normal and neoplastic tissues, it has been
linked to aging, pro-inflammatory exposures and carcinogenic
exposures. Alterations in the known DNA methylases have not
been reproducibly linked to aberrant DNA methylation in cancer.In contrast to hypomethylation, the functional consequences
of aberrant hypermethylation are unequivocal the affected genes
are silenced, and their function is stably lost in a clonally propagated
fashion. A functional link of this silencing to the pathophysiology
of cancer was established when it was demonstrated that genes
intimately involved in carcinogenesis tumour-suppressor genes
are frequently inactivated in association with promoter CpG
island methylation. This is most evident for genes mutated in
familial cancer syndromes, such as RB1, VHL, MLH1 and CDH1.
In each case, a study of sporadic tumours of the same type as
those appearing in the familial cases reveals (i) that some sporadic
tumours have mutations of those same genes, (ii) that other
sporadic tumours have methylation of those genes, and (iii)
when both mutations and methylation are present, each molec-
ular anomaly affects distinct alleles. This shared tissue distribu-
tion and allelic exclusion of mutations and DNA methylation in spo-
radic tumours can best be explained by hypothesising that both
events have equivalent selective advantage, and that there is no
further advantage to methylating a mutated allele (or vice-
versa). By extension, if one believes mutations of those genes
functionally lead to cancer, the logical conclusion is that methyla-tion-associated transcriptional inactivation of the genes also
functionally leads to cancer. There are now hundreds of genes,
many with
tumour-suppressor function, known to be hypermethylated in
various neoplasms.
The most encouraging aspects of DNA methylation research
are their potential for translation in the clinic. Aberrant methylation
has been shown to have potential in risk-assessment, early
detection, disease classification and prognosis prediction in a
variety of cancers. More remarkably, research is moving quickly
towards targeting DNA methylation therapeutically. CpG island
methylation can be reversed physiologically through embryogenesis,
and this epigenetic reprogramming has been shown to reverse
some of the malignant features of neoplasia. Hypomethylation
early in embryogenesis is achieved, in part, by DNA replicationin the absence of functional DNA-methyltransferases. Inhibition
of these methyltransferases could, therefore, achieve a certain
degree of reprogramming in adult cells and, in fact, such
inhibitors have been shown to reactivate functional expression
of tumour-suppressor genes silenced in cancer. One of these
inhibitors, 5-azacytidine, is now FDA-approved in the U.S. for
the treatment of MDS (a type of leukemia), and another one
(5-aza-2-deoxycytidine) is showing much promise, as well.
DNA METHYLATION AND THE HISTONE CODE:
CHICKEN, EGGS AND RED HERRINGSThere is considerable interest in understanding the molecular
mechanisms of DNA methylation-associated gene silencing, if
only to design better strategies aimed at reversing it in the
clinic. Much progress over the past few years has linked DNA
methylation to a cascade of protein modifications and a distinct
histone code at silenced loci.
As a possible initial step towards silencing, DNA methylation
attracts binding of MBDs. It is not known whether different CpG
island methylation and silencing events are related to specific
methyl-binding proteins or whether they are mostly interchangeable.
It is likely, of course, that some specificity exists, but the molecular
nature of this specificity is unresolved. Next is histone deacetylation,
which is now thought to be a pre-requisite for DNA methylation-
associated gene silencing. MBD proteins are part of complexes
that include histone deacetylases (HDACs), and inhibition of
histone deacetylation prior to the establishment of silencing
aborts DNA-methylation mediated suppression of gene expression.
There are numerous HDACs, and their preferences for specific
genes or specific MBDs are poorly understood. In human cancers,
genes silenced in association with promoter DNA methylation
show consistently low levels of histone acetylation, particularlyat the key modification histone H3 lysine 9 (H3 Lys9). While
required for initial silencing, HDAC inhibitors do not reactivate
most genes silenced in association with DNA methylation,
implying the importance of downstream events.
Recently, altered histone methylation has emerged as key
to DNA methylation related silencing. This type of gene silencing
in cancer is accompanied by H3 Lys4 hypomethylation and an
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increase in H3 Lys9 methylation. The mechanism of the former
modification is unknown, but an H3 Lys4 demethylase (LSD1)
was recently described and, conceivably, could be targeted by
MBDs. Histone methyltransferases have been shown to be
recruited by MBDs, and this recruitment is presumed to mediate
H3 Lys9 hypermethylation locally. The ultimate mediators of
DNA-methylation associated gene silencing have not been
established with certainty, but very likely involve recruitmentof HP1 and PCG proteins by histone methylation events.
The Chicken and Egg Question
Genetic manipulation experiments in Neurospora crassa have
suggested that histone H3 Lys9 methylation leads to DNA
methylation rather than the opposite. Other experiments support
this view, although there is also evidence in some systems that
DNA methylation is required for H3 Lys9 methylation. Thus the
chicken or egg issue does DNA methylation come first, or does
gene silencing, histone code changes and H3 Lys9 methylation
come first and lead to DNA methylation, continues to be relevant.
