motif vations

THE NEWSLETTER OF ACTIVE MOTIFAPRIL 2015 | VOLUME 16 NUMBER 1

Special Cancer Epigenetics Edition

Enabling Epigenetics ResearchAntibodies, Kits and Services for Epigenetics & Gene Regulation Research

Cancer Epigenetics

IN THIS ISSUE
2

Cancer Epigenetics

4

NEW: CRISPR / Cas9 Gene Editing

4

NEW: ChIP-Seq Validated iPSC Factors

5

FOCUS: Histone H3K27me3 Antibody

6

NEW: Tools to Assess Changes in DNA
Methylation in Normal and Diseased
Samples7

NEW: ChIP Normalization Reagents
Reduce Effects of Technical Variation
and Reveal Subtle Biological Changes

8

NEW: Tag-ChIP-IT® Enables ChIP
Without Protein-specific Antibodies

9

NEW: Histone Acetylation Analysis –Finding the Right Tools For Your Research

10 Tools to Enable Insight into theCancer Epigenome
12 NEW: Study Regulation of lncRNA
Promoters
13 Dysregulation of miRNAs in Cancer
14 Enzymes & Substrates for Drug
Discovery
15 NEW: Epigenetic Services: RIME for
Identification of Transcriptional
Networks Associated with Disease2

For over a decade, the number of known associations between
epigenetic changes and cancer has been growing. During tumor
initiation and progression, the epigenome goes through multiple
alterations, including a genome-wide loss of DNA methylation,
frequent increases in promoter methylation of CpG islands, changes
in nucleosome occupancy and shifts in modification profiles. As
researchers investigate the role of epigenetics in cancer, a growing
number of tools and techniques are available to aid in their research.

Cancer-linked epigenetic modifications
are found genome wide
Epigenetic alterations in cancer
include changes in DNA methylation
and associated histone modifications
that influence the chromatin state andimpact gene expression. The development of methods, such as chromatin
immunoprecipitation (ChIP) and DNA
methylation enrichment techniques,along with Next-Generation Sequencing
tools, is enabling researchers to geta better picture of the genome-widechanges associated with cancer andother diseases.
Not every antibody is suitable for
epigenetic techniques
One of the greatest challenges for
epigenetic-based research has been
the lack of antibodies displaying theproper specificity that have beenvalidated for use in techniques suchas ChIP and ChIP-Seq. The problem
has been compounded by numerous
antibody suppliers who do not manufacture or test the antibodies they sell,
and who sell them to one another andthen to researchers.

Developing and manufacturing histonespecific antibodies is challenging
because histones have multiple posttranslational modifications along their“tail” portion that are involved in gene
regulation and disease. Effective
antibodies must clearly differentiate
between subtle variances in thesemodifications and also be able to perform
in demanding epigenetic techniques.
What makes a good antibody?
Active Motif has established a validation
program for its antibodies to qualify
them for their intended use. This
includes ChIP-Seq testing, ChIP
validation, as well as testing thespecificity of our histone antibodies
with a unique MODified™ Histone
Peptide Array. For a complete list of
our epigenetics antibodies, please
visit www.activemotif.com/antibodies.

“The epigenetic state iscontrolled by the activity ofproteins that add and remove
small chemical modifications tohistones and directly to DNA”

www.activemotif.com

Epigenetics of Cancer

Multi-Kac

√

K18ac

√

K9me1-3+

K9ac

bromodomain
chromodomain
PHD finger
WD40 repeat

K4me1-3acetylases
methylases
kinasesMulti-Kac

READERSWRITERS

Breast√K20me3The central role of ChIP in
epigenetic studies
ChIP is the principal technique used
to map epigenetic marks to individualloci in the genome. By using ChIP,
researchers can determine the relationships between DNA and histone
modifications or proteins such astranscription factors, and gene
expression.

Tumor Type

Histone H4
K16ac

Tools for DNA methylation analysis
Due to the roles it plays in development
and disease, much research depends
on the ability to accurately detect andquantify DNA methylation. Active Motif
offers a number of products specificfor this area of research, including
kits and antibodies that enrich for
DNA fragments that contain 5-mC and5-hmC. Many of our DNA methylation
antibodies have been validated for use
in ChIP, methylated DNA immunoprecipitation, or MeDIP, and/or immunofluorescence. For complete details on
our DNA methylation products, please
visit www.activemotif.com/dnamt.

