S P R I N G 20 0 5

P R O D U C T

G U I D E

Epigenetics

FEATURING
Epigenetics: The Cancer Code of Silence by Jean-Pierre Issa, M.D., Feature Article PLUS! X Chromosome Inactivation and Chromatin IP Highlight Sections Chemicon Methylation Products Upstate Products to Histone Modifications

Diagram Inside Epigenetic Silencing of a Tumour Suppressor Gene

Epigenetics
The 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

EPIGENETICS NORMAL CELLS Epigenetics refers to stable changes in gene expression that are 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 enzymatic systems to methylate DNA, RNA and proteins. Protein methylation has been intensively studied recently and is an essential posttranslational 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 the genome. 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 associated gene. 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 by other 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

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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).

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 as EZH2). 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.

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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 consequences of 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 molecular anomaly affects distinct alleles. This shared tissue distribution and allelic exclusion of mutations and DNA methylation in sporadic 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 viceversa). By extension, if one believes mutations of those genes functionally lead to cancer, the logical conclusion is that methylation-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 replication in 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 HERRINGS There 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 methylationassociated 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, particularly at 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 recruitment of 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 selfreinforcing 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 modest level 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 interplay between multiple synergistic silencing mechanisms, including promoter CpG island methylation and histone code modifications. These silencing mechanisms show cancer-specificity and are, therefore, appropriate targets for therapeutic intervention. DNA methylation 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, 2388S2392S. 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: Epigenetics www.wellcome.ac.uk/en/genome/thegenome/hg02b002.html

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PRODUCT

HIGHLIGHT

X Chromosome Inactivation
Epigenetic Silencing at the Chromosomal Level
Enrichment of Histone macroH2A on Xi Chromosome

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.

X chromosome inactivation 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 chromosomalspecific localisation in the case of the histone variant macroH2A.

Hallmarks of the Inactive X Chromosome

Increased on the Xi Decreased on the Xi

Xist RNA
H3 Methylation Staining

Histone H4 Acetylation Histone H3 K4 Methylation

DNA Methylation Histone MacroH2A Histone H3 K9 Dimethylation Histone H3 K27 Trimethylation Histone H4 K20 Monomethylation EZH2

Inactive X Chromosome
Legend Indirect immunofluorescence (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 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.

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PRODUCT

HIGHLIGHT

Chromatin IP
The Nexus of Genomics & Proteomics
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 IP Pathway

Chromatin IP Products
Cat. # 17-371 17-295 17-245 17-229 16-157 16-201 Description EZ-ChIP Chromatin Immunoprecipitation Kit Chromatin Immunoprecipitation (ChIP) Assay Kit Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit Protein A agarose/Salmon Sperm DNA Protein G Agarose/Salmon Sperm DNA Pack Size 1 kit 1 kit 1 kit 1 kit 1 mg 1 mg Price
Legend Schematic representation of the chromatin immunoprecipitation (ChIP) technique. First, the proteins are crosslinked to DNA with formaldehyde, and then the chromatin is sheared to a manageable size by sonication. Specific proteins are immunoprecipitated with antibodies, also bringing down the DNA to which the protein is cross-linked. The cross-links are reversed, the DNA purified, and the sample is interrogated for the enrichment of specific DNA sequences. The detection step can be performed most accurately by quantitative real-time PCR, but the usage of microarrays in this step is increasing.

H3 Methylation as Domain Markers

EZ-ChIP Kit (cat. #17-371)

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 Shiv Grewal, Cold Spring Harbor Laboratories.

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PRODUCT

HIGHLIGHT

CpGenome & CpG WIZ

Methylation-specific PCR* (MSP) Systems

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 the DNA, 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 treatment. 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.
S7822 CpGenome Universal Unmethylated DNA Set
184 Cat. # Description Price 193 Cat. # Description Price 235 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 Modification Kit

CpGENOME UNIVERSAL METHYLATED CONTROL DNA Chemicons 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

CpGENOME UNIVERSAL UNMETHYLATED DNA SET The 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.
Cat. # Description Price

Detection of the Methylation State of the p16 Gene

Methylation Specific PCR* (MSP) of the p16 gene in two invasive carcinomas, a squamous intraepithelial lesion (SIL), and an adenocarcinoma of the cervix. Each numbered set is paired 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 the methylation 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 adenocarcinoma sample is clearly homozygous for the unmethylated state at the p16 locus.