This issue in cancer (and mammalian cells in general) is entirely
unclear, with evidence supporting both assertions. Nevertheless,
the data does indicate that, once silencing is achieved, a self-
reinforcing loop of DNA methylation and histone modifications
ensues, which explains the remarkable stability of the system.
Histone Code or Red Herring?
The focus on a DNA-methylation associated histone code has
detracted attention somewhat from the possibility that other
mechanisms are operative in the process. Evidence against a
strict relationship includes the facts that (i) histone code
modifications (including H3 Lys9 methylation) are much more
dynamic than DNA methylation, and (ii) DNA methylation
inhibition remains the most potent (and often the only) intervention
capable of reactivating genes silenced in cancer in association
with promoter CpG island methylation. There exists the possibility
that there are histone-independent mechanisms by which DNA
methylation can silence genes, including direct inhibition of
transcription factor binding or other more theoretical mechanisms,
such as partitioning into transcriptionally inactive nuclear
compartments. But it has been shown that histone methylation
can be targeted to specific genes in vivo to achieve a modestlevel of silencing. This fact, combined with data linking a variety
of histone methyltransferases to cancer development, suggests
that the molecular mechanism by which DNA methylation
effects transcriptional silencing requires the involvement of
histone modification and the enzymes that establish and
maintain them.
CONCLUSIONS BREAKING THE CANCER CODE OF SILENCE
In parallel to vast genetic alterations, cancer cells usurp normal
gene silencing mechanisms and apply them to the functional
ablation of tumour-suppressor pathways. This is achieved via inter-
play between multiple synergistic silencing mechanisms, includ-
ing promoter CpG island methylation and histone code modifica-
tions. These silencing mechanisms show cancer-specificity and are,
therefore, appropriate targets for therapeutic intervention. DNAmethylation inhibitors and HDAC inhibitors are currently in clinical
trials for the treatment of cancer, and the field is looking forward
to the availability of inhibitors of other key components of the
pathway, such as MBPs or histone H3 Lys9 or H3K27 methylases.
Further Reading:
Bestor,T.H. (2000). The DNA methyltransferases of mammals. Hum. Mol. Genet.
9, 2395-2402.
Bird,A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev.
16, 6-21.
Egger,G., Liang,G., Aparicio,A. and Jones,P.A. (2004). Epigenetics in human disease
and prospects for epigenetic therapy. Nature 429, 457-463.
Herman,J.G. and Baylin,S.B. (2003). Gene silencing in cancer in association with
promoter hypermethylation. N. Engl. J. Med. 349, 2042-2054.
Issa,J.P. (2002). Epigenetic variation and human disease. J. Nutr. 132, 2388S-
2392S.
Jaenisch,R. and Bird,A. (2003). Epigenetic regulation of gene expression: how
the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl,
245-254.
Jenuwein,T. and Allis,C.D. (2001). Translating the histone code. Science 293,
1074-1080.
Kondo,Y. and Issa,J.P. (2004). Epigenetic changes in colorectal cancer. Cancer
Metastasis Rev. 23, 29-39.
Lachner,M. and Jenuwein,T. (2002). The many faces of histone lysine methylation.
Curr. Opin. Cell Biol. 14, 286-298.Santini,V., Kantarjian,H.M., and Issa,J.P. (2001). Changes in DNA methylation in
neoplasia: pathophysiology and therapeutic implications. Ann. Intern. Med. 134,
573-586.
Web Resources:
Science Magazine: Epigenetics
www.sciencemag.org/feature/plus/sfg/resources/res_epigenetics.shtml
The University of Texas MD Anderson Cancer Center: DNA Methylation in Cancer
www.mdanderson.org/departments/methylation/
The Wellcome Trust: Epigenetic s
www.wellcome.ac.uk/en/genome/thegenome/hg02b002.html
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P R O D U C T H I G H L I G H T
X chromosome inactivat ion is a mechanism whereby mammals equalise X-linked
gene expression between males and females. Early in development in female embryos,
a choice is made between the two X chromosomes, resulting in expression of the Xist
gene from only the X chromosome destined to be inactivated (the Xi). Xist encodes
a large untranslated RNA that decorates only the X chromosome from which it is
expressed, which leads to inactivation of that chromosome. Xist RNA is required for
recruitment to the Xi of many of the other factors that contribute to the establishment
and maintenance of inactivation (see table below). Many of these changes involve
histones, be it an increase or decrease of a particular modification, or chromosomal-
specific localisation in the case of the histone variant macroH2A.
X Chromosome InactivationEpigenetic Silencing at the Chromosomal Level
Enrichment of HistonemacroH2A on Xi Chromosome
Hallmarks of the Inactive X Chromosome
6
Increased on the Xi
Inactive X ChromosomeLegend Indirect immuno-
fluorescence (IF) to detect
enrichment on the Xi of
histone H3 trimethyl lysine 27
(top left, using cat. #07-449),
histone H4 monomethyl
lysine 20 (top middle, using
cat. #07-440) & the EZH2
methyltransferase (top right)
on interphase chromosomes
of undifferentiated mouse
embryonic stem cells that
express Xist. Subsequent
RNA FISH shows the Xist RNA
on the inactive X chromosome
(red, bottom panels). From
Kohlmaier et al, 2004. PLoS
Biol. 2: 991-1003.