Histone H3

K12ac

Regulating the histone code
The “histone code” is comprised of post-translational
modifications that occur
on the histone tails. These
modifications are generated,interpreted and edited byproteins coined “Writers”,“Readers” and “Erasers”.
Aberrant epigenetic regulation
can lead to changes in gene
expression and the development of cancer (Table 1).

R3me2

DNA methylation andhydroxymethylation
Aberrant changes in DNA methylation
have been well characterized in avariety of cancers. In general, thecancer epigenome is marked by global
DNA hypomethylation and promoterspecific DNA hypermethylation, which
often leads to silencing of tumor
suppressor genes. Also, the recently
characterized DNA methylation variant
5-hydroxymethylcytosine (5-hmC) has
also been linked to cancer.

√

√

√

√

Colorectal
Esophageal

√
√

√

Lung

√

Pancreatic√

Prostate

√

√

√

√

√

√

√

Hematological
Kidney

√

√

√
√*

√

√

√

√

√

√

Table 1: Histone modifications exhibiting altered levels in neoplastic tissue.

Using the right tools for ChIP
ChIP is a technically challenging
method, and researchers who are notexperts are best served using wellvalidated and reliable kits to performthese assays. Active Motif has developed a number of kits and accessory
reagents tailored to help researcherswith their ChIP experiments. They
provide all the critical components ina single kit along with easy-to-follow
instructions. These kits and associatedantibodies have been used in hundredsof labs and cited in thousands ofpapers in peer reviewed journals. For
a complete list of available kits andreagents for ChIP, please visit us atwww.activemotif.com/chip.

Let us do the work for you:
Active Motif Epigenetic Services
Reproducibly generating high-quality,interpretable data from ChIP experiments can be challenging as it
requires prior knowledge of working
antibodies, optimized protocols forvarious cell types and knowledge ofcell type-specific binding sites. Addin the technical and bioinformatics
challenges associated with generating whole-genome data sets, and
ChIP-Seq may literally be beyond
your reach. That is why our Epigenetic
Services team provides a wide varietyof ChIP services, making it possible
for you to utilize our expertise andresearch tools without having to be an
expert in the techniques yourself. To
find out more, or to get a quote, go towww.activemotif.com/services.

ERASERS
deacetylases
demethylases
phosphatases“The link between epigenetics and cancer has been substantiatedthrough the identification of mutations in, or altered expression of,
epigenetic regulator proteins in many different types of cancer”

sales@activemotif.com

3

Cas9 and Stem Cell Antibodies

CRISPR / Cas9 Gene Editing
with Active Motif’s Cas9 Antibody
Cas9 is part of the CRISPR/Cas9
system that has revolutionizedgenome engineering by providing amore efficient and robust method fortargeted genome editing. Cas9 can betargeted to particular DNA sequences
through a guide RNA. This RNA-guided
system directs Cas9 to a specificlocation in the genome where it can edit
sequences or alter epigenetic modifications & gene expression.
While Cas9 is a component of the
CRISPR/Cas Streptococcus pyogenes
prokaryotic immune system thatprovides protection against mobile
genetic elements (viruses, transpos-

able elements, plasmids, etc.), CRISPR/
Cas9 has been adapted by scientists
as a useful tool to insert, delete, ormodify sequences at any specificlocation in a cell’s genome.
Active Motif’s new Cas9 Monoclonal
Antibody enables you to monitor
targeting efficiency and specificityof Cas9 endonuclease during yourCRISPR/Cas9 experiments. Cas9
antibody is validated for use in immunoprecipitation, immunofluorescenceand Western blot applications. This
antibody can be used to verify expressionof Cas9 and dCas9 based on its
antigen design.

Product

FormatCatalog No.

Cas9 antibody (pAb)

100 µg61577

ChIP-Seq Validated iPSC Factors
Active Motif now offers ChIP-Seq
validated antibodies specific forstem cell pluripotency factors.
Name

4

Catalog No.