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Step 1. Bisulfite Treatment The first step in MSP involves the chemical conversion of all unmethylated cytosines 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 treatment will be different depending on the original methylation state 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 mispriming between 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 include primers (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 gel stained to visualize the DNA. If the sample DNA was originally unmethylated prior to modification, 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 Kits

Cat. # Description Price 325 325 325 325 325 325 325 325 325 325 Cat. # Description Price 325 325 325 325 325 325 325 325 325 325

S7800 S7801 S7802 S7803 S7804 S7805 S7806 S7807 S7808 S7809

CpG WIZ p16 Amplification Kit CpG WIZ DAP Kinase Amplification Kit CpG WIZ p15 Amplification Kit CpG WIZ MGMT Amplification Kit CpG WIZ E-cadherin Amplification Kit CpG WIZ VHL Amplification Kit CpG WIZ Prader-Willi/Angelman Amplification Kit CpG WIZ Fragile X Amplification Kit CpG WIZ GST-pi Amplification Kit CpG WIZ SOCS1 Amplification Kit

S7810 S7811 S7812 S7813 S7814 S7815 S7816 S7817 S7818 S7830

CpG WIZ RB1 Amplification Kit CpG WIZ hMLH1 Amplification Kit CpG WIZ APC Amplification Kit CpG WIZ RASSF1A Amplification Kit CpG WIZ RAR1 Amplification Kit CpG WIZ ER Amplification Kit

CpG WIZ HIN Amplification Kit CpG WIZ p14/ARF Amplification Kit CpG WIZ TMS1/ASC Amplification Kit CpG WIZ BRCA1 Amplification Kit

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Epigenetics
Product List
Modification
..........acetylation .........methylation

Tested Applications

(see note below) HMT..........histone methyltransferase assay IAP.............immunoaffinity purification ICC ............immunocytochemistry IF ...............immunofluorescence IHC............immunohistochemistry IP ...............immunoprecipitation IPK ............IP-kinase assay KA .............kinase assay MET ..........methylation assay N ...............neutralisation PIA.............peptide inhibition assay PA ..............phosphatase assay RIA ............radio immunoassay TFX ............transfection WB ............Western blotting

Tested Species Reactivity

Am ............amphibian Av ..............avian 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

(see note below)

APA ...........affinity precipitation BA .............biologically active BD.............Beadlyte assay CC .............cell culture ChIP ..........chromatin IP 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

Mi ..............mink 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

Note: Additional species and applications may apply. Call Tech Support at 0805 0190 555 for more information.

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. Supplied by Chemicon International

Epigenetics Products
Description Tested Applications Species Reactivity Cat. # Pack Size Pos. Cntrl. Price

Antibodies to Tumour Suppressors

cer APC Anti-APC DAPK2 Anti-DAP Kinase 2 E-cadherin Anti-E-cadherin, clone 674A FMRP Anti-FMRP ING Anti-ING1, clone CAb3 MLH1 Anti-hMLH1 MGMT Anti-MGMT, clone MT3.1 p16 Anti-p16, clone D25 Anti-p16, clone ZI11 p19 ARF Anti-p19 ARF p53 Anti-p53, clone BP53-12 Anti-acetyl-p53 (Lys320) Anti-acetyl-p53 (Lys373) Anti-acetyl-p53 (Lys373, Lys382)

Important regulators of cell division whose loss of function by mutation or silencing can contribute to can-

IHC IP FC WB WB FC WB IHC ELISA FC WB IP ICC WB IP WB IHC IP FC WB IHC WB IHC IF WB IP WB IP WB IP WB IP WB

H HMR H H H H H H H MR H H H WR

AB4063 AB3606 MAB3199 MAB2160 05-720 AB3902 MAB16200 MAB4133 MAB88057 07-543 05-224 06-915 06-916 06-758

100 g 100 g 100 g 100 l 100 g 50 g 100 g 100 g 50 g 200 l 200 200 200 200 l l l l

193

180

197

287

217

137

197

197 146

204

224 202 202 231

10

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Description

Tested Applications

Species Reactivity

Cat. #

Pack Size

Pos. Cntrl.