Legend Detection of histone macroH2A on
the inactive X chromosome in female human
fibroblast using Anti-Histone macroH2A1
(cat. #07-219). Image courtesy of Dr.
Barbara Panning, University of California
at San Francisco.
H3 Methylation Staining
Legend Human female metaphase
chromosome spreads stained with DAPI
(blue) and either Anti-H3 K9 Me (red, top
panel, cat. #07-212) or Anti-H3 K4 Me
(red, bottom panel, cat. #07-030). The
inactive X chromosome is indicated with
an arrow in each panel. Image courtesy of
Barbie Boggs, Baylor College of Medicine.
Decreased on the Xi
Xist RNA
DNA Methylation
Histone MacroH2A
Histone H3 K9 Dimethylation
Histone H3 K27 Trimethylation
Histone H4 K20 Monomethylation
EZH2
Histone H4 Acetylation
Histone H3 K4 Methylation
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P R O D U C T H I G H L I G H T
If you are studying transcriptional regulation, then it is likely that you are interested in the
binding of a transcription factor to a particular gene regulatory element. Or, for that
matter, the changes in histone modification that accompany gene activation and silencing.
Chromatin immunoprecipitation (ChIP) is a powerful technique for looking at protein
distribution throughout the genome. It works well for identifying in vivo protein:DNA
interactions, and its the only method for quantifying levels of histone modifications
at specific loci.
Until recently, researchers who wanted to interrogate a DNA sequence for specific
protein interactions needed to design their own protocols. With Upstates Chromatin
Immunoprecipitation (ChIP) Assay Kits, however, protocol design is no longer an issue.
In addition, Upstate offers the widest range of ChIP validated antibodies, saving
researchers the time and tedium of repeatedly running the assay to validate the
antibodies themselves.
Using Upstates ChIP Assay, the study of transcriptional regulation can proceed more
quickly and conveniently than ever. Visit us online at www.upstate.com/chromatin
for a complete listing of products for chromatin research.
Chromatin IPThe Nexus of Genomics & Proteomics
Chromatin IP Products
Pack
Cat. # Description Size Price
7
Chromatin IP Pathway
Legend Schematic representation of
the chromatin immunoprecipitation (ChIP)technique. First, the proteins are cross-
linked to DNA with formaldehyde, and thenthe chromatin is sheared to a manageable
size by sonication. Specific proteins areimmunoprecipitated with antibodies,also bringing down the DNA to which the
protein is cross-linked. The cross-links arereversed, the DNA purified, and the sample
is interrogated for the enrichment of specificDNA sequences. The detection step can be
performed most accurately by quantitativereal-time PCR, but the usage of microarraysin this step is increasing.
17-371 EZ-ChIP Chromatin Immunoprecipitation Kit 1 kit
17-295 Chromatin Immunoprecipitation (ChIP) Assay Kit 1 kit
17-245 Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit 1 kit
17-229 Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit 1 kit
16-157 Protein A agarose/Salmon Sperm DNA 1 mg
16-201 Protein G Agarose/Salmon Sperm DNA 1 mg
Legend Histone H3 at lysine 4 is highly correlated with transcriptional competence, and Histone H3
methylation at lysine 9 is highly correlated with transcriptional silencing. Image courtesy of ShivGrewal, Cold Spring Harbor Laboratories.
H3Methylation
asDomainMarkers
EZ-ChIP Kit (cat. #17-371)
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P R O D U C T H I G H L I G H T
CpGenome & CpG WIZ
Methylation-specific PCR* (MSP) Systems
8
Cat. # Description Price
Cat. # Description Price
Detection of the Methylation State of the p16 GeneMethylation Specific PCR* (MSP) of the p16 gene in two invasive carcinomas, a squamousintraepithelial lesion (SIL), and an adenocarcinoma of the cervix. Each numbered set ispaired MSP reactions specific for both the unmethylated (U) and methylated (M) alleles of
the p16 CpG island. The presence or absence of theMMSP amplicon is indicative of themethylation state of the p16 gene in the sample. The results indicate that both invasive
carcinomas and the SIL sample are heterozygous for methylation while the adenocarcinomasample is clearly homozygous for the unmethylated state at the p16 locus.
Cat. # Description Price
The CpGenome and CpG WIZ Systems quickly and easily
detect the methylation state of a gene using methylation-specific
PCR* (MSP). This advanced technique enables the precise
mapping of methylation patterns in the CpG islands of genomic
DNA. Methylation-specific PCR* is a technology for the sensitive
detection of abnormal gene methylation utilising small amounts
of DNA1. The procedure is capable of detecting methylated
sequences in mixed cell populations (methylated and unmethylated).
This process employs an initial bisulfite reaction to modify theDNA, followed by PCR* amplification with specific primers
designed to distinguish methylated from unmethylated DNA
(see chart on page 9).