KLF5 antibody (pAb)

61099

Nanog antibody (pAb)61419

Oct-4 antibody (pAb)39811

Sox2 antibody (pAb)

39843

TECFL1/TCF3 antibody (pAb)61125

Tet1 antibody (pAb)61443

www.activemotif.com

Histone H3K27me3 Antibody

Stem cell biology is an importantarea of research, especially with regard
to translational research and the huge
potential of regenerative medicine tooffer solutions for re-engineering tissue
and organs and for treating and preventing disease. Epigenetics continues
to guide much of this area of research
given that stem cell renewal and differentiation are dependent on chromatin
structure and gene transcription.

Histone H3K27me3 Antibody

Recently Published

Catalog No. 39155
Introduction Date: 2007
Publications: 55

Epigenomic Analysis of Multilineage
Differentiation of Human Embryonic
Stem Cells.

Modification role:“To investigate epigenetic regulationof embryonic development, we
differentiated human embryonicstem cells into mesendoderm,neural progenitor cells, trophoblastlike cells, and mesenchymal
stem cells and systematicallycharacterized DNA methylation,
chromatin modifications, and thetranscriptome in each lineage.”Transcription repressor
Active Motif Validated Applications:

In the case of histone modifications,the repressive mark H3K27me3 forms
a bivalent domain with the activating
mark H3K4me3. This domain is criticalto gene expression during embronic
stem (ES) cell differentiation. Researchsuggests a link between these bivalentdomains in ES cells and aberrant gene
regulation in cancer. Being able toidentify these links means having theright tools.
For this reason Active Motif has
established a validation program for
its antibodies to qualify them for theirintended use. This includes ChIP-Seq
testing, ChIP validation, as well as
testing the specificity of our histone
antibodies with our unique MODified™
Histone Peptide Array. For a complete
up-to-date list of available
epigenetics antibodies, please visit
www.activemotif.com/antibodies.

ChIP

ChIPSeq

DB

WB

ICC

IF

Xie, W. et al. (2013) Cell. 153, 1134-48.

modENCODE validation
The modENCODE and NIH Roadmap
Epigenomics Mapping Consortiums
have implemented rigorous standardization criteria for all assays andreagents to be used. As part of thisinitiative, antibody specificity testing
and the ability of the antibodies towork in ChIP-Seq were assessed ina large-scale study. Our Histone H3
trimethyl Lys27 antibody was validated
for ChIP-Seq in the study (Figure 1).

X Chromosome Reactivation
Dynamics Reveal Stages of
Reprogramming to Pluripotency.
“Reprogramming to iPSCs resets
the epigenome of somatic cells,
including the reversal of X
chromosome inactivation. We
sought to gain insight into the steps
underlying the reprogramming
process by examining the meansby which reprogramming leads to
X chromosome reactivation (XCR).”
Pasque, V. et al. (2014) Cell. 159, 1681-97.

Egelhofer T.A. et al. (2011) Nat Struct Mol Biol. 18, 91-3.

Latent Enhancers Activated by
Stimulation in Differentiated Cells.

HOXC4
HOXC5

HOXC13HOXC6“Here, we describe latent enhancers,
defined as regions of the genomethat in terminally differentiatedcells are unbound by TFs and lackthe histone marks characteristicof enhancers but acquire thesefeatures in response to stimulation.”
Ostuni, R. et al. (2013) Cell. 152,157-71.

Figure 1: Histone H3 trimethyl Lys27 antibody tested in ChIP-Seq by Active Motif’s Epigenetic Services.
ChIP was performed using chromatin from the human iPS 19.11 cell line. ChIP DNA was sequenced on the Illumina GA II andsequence tags were mapped to identify H3K27me3 binding. The image shows H3K27me3 binding across a 140,000 bp region of the
HOX gene cluster on chromosome 12.

sales@activemotif.com

5

DNA Modification Products

NEW

Tools to Assess Changes in DNA Methylation in
Normal and Diseased Samples
The cancer epigenome is marked by alterations in a myriad of epigenetic features,
including DNA methylation, nucleosome positioning, and histone post-translational
modifications. These changes influence gene regulation and can serve as signaturesof cancer. To better understand how changes in DNA modifications influence normal
and diseased states, Active Motif offers reagents to perform comparative studies ofglobal changes in methylcytosine (5-mC) levels across samples, as well as for mapping
and profiling changes in hydroxymethylcytosine (5-hmC) across the genome.