Price

p73 Anti-p73, / Anti-p73, clone GC15 Rb Anti-Rb, clone XZ-77 Anti-Rb1 Anti-Rb2 (p130) Anti-Rb protein, underphosphorylated, clone MAB549 WT1 Anti-WT1, clone 6F-H2 WB ICC IHC H 05-753 200 g
217

WB IP IHC WB IP EMSA WB IP IP WB WB IP

H H Ht B Mk H Av H HMR HM

AB7824 05-509 05-377 CBL447 07-282 MAB3187

100 g 200 g 200 100 100 50 g g l g

244 224

224 188 202 205

DNA Methylation Kits & Reagents

Base Kits CpGenome CpGenome CpGenome CpGenome Target-Specific Kits CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ CpG WIZ Universal Universal Universal Fast DNA

Tools for measuring levels of silencing-associated CpG methylation in DNA at specific genes MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET S7820 S7821 S7822 S7824 S7812 S7830 S7801 S7804 S7815 S7807 S7808 S7816 S7803 S7811 S7817 S7802 S7800 S7806 S7813 S7814 S7810 S7809 S7818 S7805 100 10 5 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 assays g g assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays assays
235 193 184 128

DNA Modification Kit Methylated Control DNA Unmethylated Control DNA Set Modification Kit

APC Amplification Kit BRCA1 Amplification Kit DAP Kinase Amplification Kit E-cadherin Amplification Kit ER Amplification Kit Fragile X Amplification Kit GST-pi Amplification Kit HIN Amplification Kit MGMT Amplification Kit hMLH1 Amplification Kit p14/ARF Amplification Kit p15 Amplification Kit p16 Amplification Kit Prader-Willi/Angelman Amplification Kit RASSF1A Amplification Kit RAR1 Amplification Kit RB1 Amplification Kit SOCS1 Amplification Kit TMS1/ASC Amplification Kit VHL Amplification Kit

325 325 325 325 325 325 325 325 325 325 325 325 325 325 325 325 325 325 325 325

Antibodies to Methyl DNA Binding Proteins

Kaiso Anti-Kaiso, clone 6F MBD Anti-MBD2 Anti-MBD2/3 MeCP2 Anti-MeCP2 Anti-Methyl CpG Binding Protein 2

Bind to methylated CpG islands and recruit other proteins, such as HDACs and HMTs WB IP IHC EMSA WB IP EMSA ChIP WB WB WB WR HM H HMR H 05-659 07-198 07-199 07-013 AB3469 200 g 200 g 200 g 200 g 100 g

224

202 224

224 265

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Epigenetics Products continued

Description Tested Applications Species Reactivity Cat. # Pack Size Pos. Cntrl. Price

Enzyme Assay Kits

ChIP

Used to detect levels of specific post-translational modifications on histones ChIP ChIP HDAC HDAC IP ChIP IP ChIP HMT ELISA HAT HAT 17-295 17-371 17-356 17-320 17-245 17-229 17-330 17-289 17-329 17-284 22 assays 22 assays 96 assays 100 assays 22 assays 22 assays 100 assays 96 assays 100 assays 40 assays
201 232

Chromatin Immunoprecipitation (ChIP) Assay Kit EZ-ChIP HDAC HDAC Assay Kit (Fluorometric Detection) Histone Deacetylase Assay Kit (HDAC) Histone H3 Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit Histone H4 Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit HMT Histone Methyltransferase Assay Reagent Kit HAT HAT Assay Kit HAT Assay Reagent Kit p300 p300/CBP Immunoprecipitation HAT Assay Kit