CpGENOME DNA MODIFICATION KIT
Chemicons CpGenome DNA Modification Kit is used to
create methylation-specific sequence alterations in any DNA
sample. Using a bisulfite treatment process, all unmethylated
cytosines are converted to uracils; methylated cytosines remain
unaltered. Thus, the methylation state of the DNA in vivo will
determine the sequence of the DNA following bisulfite treat-
ment. Methylation-specific PCR* (MSP) is then employed to
detect which sequence is now present by using primers specific
to each possible sequence. These primers can be designed
using CpG WARE or other software; alternatively, CpG WIZ
kits are available that supply the necessary reagents (including
primers) to detect the methylation state of specific targets.
The CpGenome DNA Modification Kit contains the reagents
and the protocol needed to perform bisulfite treatment on
100 DNA samples.
CpGENOME DNA Modification Kit Advantages
Flexible: Universal method for any gene of interest
Sensitive: Modify as little as 1 ng of DNA
Easy: No restriction digests or Southern blots required
S7820 CpGenome DNA Modif icat ion Kit 235
CpGENOME UNIVERSAL METHYLATED CONTROL DNAChemicons CpGenome Universal Methylated Control DNA is
a positive control for investigating the methylation status of any
human gene. This control consists of methylated in vitro human
male genomic DNA and has been qualified for use with the
CpGenome Universal DNA Modification Kit (cat. #S7820).
S7821 CpGenome Universal Methylated Control DNA 193
CpGENOME UNIVERSAL UNMETHYLATED DNA SETThe CpGenome Universal Unmethylated DNA Set provides two
separate genomic DNA controls (5 g each) for gene methylation
studies. One is genomic DNA from human tissue, and the other
is genomic DNA from a primary human foetal cell line. While
both DNAs are known to be primarily unmethylated, one of
the two may be a better unmethylated control for certain
genes.
S7822 CpGenome Universal Unmethylated DNA Set 184
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Step 1. Bisulfite Treatment The fi rst step in MSP involves the chemical conversion of al l unmethylated cy tosines to uracil using the reagents in the CpGenome
Universal DNA Modification Kit (cat. #S7820). Methylated cytosines remain unaltered in the process. Thus, the sequence of the DNA after bisulfite treatmentwill be different depending on the orig inal methy lation s tate of the DNA.
Step 2. PCR* with Methylation Specific Primer Sets Methylation specific PCR* is used to determine which sequence is present after bisulfite treatment. Primers
to the unmethylated and methylated sequences must be designed, such that mismatches are created depending on which sequence is present to prevent misprimingbetween the primer sets and the undesired target DNA. A typical experiment will involve performing 2 PCR* reactions using the same bisulfite-treated template
DNA. One reaction uses primers (U primer set) designed to anneal to the sequence present if the DNA is unmethylated, and the other reaction will includeprimers (M primer set) designed to anneal to the sequence if the DNA is methylated.
Step 3. Gel Analysis The PCR* products are run on an agarose ge l s tained to visua lize the DNA. If the sample DNA was or iginally unmethylated prior tomodification, only the U primer set will produce an amplification product. Conversely, a product will only be produced with the M primer set if the DNA
was originally methylated.
CgG WIZ
Amplification KitsCat. # Description Price Cat. # Description Price
S7800 CpG WIZ p16 Amplification Kit 325
S7801 CpG WIZ DAP Kinase Amplification Kit 325
S7802 CpG WIZ p15 Amplification Kit 325
S7803 CpG WIZ MGMT Amplification Kit 325
S7804 CpG WIZ E-cadherin Amplification Kit 325
S7805 CpG WIZ VHL Amplification Kit 325
S7806 CpG WIZ Prader-Willi/Angelman Amplification Kit 325
S7807 CpG WIZ Fragile X Amplification Kit 325
S7808 CpG WIZ GST-pi Amplification Kit 325
S7809 CpG WIZ SOCS1 Amplification Kit 325
S7810 CpG WIZ RB1 Amplification Kit 325
S7811 CpG WIZ hMLH1 Amplification Kit 325
S7812 CpG WIZ APC Amplification Kit 325
S7813 CpG WIZ RASSF1A Amplification Kit 325
S7814 CpG WIZ RAR1 Amplification Kit 325
S7815 CpG WIZ ER Amplification Kit 325
