epigenetics - the cancer code of silence

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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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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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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CpGenome & CpG WIZ

Methylation-specific PCR* (MSP) Systems

8

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.


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



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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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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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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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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



15/16

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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



16/16

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.

www.upst ate.comUK

Fischer Building

Gemini Crescent, Dundee Technology Park

Dundee, DD2 1SW, UK

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]

For tech support:

tel +44 (0) 1382 560813

freephone (UK only) 0800 0190 555


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Charlottesville, Virginia 22903-5231

To place an order:

tel 434 975 4300 toll free 800 233 3991fax 434 220 0480 toll free fax 866 831 3991


For tech support:

tel 800 548 7853


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.

www.chemicon.com

CHEMICON International, Inc.

28820 Single Oak Drive

Temecula, CA 92590

To place an o rder:

tel 951 676 8080 toll free 800 437 7500

fax 951 676 9209 toll free fax 800 437 7502

CHEMICON Australia Pty. Ltd.

34 Wadhurst Drive

Boronia, Victoria 3155, AustraliaTo place an o rder:

tel 03 9839 2000 toll free (Australia only) 800 252 265

fax 03 9887 3912


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The Science Centre

Eagle Close, Chandlers Ford

Hampshire, SO53 4NF, UK

tel 023 8026 2233

fax 023 8025 2508


epigenetics - the cancer code of silence

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