262 199

390

393

180

514 72

381

Histone Modifying Enzymes & Proteins

CARM 1 CARM1, active HAT Hat1, active HDAC HDAC8, active Histone Core Histones Histone H1 Histone H1 Histone H2A Histone H2A, human Histone H2A.X Histone H2A.X Histone H2A.Z Histone H2A.Z Histone H2B Histone H2B, human Histone H3 Histone H3, human Histone H4 Histone H4 HOS3 HOS3, yeast, active MOZ MOZ, active p300 p300, HAT Domain PCAF PCAF, active PRMT PRMT 1, active

Add or subtract modifications to histone proteins and help regulate transcription HMT KA HDAC HAT KA EA KA KA EA EA EA HDAC EA HAT HAT HMT 14-575 14-580 14-609 13-107 14-155 14-493 14-576 14-597 14-491 14-494 14-412 14-472 14-631 14-418 14-309 14-474 10 g 20 g 50 g 1 mg 20 mg 100 g 100 g 100 g 100 g 100 g 100 g 250 g 25 g 50 g 50 g 25 g
232

217

239

112

129

224

217

217

224

224

224

224

239

239

239

224

12

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Description

Tested Applications

Species Reactivity

Cat. #

Pack Size

Pos. Cntrl.

Price

SET PR-SET7, active SET9, active SIRT / SIR2 SIRT1 Deacetylase HMT EA HDAC 14-539 14-469 17-370 100 g 100 g 1 kit
224 239

232

Antibodies to Histone Modifications

Histone modifications are reversible events involved in the regulation of transcription. WB WB WB WB WB WB WB WB WB WB WR WB WB WB WB WB WB WB WB WR WB WB WB WB WR WB WB WB WB WB WB ICC ChIP ICC ChIP DB ChIP ChIP IF ChIP HMR H WR HMT Eu HMBY H Y WR H WR H H H M Ch WR H WR H Ch WR H HT H Ch H Ch Xn H H Ch Y WR H M Ch Y H WR H WR WR H H H WR H H H M WR H 06-755 05-499 06-599 06-942 07-593 07-360 07-436 05-713 07-395 07-450 07-448 05-791 05-684 05-790 07-030 07-370 05-685 05-768 07-212 07-441 07-521 16-187 07-322 07-421 07-452 05-745 07-473 07-442 07-523 05-851 07-449 200 200 200 200 200 100 200 100 100 100 200 100 400 100 200 100 200 100 100 100 200 100 200 100 200 100 200 100 200 100 200 g g g g l l g l l g g l l l l l l l l g l l g l g g l g l l g
224 226 236 236 217 231 224 239 216 210 217 217 216 232 232 216 216 232 190 210 224 246 194 232 217 232 216 210 217 211 217

Histone H3 Anti-Histone H3 Anti-Histone H3 Anti-acetyl-Histone H3 Anti-acetyl-Histone H3 (Lys9) Anti-acetyl-Histone H3 (Lys9/18) Anti-acetyl-Histone H3 (Lys27) Anti-monomethyl-Histone H3 (Lys4) Anti-monomethyl-Histone H3 (Lys9), clone RR103 Anti-monomethyl-Histone H3 (Lys9) Anti-monomethyl-Histone H3 (Lys9) Anti-monomethyl-Histone H3 (Lys27) Anti-mono/di/trimethyl-Histone H3 (Lys4), clone AW304 Anti-dimethyl-Histone H3 (Lys4), clone RR302 Anti-dimethyl-Histone H3 (Lys4), clone AW30 Anti-dimethyl-Histone H3 (Lys4) Anti-dimethyl (Lys4) dimethyl (Lys9) Histone H3 Anti-dimethyl-Histone H3 (Lys9), clone RR202 Anti-dimethyl-Histone H3 (Lys9), clone MC554 Anti-dimethyl-Histone H3 (Lys9) Anti-dimethyl-Histone H3 (Lys9) Anti-dimethyl-Histone H3 (Lys9) Anti-dimethyl-Histone H3 (Lys9), biotin conjugated Anti-dimethyl-Histone H3 (Lys27) Anti-dimethyl-Histone H3 (Lys27) Anti-dimethyl-Histone H3 (Lys27) Anti-trimethyl-Histone H3 (Lys4), clone MC315 Anti-trimethyl-Histone H3 (Lys4) Anti-trimethyl-Histone H3 (Lys9) Anti-trimethyl-Histone H3 (Lys9) Anti-trimethyl-Histone H3 (Lys27) Anti-trimethyl-Histone H3 (Lys27)