Quantify changes in global DNA
methylation levels
In mammals, DNA methylation ischaracterized by the covalent additionof a methyl group within the cytosineof a CpG dinucleotide. These CpG
sites are often found within repetitive
regions and are normally methylated torepress transcription. Hypomethylationof these elements is observed in cancer.
Active Motif’s Global DNA Methylation– LINE-1 Kit provides a quantitative
method to screen for changes in 5-mC
levels across samples for correlative
studies to assess changes induced byvariable treatments, environmental orsocial exposures, or other factors The
assay uses a consensus sequence
within the Long Interspersed Nucleotide
Element 1 (LINE-1) as a surrogate
readout for global DNA methylation.
The kit includes methylated and nonmethylated standards that can beutilized to generate a standard curvefor determination of the % 5-mC.
Profile changes in 5-hmC in cancer
Hydroxymethylcytosine results fromthe oxidation of methylcytosine by
the Ten Eleven Translocation (TET)
enzymes. Hydroxymethylcytosine has
been observed to be redistributed incancer tissues and highly enriched at

oncogenic promoters.
Active Motif’s new Hydroxymethyl
Collector™-Seq Kit is designed to
specifically enrich genomic DNA fragments containing 5-hydroxymethylcytosine, or 5-hmC. This is accomplishedby appending a glucose moiety to
5-hmC. Following biotin conjugation,
the glucosyl-hydroxymethylcytosine
residues are captured with streptavidin
beads. High stringency washes are
performed to reduce non-specific binding before the DNA is eluted. Enriched
DNA can be used for genome-wide
analysis to determine the localizationof 5-hmC within the sample.

Input

Global DNA Methylation – LINE-1
2,5

Brain

OD 450nm

2

Kidney

1,5
1

Liver

0,5

HeLa

HeLa + Aza

STD 75

STD 100

STD 50

STD 30STD 20

STD 0

STD 10BLANK

0DNA (100 ng/well)Figure 1: Global DNA Methylation Assay results showing
a decrease in 5-methylcytosine levels resulting fromtreatment of HeLa cells with 5-azacytidine (Aza), a DNA
methyltransferase inhibitor.

6

Figure 2: Hydroxymethyl Collector-Seq was performed on 5 µg each of mouse brain, mouse kidney and mouse liver genomic DNA
and submitted for Next-Generation Sequencing to identify the localization of 5-hmC within each sample.

Product

FormatCatalog No.

Global DNA Methylation – LINE-1 Kit

1 x 96 rxns

55017

25 rxns

55019

Hydroxymethyl Collector™-Seq

www.activemotif.com

ChIP-Seq Normalization

NEW

ChIP Normalization Reagents Reduce Effects ofTechnical Variation and Reveal Subtle Biological Changes
ChIP is a multi-step process in which variations caused by sample loss during
immunoprecipitation and library preparation, uneven sequencing read depth orhand-to-hand differences can lead to results that are difficult to interpret. To overcome
this challenge, Active Motif has developed a spike-in strategy that utilizes Drosophila
chromatin and a Drosophila-specific antibody for normalization of technical variation andsample processing bias. Additionally, the normalization strategy can be used for monitoring
conditional effects, such as those induced by compounds or mutants, on your experiments.

How does it work?
Active Motif’s Spike-In Normalization
Strategy works with both ChIP-qPCR
and ChIP-Seq analysis to eliminate
bias and reveal latent biologicalchanges in your samples (Figure 1).
ChIP normalization can easily be
implemented simply by integrating
our Spike-in reagents into yourstandard ChIP protocol.

“spiked-in”, to each reaction as a minor
fraction of total chromatin. An antibody
that recognizes the Drosophila-specifichistone variant, H2Av, is also added
to the reaction. The Spike-in Antibody
provides a mechanism to reliably pull
down a small fraction of Drosophila
chromatin that is consistent across all

samples (see Workflow below).
Since variation introduced during theChIP procedure will also occur with
the Spike-in Chromatin, a normalization
factor can be created based on the
Drosophila signal and applied to thesample genome.