S7816 CpG WIZ HIN Amplification Kit 325
S7817 CpG WIZ p14/ARF Amplification Kit 325
S7818 CpG WIZTMS1/ASC Amplification Kit 325
S7830 CpG WIZ BRCA1 Amplification Kit 325
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Epigenetics Products
Tested Species Pack Pos.Description Applications Reactivity Cat. # Size Cntrl. Price
EpigeneticsProduct List
Antibodies to Tumour Suppressors Important regulators of cell division whose loss of function by mutation or silencing can contribute to can-
cerAPC
Anti-APC IHC IP FC H AB4063 100 g 193
DAPK2
Anti-DAP Kinase 2 WB H M R AB3606 100 g 180
E-cadherin
Anti-E-cadherin, clone 674A WB FC H MAB3199 100 g 197
FMRP
Anti-FMRP WB IHC ELISA FC H MAB2160 100 l 287
ING
Anti-ING1, clone CAb3 WB IP ICC H 05-720 100 g 217
MLH1
Anti-hMLH1 WB IP H AB3902 50 g 137MGMT
Anti-MGMT, clone MT3.1 WB IHC IP FC H MAB16200 100 g 197
p16
Anti-p16, clone D25 WB IHC H MAB4133 100 g 197
Anti-p16, clone ZI11 WB IHC IF H MAB88057 50 g 146
p19 ARF
Anti-p19 ARF WB IP M R 07-543 200 l 204
p53
Anti-p53, clone BP53-12 WB IP H 05-224 200 l 224
Anti-acetyl-p53 (Lys320) WB IP H 06-915 200 l 202
Anti-acetyl-p53 (Lys373) WB IP H 06-916 200 l 202
Anti-acetyl-p53 (Lys373, Lys382) WB WR 06-758 200 l 231
Am ..... .... ...amphib ian
Av ... .... .... ...a vian
B................bovine
Ca .............canine
Ce..............C. elegans
Ch .............chicken
Di ..............dictyostelium
Dr..............drosophila
Eu..............eukaryotes
Ft...............ferret
Gp.............guinea pig
H ...............human
Ht ..............hamster
M ... .... .... ....mouse
Ma.. .... .... ...mammal
Mi .... .... .... ..mi nk
Mk .... .... .... .monkey
Pl ...............plant
Po..............porcine
R................rat
Rb..............rabbit
Sh. .... .... .... .sheep
Sp. .... .... .... .S. pombe
T ................Tetrahymena
WR ............wide range
Xn. .... .... .... .Xenopus
Y ................yeast
Ze .... .... .... ..zebra fish
* predicted
Tested Species Reactivity (see note below)Tested Applications (see note below)
Note:Additional species and applications may apply. Call Tech Support at 0805 0190 555 for more information.
.... .... ..ace tyla tion
.... .... .meth ylat ion
Modification
APA ..... ... ...a ffin ity precipitat ion
BA .............biologically active
BD.............Beadlyte assay
CC.............cell culture
ChIP..........chromatin I P
DB.............dot blot
EA..............enzyme assay
EA-IB.........enzyme assay-immunoblot
ELISA ........ enzyme-linked immunosorbent assay
EMSA........electrophoretic mobility shift assay
FACS. ........ fluorescent activated cell sorting
FC..............flow cytometry
GK.............gene knockdown
HAT... ........ histone acetyltransferase assay
HDAC .......HDAC assay
HMT..........histone methyltransferase assay
IAP.............immunoaffinity purification
ICC............immunocytochemistry
IF ...............immunofluorescence
IHC............immunohistochemistry
IP...............immunoprecipitation
IPK ............IP-kinase assay
KA .............kinase assay
MET .... .... ..me thyla tion assay
N ...............neutralisation
PIA.............peptide inhibition assay
PA..............phosphatase assay
RIA ............radio immunoassay
TFX............transfection
WB ............Western blotting
Upstate and Chemicon are both part of the Serologicals family of companies. In this brochure, you will find Chemicons impressive array of DNA
methylation products, rounding out Upstates lineup of products for epigenetics research.
For your convenience, you can order any of our products and get complete technical support from either company.
Suppl ied by Chemicon International
sales orders t: 0800 0190 333 f: 0800 0190 444 tech support: 0800 0190 555 calls outside the UK: +44 (0)1382 560812
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Tested Species Pack Pos.
Description Applications Reactivity Cat. # Size Cntrl. Price
p73
Anti-p73, / WB IP IHC H AB7824 100 g 244
Anti-p73, clone GC15 WB IP EMSA H Ht B Mk 05-509 200 g 224
Rb
Anti-Rb, clone XZ-77 WB IP H Av 05-377 200 g 224
Anti-Rb1 IP H CBL447 100 g 188
Anti-Rb2 (p130) WB H M R 07-282 100 l 202
Anti-Rb protein, underphosphorylated, clone MAB549 WB IP H M MAB3187 50 g 205
WT1