ICC ChIP PIA ChIP BD BD ICC DB IF ChIP IP ICC ELISA IF ICC BD ICC ChIP ChIP DB ChIP DB IF ChIP ICC ChIP PIA ICC ChIP PIA

Antibodies to Histone Modifying Enzymes

ACTR Anti-ACTR/AIB1, clone AX15 CARM1 Anti-CARM1 CBP Anti-CBP NT ESET Anti-ESET/SetDB1 G9a Anti-G9a GCN5 Anti-GCN5, Histone Acetyltransferase HDAC Anti-HDAC1 Anti-HDAC1, clone 2E10 Anti-HDAC2 Anti-HDAC2, clone 3F3 Anti-HDAC3

Add or subtract modifications to histone proteins and help regulate transcription WB IP WB IP WB WB WB WB IHC ELISA WB WB WB WB WB IP IC IP IC ChIP IP IC HDAC IP HDAC H HMR HMR HM H H H H H H H MR M M M Ht B 05-490 07-080 06-297 07-378 07-551 MAB3622 06-720 05-614 07-222 05-814 07-522 150 l 100 l 200 g 100 l 200 g 100 l 200 200 200 200 200 g g l g g
224

202

202

194

210

338

226 224 202 217 210

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13

Epigenetics Products continued

Description Tested Applications Species Reactivity Cat. # Pack Size Pos. Cntrl. Price 202 217 224 224 202

Anti-HDAC3 Anti-HDAC3, clone 3G6 Anti-HDAC4 Anti-HDAC5 Anti-HDAC8 MLL/HRX Anti-MLL/HRX, C-term., clone 9-12 Anti-MLL/HRX, N-term., clone N4.4 MOZ Anti-MOZ; MYST Histone Acetyltransferase 3 p300 Anti-p300 CT, clone RW 128 Anti-dimethyl-p300 (Arg2142) PRMT Anti-PRMT1 Anti-PRMT3 Anti-PRMT5 Anti-PRMT7 SET Anti-SET07 (Histone H4-K20 Methyltransferase) Anti-hPR-SET7 Anti-SET9 SIRT/SIR2 Anti-Sirt1, clone 2G1/F7 Anti-Sir2 SUV39H1 Anti-SUV39H1 Anti-SUV39H1, clone MG44 Anti-SUV39H1, Histone H3-K9 Methyltransferase 1 Tip60 Anti-Tip60

WB WB IP IC HDAC WB IC WB WB IP WB IP WB IP WB WB IP ChIP WB WB WB WB IP WB WB ELISA WB IC WB WB IP WB IP ICC WB WB IP ChIP WB ICC ELISA WB

H H H H H

MR M Ht B M MR M

06-890 05-813 07-040 07-045 07-505 05-765 05-764 AB4141 05-257 07-656 07-404 07-256 07-405 07-639 AB3351 07-316 07-314 05-707 07-131 07-550 05-615 AB3353 07-038

200 200 200 200 100

g g g g l

HM MH HMR HMR H H H H H H H H H HM HM HM H HM M MR M M

200 g 200 g 100 g 200 g 100 l 200 200 200 200 g g g l

217 217

193

244 211

194 202 202 204

100 g 100 l 200 g 200 g 200 l 200 g 200 l 100 l 200 g

201 210 202

224 202

210 224 210

224

Antibodies to Regulators of Chromatin Function

Bmi-1 Anti-Bmi-1, clone F6 EED Anti-EED EZH2 Anti-EZH2 HP1 Anti-HP1 Anti-HP1, clone15.19s2 Anti-HP1 Anti-HP1 Anti-HP1 Anti-HP1, clone 42s2 Mi-2 Anti-Mi-2 p66 Anti-p66 (MeCP1 repressor component) SNF Anti-hSNF2, clone 1B9/D12 Anti-hSNF2H Anti-SNF2/BRG1 Anti-SNF2p (yeast specific) Anti-SNF5p (yeast specific)