A standard ChIP reaction is set up
using experimental chromatin (e.g.
human) and an antibody of interest. In
addition, Drosophila melanogaster
Spike-in Chromatin is added, or
ChIP-Seq Normalization Workflow

Experimental
Chromatin

Spike-in
Chromatin
Spike-in
Antibody

Antibody ofinterest

Figure 1: ChIP-Seq Spike-in Normalization Strategy reveals changes in H3K27me3 levels following treatment with
EZH2 inhibitor compound. Cells treated with a small molecule inhibitor of EZH2 methyltransferase have dramatic
reductions in global H3K27me3 levels. However, H3K27me3 ChIP-Seq using standard ChIP-Seq protocols (-) does not

Chromatin Immunoprecipitation

detect these differences. Incorporation of Active Motif’s ChIP-Seq Spike-in Strategy (+) reveals the expected decrease in
H3K27me3 ChIP-Seq signal.
Sequencing

Map to experimental
genomeNormalize sample
tag counts by same ratio

sales@activemotif.com

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

Product

FormatCatalog No.

Spike-in Chromatin

10 µg

53083

Spike-in Antibody

10 μg

61686

Drosophila Positive Control Primer Set Pbgs

96 rxns

71037

Drosophila Negative Control Primer Set 1

96 rxns

71028

7

Tag-ChIPNEW

Tag-ChIP-IT® Enables ChIP Without
Protein-specific Antibodies
Interested in studying transcription factor sequence variants, mutations or
truncations? Can’t find a suitable antibody for ChIP? Don’t limit your ChIP
experiments based on antibody availability. Active Motif’s new Tag-ChIP-IT systemenables you to create a fusion protein expressing Active Motif’s unique AM-tag
sequence that was specifically designed to work in ChIP. Simply clone your protein
of interest in-frame with the C-terminal AM-tag. Transient or stable transfections of
the fusion protein can be analyzed by ChIP with an antibody directed against the AM-tag.

No ChIP-validated antibody for yourprotein of interest? No problem!
Transcription factor ChIP is often
challenging due to a lack of available
antibodies that are capable of recognizing target-bound protein of interest
post-fixation, or the inability of available
antibodies to distinguish between
protein isoforms. These limitations
make it difficult to study the effects
of sequence variants, mutations andtruncations on gene regulation.
To overcome these challenges, Active
Motif has developed the Tag-ChIP-IT Kit toenable ChIP without the use of a targetspecific antibody. Tag-ChIP-IT utilizes aunique AM-tag specifically designed tominimize cross-reactivity with mammaliansamples for reduced background signal.
Additionally, the design maximizes exposure of the AM-tag during the immunoprecipitation reaction to increase theenrichment efficiency of low abundance
transcription factors for more reliable
and consistent ChIP results (Figure 1).
Tag-ChIP-IT advantages

Tag-ChIP-IT is Used to Identify Estrogen Receptor (ER) Binding Motifs
Input
Endogenous ER
AM-tag ER#1

SampleAM-tag ER#1

AM-tag ER#2

AM-tag ER#2

Figure 1: ER cDNA was cloned into pAM_1C Empty Vector and transiently transfected into cells. Cells were induced with estradiol
and chromatin was harvested and Tag-ChIP performed using the Tag-ChIP-IT Kit. Following cross-link reversal, enriched DNA was
submitted for Next-Generation Sequencing. Data was compared to published ChIP-Seq results using an anti-ER antibody in thesame cell line and induction conditions. ChIP-Seq data shows the same ER peak profile with the AM-tag ChIP as endogenous ER.
Detected binding sites were further evaluated for binding motifs and results show that the ER motif was identified in both Tag-ChIP-IT samples.

How does it work?
Use the pAM_1C Empty Vector to clone
your protein of interest in-frame with
the C-terminal AM-tag. Alternatively,
the AM-tag sequence can be cloned
into your expression vector of choice.
Following transfection and expressionof your tagged protein, the Tag-ChIP-IT
Kit can be used to isolate chromatin
and perform immunoprecipitations using
AM-Tag antibody specific for the AM-tag.