Anti-WT1, clone 6F-H2 WB ICC IHC H 05-753 200 g 217
DNA Methylation Kits & Reagents Tools for measuring levels of silencing-associated CpG methylation in DNA at specific genes
Base Kits
CpGenome Universal DNA Modification Kit MET S7820 100 assays 235
CpGenome Universal Methylated Control DNA MET S7821 10 g 193
CpGenome Universal Unmethylated Control DNA Set MET S7822 5 g 184
CpGenome Fast DNA Modification Kit MET S7824 25 assays 128
Target-Specific Kits
CpG WIZ APC Amplification Kit MET S7812 25 assays 325
CpG WIZ BRCA1 Amplification Kit MET S7830 25 assays 325
CpG WIZ DAP Kinase Amplification Kit MET S7801 25 assays 325
CpG WIZ E-cadherin Amplification Kit MET S7804 25 assays 325
CpG WIZ ER Amplification Kit MET S7815 25 assays 325
CpG WIZ Fragile X Amplification Kit MET S7807 25 assays 325
CpG WIZ GST-pi Amplification Kit MET S7808 25 assays 325CpG WIZ HIN Amplification Kit MET S7816 25 assays 325
CpG WIZ MGMT Amplification Kit MET S7803 25 assays 325
CpG WIZ hMLH1 Amplification Kit MET S7811 25 assays 325
CpG WIZ p14/ARF Amplification Kit MET S7817 25 assays 325
CpG WIZ p15 Amplification Kit MET S7802 25 assays 325
CpG WIZ p16 Amplification Kit MET S7800 25 assays 325
CpG WIZ Prader-Willi/Angelman Amplification Kit MET S7806 25 assays 325
CpG WIZ RASSF1A Amplification Kit MET S7813 25 assays 325
CpG WIZ RAR1 Amplification Kit MET S7814 25 assays 325
CpG WIZ RB1 Amplification Kit MET S7810 25 assays 325
CpG WIZ SOCS1 Amplification Kit MET S7809 25 assays 325
CpG WIZTMS1/ASC Amplification Kit MET S7818 25 assays 325
CpG WIZ VHL Amplification Kit MET S7805 25 assays 325
Antibodies to Methyl DNA Binding Proteins Bind to methylated CpG islands and recruit other proteins, such as HDACs and HMTs
Kaiso
Anti-Kaiso, clone 6F WB IP IHC EMSA WR 05-659 200 g 224
MBD
Anti-MBD2 WB IP EMSA ChIP H M 07-198 200 g 202Anti-MBD2/3 WB H 07-199 200 g 224
MeCP2
Anti-MeCP2 WB H M R 07-013 200 g 224
Anti-Methyl CpG Binding Protein 2 WB H AB3469 100 g 265
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Tested Species Pack Pos.Description Applications Reactivity Cat. # Size Cntrl. Price
Epigenetics Products continued
Enzyme Assay Kits Used to detect levels of specific post-translational modifications on histones
ChIP
Chromatin Immunoprecipitation (ChIP) Assay Kit ChIP 17-295 22 assays 201
EZ-ChIP ChIP 17-371 22 assays 232
HDAC
HDAC Assay Kit (Fluorometric Detection) HDAC 17-356 96 assays 262
Histone Deacetylase Assay Kit (HDAC) HDAC 17-320 100 assays 199
Histone H3
Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit IP ChIP 17-245 22 assays 390
Histone H4
Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit IP ChIP 17-229 22 assays 393
HMT
Histone Methyltransferase Assay Reagent Kit HMT 17-330 100 assays 180
HAT
HAT Assay Kit ELISA 17-289 96 assays 514
HAT Assay Reagent Kit HAT 17-329 100 assays 72
p300
p300/CBP Immunoprecipitation HAT Assay Kit HAT 17-284 40 assays 381
Histone Modifying Enzymes & Proteins Add or subtract modif icat ions to histone proteins and help regulate transcription
CARM 1
CARM1, active HMT 14-575 10 g 232
HAT
Hat1, active KA 14-580 20 g 217
HDAC
HDAC8, active HDAC 14-609 50 g 239
Histone
Core Histones HAT 13-107 1 mg 112
Histone H1
Histone H1 KA 14-155 20 mg 129
Histone H2A
Histone H2A, human EA 14-493 100 g 224
Histone H2A.X
Histone H2A.X KA 14-576 100 g 217
Histone H2A.Z
Histone H2A.Z KA 14-597 100 g 217
Histone H2B
Histone H2B, human EA 14-491 100 g 224
Histone H3
Histone H3, human EA 14-494 100 g 224
Histone H4
Histone H4 EA 14-412 100 g 224
HOS3
HOS3, yeast, active HDAC 14-472 250 g 224
MOZ
MOZ, active EA 14-631 25 g 239
p300
p300, HAT Domain HAT 14-418 50 g 239
PCAF
PCAF, active HAT 14-309 50 g 239
PRMT
PRMT 1, active HMT 14-474 25 g 224
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Tested Species Pack Pos.Description Applications Reactivity Cat. # Size Cntrl. Price
SET
PR-SET7, active HMT 14-539 100 g 224
SET9, active EA 14-469 100 g 239
SIRT / SIR2
SIRT1 Deacetylase HDAC 17-370 1 kit 232
Antibodies to Histone Modifications Histone modifications are reversible events involved in the regulation of transcription.Histone H3
Anti-Histone H3 WB ICC ChIP H M R 06-755 200 g 224
Anti-Histone H3 WB H WR 05-499 200 g 226