Proteins that influence transcription and silencing through effects on chromatin structure WB IP ICC WB WB ICC WB WB IHC ChIP WB WB IHC ELISA IF WB WB IHC ChIP WB WB WB WB WB WB WB H M R Rb HM H M R Rb H H H H H H H HM HM H HM Y Y M M WR M M M M 05-637 07-368 07-400 07-346 05-689 07-333 MAB3448 07-332 05-690 06-878 07-365 05-698 07-624 07-478 07-319 07-320 100 g 100 l 200 l 200 200 200 100 200 200 l g g l g g
224

194

194

202 216 202 338 202 216

200 g 200 g 100 100 200 200 200 g l l g g

202

194

IP ChIP IP ICC IP IP

202 188 202 202 202

14

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tech support: 0800 0190 555

calls outside the UK: +44 (0)1382 560812

Description

Tested Applications

Species Reactivity

Cat. #

Pack Size

Pos. Cntrl.

Price

SRC-1 Anti-SRC-1, clone 1135 SUZ12 Anti-SUZ12 TRF2 Anti-TRF2, clone 4A794 WB ICC HR 05-521 100 g WB HM 07-379 100 l WB IP HMR 05-522 100 g

224

194

224

ChIP-Qualified Antibodies to Study Histone Methylation

Antibodies specific for mono-, di- and tri-methylated histones
Histone methylation is an important phenomenon that is 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 out www.upstate.com/chip for all available ChIPqualified antibodies.

Histone H3 Methylation in Mouse Embryonic Fibroblasts

Histone Methylation Site

Mono Cat. #

Di Cat. #

Tri Cat. #

Histone H3 (Lys4) Histone H3 (Lys9) Histone H3 (Lys27) Histone H4 (Lys20)

07-436 07-450 07-448 07-440

07-030 07-441 07-452 07-367

05-745, 07-473 07-442 05-581, 07-449 07-463

Legend Mouse embryonic fibroblasts stained with polyclonal antibodies that recognise monomethyl (left panels), dimethyl (middle panels) or trimethyl (right panels) histone H3. Top panels (green): antibodies specific for H3 lysine 27 methylation. Bottom panels (red): antibodies specific for H3 lysine 9 methylation were employed. Note: the characteristic localization of trimethyl H3 (Lys27) to the inactive X-chromosome (Xi, upper right panel). Consult the chart to the left for catalog numbers of the products used. Images courtesy Dr. Thomas Jenuwein, Institute for Molecular Pathology, Vienna.

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15

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.

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.

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www.chemicon.com CHEMICON International, Inc. 28820 Single Oak Drive Temecula, CA 92590 To place an order: 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, Australia To place an order: tel 03 9839 2000 toll free (Australia only) 800 252 265 fax 03 9887 3912 e-mail custserv@chemicon.com.au CHEMICON Europe, Ltd. The Science Centre Eagle Close, Chandlers Ford Hampshire, SO53 4NF, UK tel 023 8026 2233 fax 023 8025 2508 e-mail uk@chemicon.com

www.upstate.com UK 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 ukinfo@upstate.com For tech support: tel +44 (0) 1382 560813 freephone (UK only) 0800 0190 555 e-mail uktechserv@upstate.com United States 706 Forest Street Charlottesville, Virginia 22903-5231 To place an order: tel 434 975 4300 toll free 800 233 3991 fax 434 220 0480 toll free fax 866 831 3991 e-mail info@upstate.com For tech support: tel 800 548 7853 e-mail techserv@upstate.com
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