• Ideal for targets lacking ChIP Abs

Product• Distinguish between protein
isoforms and mutants• High specificity due to low
cross-reactivity of AM-tag withmammalian samples

8

ER Motif Identified

What’s in the box?
The Tag-ChIP-IT Kit contains the same
optimized reagents as our ChIP-IT®
High Sensitivity Kit to enable specificdetection of low abundance proteins.
The kit includes buffers, proteaseinhibitors, Protein G agarose beads,
DNA purification columns and the
AM-Tag antibody. For cloning, thepAM_1C Empty Vector is available
separately. For more information, visit
www.activemotif.com/tagchip.
FormatCatalog No.

Tag-ChIP-IT Kit

16 rxns53022

pAM_1C Empty Vector

20 μg

53023

pAM_1C_JunD Vector

50 μg

53044

AM-Tag antibody

100 μg

61677

FuGENE HD Transfection Reagent

0.2 ml

32042

®

®

www.activemotif.com

Histone Acetylation

Histone Acetylation Analysis – Finding the Right
Tools For Your Research
Histone acetylation plays an important role in chromatin decondensation. Decondensation isa prerequisite of gene activity as it enables regulatory proteins, such as transcription factors
and DNA repair elements, to access the underlying DNA. Histone acetylation is regulated by the“Writers”, “Erasers” and “Readers” of the Histone Code. These include histone acetyltransferases(HATs) and deacetylases (HDACs), and bromodomain-containing proteins that bind acetylated
lysine residues and recruit other regulatory factors to the acetylated sites. Because of their role intranscriptional control, these regulators are often the focus of drug targets and inhibitor screens.
Active Motif offers a broad portfolio of kits and reagents for the study of histone acetylation.

Multiplex histone modification analysis
To enable screening of multiple histone
modifications in a single sample, Active
Motif’s Histone H3 PTM Multiplex Kit
offers the first-of-its-kind multiplex
epigenetic assay for use with Luminex®
instruments. The kit enables you tosimultaneously measure changes in
the relative levels of multiple histone
modifications in a single well using
only nanogram amounts of sample
material. Antibody-conjugated bead sets
are available to study Histone H3 panacetyl, K9ac, K27ac and K56ac. A Total
H3 bead can be used for normalization.

sales@activemotif.com

To learn more, and to see thecomplete list of available antibodyconjugated bead sets, visit
www.activemotif.com/luminex.

are compatible for use in Western blot,
mass spectrometry or with Active
Motif’s Histone Modification ELISAs
and Histone H3 PTM Multiplex Assay.

For more information, please visit
Histone purificationwww.activemotif.com/histonepur.
Active Motif’s Histone Purification Kits
are the only commercially
Histone H3 acetyl Lys9 ELISA
available assays for
3.5
isolation of purified core
Recombinant Histone H3
3
histones from cell or tissue
Recombinant Histone H3 monomethyl Lys9
samples. Unlike standard
Recombinant Histone H3 dimethyl Lys9
2.5
Recombinant Histone H3 trimethyl Lys9
acid extraction techniques,2
Recombinant Histone H3 acetyl Lys9
purification better preserves
1.5
Recombinant Histone H3 acetyl Lys14
post-translational modifica1
tions, and histones can be
0.5
isolated either as a single
fraction or separate H2A/
0
10
5
2.5
1.25
0.625
0.313
0.156
0
H2B, H3/H4 fractions. The
Recombinant proteins (ng/well)kits are available in threedifferent formats to support
Figure 1: Histone H3 acetyl Lys9 ELISA. Histone H3 acetyl Lys9 ELISA includes
a recombinant histone for quantitation of H3K9ac in acid extracted or purified
low, medium or highhistone samples. When tested in the presence of other histone modifications,throughput sample prothe assay shows specificity for H3K9ac.
cessing. Purified histones
Histone H3 acetyl Lys9 (OD 450nm)

Quantify histone acetylation levels
The Histone Modification ELISA Kits
offer a quick and easy way to screenand quantify specific histone modifications within your sample (Figure 1).
The kits are designed as sandwich
ELISAs that utilize a histone H3 capture
antibody and a detecting antibody thatis specific for the modification of interest. A recombinant protein standard isincluded in each kit for quantitation,and the Total Histone H3 ELISA canbe used to normalize histone levels
across samples. In addition to Active
Motif’s new Histone H3 acetyl Lys27
ELISA, which marks active enhancers,
assays are also available to study
acetylation of Lys9, Lys14, methylationand phosphorylation. For available
kits and more information, please visit
www.activemotif.com/hiselisa.