Anti-acetyl-Histone H3 WB ICC ChIP H M T 06-599 200 g 236
Anti-acetyl-Histone H3 (Lys9) WB DB Eu 06-942 200 g 236
Anti-acetyl-Histone H3 (Lys9/18) WB ChIP H M B Y 07-593 200 l 217
Anti-acetyl-Histone H3 (Lys27) WB ChIP H Y WR 07-360 100 l 231
Anti-monomethyl-Histone H3 (Lys4) WB IF ChIP H WR 07-436 200 g 224
Anti-monomethyl-Histone H3 (Lys9), clone RR103 WB H 05-713 100 l 239
Anti-monomethyl-Histone H3 (Lys9) WB H 07-395 100 l 216
Anti -monomethyl -Histone H3 (Lys9) WB ICC ChIP P IA H M Ch 07-450 100 g 210
Anti-monomethyl-Histone H3 (Lys27) WR WR 07-448 200 g 217
Anti-mono/di/trimethyl-Histone H3 (Lys4), clone AW304 WB ChIP BD H WR 05-791 100 l 217
Anti-dimethyl-Histone H3 (Lys4), clone RR302 WB H Ch WR 05-684 400 l 216
Anti-dimethyl-Histone H3 (Lys4), clone AW30 WB BD H 05-790 100 l 232
Anti-dimethyl-Histone H3 (Lys4) WB ICC DB IF ChIP H T 07-030 200 l 232
Anti-dimethyl (Lys4) dimethyl (Lys9) Histone H3 WB IP ICC H Ch 07-370 100 l 216
Anti-dimethyl-Histone H3 (Lys9), clone RR202 WB ELISA H Ch Xn 05-685 200 l 216
Anti-dimethyl-Histone H3 (Lys9), clone MC554 WB IF H 05-768 100 l 232
Anti-dimethyl-Histone H3 (Lys9) WB ICC H Ch Y WR 07-212 100 l 190
Anti-dimethyl-Histone H3 (Lys9) WR H M Ch Y 07-441 100 g 210
Anti-dimethyl-Histone H3 (Lys9) WB BD H WR 07-521 200 l 224
Anti-dimethyl-Histone H3 (Lys9), biotin conjugated WB ICC 16-187 100 l 246
Anti-dimethyl-Histone H3 (Lys27) WB H WR 07-322 200 g 194
Anti-dimethyl-Histone H3 (Lys27) WB ChIP WR 07-421 100 l 232
Anti-dimethyl-Histone H3 (Lys27) WR H 07-452 200 g 217
Anti-trimethyl-Histone H3 (Lys4), clone MC315 WB ChIP H 05-745 100 g 232
Anti-trimethyl-Histone H3 (Lys4) WB DB ChIP H WR 07-473 200 l 216Anti-trimethyl-Histone H3 (Lys9) WB DB IF ChIP H 07-442 100 g 210
Anti-trimethyl-Histone H3 (Lys9) WB H 07-523 200 l 217
Anti-trimethyl-Histone H3 (Lys27) WB ICC ChIP PIA H M WR 05-851 100 l 211
Anti-trimethyl-Histone H3 (Lys27) WB ICC ChIP PIA H 07-449 200 g 217
Antibodies to Histone Modifying Enzymes Add or subtract modifi cations to histone proteins and help regulate transcription
ACTR
Anti-ACTR/AIB1, clone AX15 WB IP H 05-490 150 l 224
CARM1
Anti-CARM1 WB IP H M R 07-080 100 l 202
CBP
Anti-CBP NT WB H M R 06-297 200 g 202
ESET
Anti-ESET/SetDB1 WB H M 07-378 100 l 194
G9a
Anti-G9a WB H 07-551 200 g 210
GCN5
Anti-GCN5, Histone Acetyltransferase WB IHC ELISA H MAB3622 100 l 338
HDAC
Anti-HDAC1 WB IP IC H M R 06-720 200 g 226
Anti-HDAC1, clone 2E10 WB IP IC ChIP H M 05-614 200 g 224Anti-HDAC2 WB H M 07-222 200 l 202
Anti-HDAC2, clone 3F3 WB IP IC HDAC H M Ht B 05-814 200 g 217
Anti-HDAC3 WB IP HDAC H 07-522 200 g 210
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Tested Species Pack Pos.Description Applications Reactivity Cat. # Size Cntrl. Price
Anti-HDAC3 WB H M R 06-890 200 g 202
Anti-HDAC3, clone 3G6 WB IP IC HDAC H M Ht B 05-813 200 g 217
Anti-HDAC4 WB IC H M 07-040 200 g 224
Anti-HDAC5 WB H M R 07-045 200 g 224Anti-HDAC8 WB IP H M 07-505 100 l 202
MLL/HRX
Anti-MLL/HRX, C-term., clone 9-12 WB IP H M 05-765 200 g 217
Anti-MLL/HRX, N-term., clone N4.4 WB IP M H 05-764 200 g 217
MOZ
Anti-MOZ; MYST Histone Acetyltransferase 3 WB H M R AB4141 100 g 193
p300
Anti-p300 CT, clone RW 128 WB IP ChIP H M R 05-257 200 g 244
Anti-dimethyl-p300 (Arg2142) WB H 07-656 100 l 211
PRMT
Anti-PRMT1 WB H M 07-404 200 g 194Anti-PRMT3 WB H M R 07-256 200 g 202
Anti-PRMT5 WB IP H M 07-405 200 g 202Anti-PRMT7 WB H M 07-639 200 l 204
SET
Anti-SET07 (Histone H4-K20 Methyltransferase) WB ELISA H AB3351 100 g 201
Anti-hPR-SET7 WB IC H 07-316 100 l 210
Anti-SET9 WB H 07-314 200 g 202
SIRT/SIR2
Anti-Sirt1, clone 2G1/F7 WB IP H 05-707 200 g 224
Anti-Sir2 WB IP ICC H M 07-131 200 l 202
SUV39H1
Anti-SUV39H1 WB H M 07-550 200 g 210
Anti-SUV39H1, clone MG44 WB IP ChIP H M 05-615 200 l 224
Anti-SUV39H1, Histone H3-K9 Methyltransferase 1 WB ICC ELISA H AB3353 100 l 210
Tip60
Anti-Tip60 WB H M 07-038 200 g 224
Antibodies to Regulators of Chromatin Function Proteins that influence transcription and silencing through effects on chromatin structure
Bmi-1
Anti-Bmi-1, clone F6 WB IP ICC H M R Rb 05-637 100 g 224
EED
Anti-EED WB H M 07-368 100 l 194
EZH2
Anti-EZH2 WB ICC H M R Rb 07-400 200 l 194
HP1
Anti-HP1 WB H M 07-346 200 l 202
Anti-HP1, clone15.19s2 WB IHC ChIP H M WR 05-689 200 g 216
Anti-HP1 WB H M 07-333 200 g 202Anti-HP1 WB IHC ELISA IF H M MAB3448 100 l 338
Anti-HP1 WB H M 07-332 200 g 202Anti-HP1, clone 42s2 WB IHC ChIP H M 05-690 200 g 216
Mi-2
Anti-Mi-2 WB H 06-878 200 g 202
p66
Anti-p66 (MeCP1 repressor component) WB H M 07-365 200 g 194
SNF
Anti-hSNF2, clone 1B9/D12 WB H M 05-698 100 g 202
Anti-hSNF2H WB IP ChIP H 07-624 100 l 188Anti-SNF2/BRG1 WB I P ICC H M 07-478 200 l 202
Anti-SNF2p (yeast specific) WB IP Y 07-319 200 g 202
Anti-SNF5p (yeast specific) WB IP Y 07-320 200 g 202
Epigenetics Products continued
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Tested Species Pack Pos.Description Applications Reactivity Cat. # Size Cntrl. Price
SRC-1
Anti-SRC-1, clone 1135 WB IP H M R 05-522 100 g 224
SUZ12
Anti-SUZ12 WB H M 07-379 100 l 194
TRF2
Anti-TRF2, clone 4A794 WB ICC H R 05-521 100 g 224
ChIP-Qualified Antibodies toStudy Histone MethylationAntibodies specific for mono-, di- and tri-methylated histones
Histone methylation is an important phenomenon thatis involved in regulating access to specific regions of the
genome. Upstate has developed a panel of antibodies that
recognise each of the methylated versions (mono, di and tri)
of all the widely studied and biologically relevant methylation
sites on histones H3 and H4. They are qualified for use in
chromatin immunoprecipitation (ChIP) and most work well
in a variety of other applications, like Western blotting,
immunofluorescence and immunohistochemistry.
Check outwww.upstate.com/chip for all available ChIP-
qualified antibodies.
Legend Mouse embryonic fibroblasts stained with polyclonal antibodiesthat recognise monomethyl (left panels), dimethyl (middle panels) or
trimethyl (right panels) histone H3. Top panels (green): antibodiesspecific for H3 lysine 27 methylation. Bottom panels (red): antibodies
specific for H3 lysine 9 methylation were employed. Note: the characteristiclocalization of trimethyl H3 (Lys27) to the inactive X-chromosome (Xi,
upper right panel). Consult the chart to the left for catalog numbers ofthe products used. Images courtesy Dr. Thomas Jenuwein, Institute forMolecular Pathology, Vienna.
Histone H3 Methylationin Mouse Embryonic Fibroblasts
Mono Di TriHistone Methylation Site Cat. # Cat. # Cat. #
Histone H3 (Lys4) 07-436 07-030 05-745, 07-473
Histone H3 (Lys9) 07-450 07-441 07-442
Histone H3 (Lys27) 07-448 07-452 05-581, 07-449
Histone H4 (Lys20) 07-440 07-367 07-463
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opyright,2005
Upstate
Group
LLC.
AllRightsReserved.
FBC-FE4
Our commitment to quality
comes from two simple
beliefs beliefs we are
sure you share.
Good reagents are those that
produce meaningful resul ts
Your time is valuable
This commitment to quality is reflected in our no
risk guarantee: if any Upstate product fails to meet
the physical criteria listed on the accompanying
certificate of analysis, just let us know, and we'll
replace the product or refund your money.
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To place an order:
tel +44 (0) 1382 560812 freephone (UK only) 0800 0190 333
fax +44 (0) 1382 560802 freefax (UK only) 0800 0190 444
e-mail [email protected]
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To place an order:
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e-mail [email protected]
For tech support:
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Upstate offers over 2,700 antibodies, kinases, phosphatases,
siRNA kits, assay systems, substrates, inhibitors, cDNAs,
cell growth and multiplexing products for cell signalling.
Visit us at www.upstate.com to find out more.
CpGenome is a trademark of Chemicon International, Inc.
CpG WARE is a trademark of Chemicon International, Inc.
CpG WIZ is a registered trademark of Chemicon International, Inc.
IHC Select is a registered trademark of Chemicon International, Inc.
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CHEMICON International, Inc.
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To place an o rder:
tel 951 676 8080 toll free 800 437 7500
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fax 03 9887 3912
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