Product

Web page

Histone Purification Kits

www.activemotif.com/histonepur

Histone Modification ELISAs

www.activemotif.com/hiselisa

Histone H3 PTM Multiplex Kit

www.activemotif.com/luminex

Histone Peptide Array

www.activemotif.com/modified

HAT & HDAC Assays

www.activemotif.com/histone

Recomb. HATs, HDACs, BRDs, Histones & Nucleosomes www.activemotif.com/hisprot
Epigenetic Activators / Inhibitors

www.activemotif.com/smallmol

Histone Antibodies

www.activemotif.com/histoneabs

9

Products to Enable Cancer ResearchTools to Enable Insight into the Cancer Epigenome
Epigenetic changes are hallmarks of human cancers. Changes in DNA methylation, as well as
histone modifications, have been found in every tumor type studied to date, both benign andmalignant, and have been associated with the progression of colorectal cancer. Thus, gaining
further insight into these complex regulatory mechanisms is crucial to understanding diseasesusceptibility, initiation and progression. In a recent study performed by the laboratory of
Dr. Manuel Perucho at the IMPPC in Barcelona, numerous assays from the Active Motif productline were utilized in colorectal cancer studies to analyze the role of epigenetic aberrations, inparticular global hypomethylation, in the development of cancer.

As LINE-1 demethylation is a commonlyused marker of global hypomethylation,
the LINE-1 methylation levels were
determined by the Active Motif Global
DNA Methylation – LINE-1 kit (Figure 1,
top) and bisulfite sequencing (Figure 1,
bottom) in a subset of colorectal cancer
patients in which SST1 methylationlevels had been determined. The data
reveal both assays yield similar estimated levels of LINE-1 hypomethylationin tumors compared to corresponding
normal tissue.

Because changes in global methylation
are a hallmark of many human diseases,
including cancer, simpler methods
than HPLC or bisulfite sequencing are
warranted for correlative studies thatanalyze variances in genome-wide
DNA methylation status.
Active Motif’s Global DNA Methylation– LINE-1 Assay uses a unique hybridization approach that quantitates
5-methylcytosine (5-mC) levels
at LINE-1 repeats as a surrogate
measure of global methylation. Thisapproach offers better specificity andreproducibility than other available
methods that utilize non-specificpassive adsorption (Figure 1).

Through a genome-wide analysis of
DNA methylation alterations, a specificfamily of non-coding pericentromeric
DNA repetitive elements, SST1, wasfound to exhibit a wide degree of
demethylation in colorectal tumors.

10

Methylation Level

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1

33N

33T

633N 633T 673N 673T

Further, the analyses reveal thatdemethylation occurs in both repeats,
especially in LINE-1s, in a highproportion of the patients. The results
also demonstrate that demethylationof SST1 positively correlates withdemethylation of LINE-1 (Figure 2).

Figure 1: LINE-1 methylation levels of four human colon

1.0

cancer cases, determined by the Global DNA Methylation– LINE-1 assay (top) and bisulfite sequencing (bottom).

100
80

Methylation Level

0.8% LINE-1 Methylation

Quantify changes in global DNA
methylation levels
The global hypomethylation observedin cancer cells primarily reflects
the somatic demethylation of DNA
repetitive elements. Hypomethylation
predisposes normally compacted DNA
to decondensation which can lead tochromosomal instability and tumor
development and/or progression.

1.0

p=0.0008
r2=0.4737

60
40

0.6

0.4

0.2

20
0

50

60

70

80

90

% SST1 Methylation

0.0
Caco-2 LS174 A2780 OV-90
ADR

Figure 2: Demethylation of SST1 correlates with

Figure 3: SST1 methylation levels of individual clones ana-

LINE-1 demethylation (p=0.0008), a commonly

lyzed by bisulfite sequencing in colon and ovarian cancer

accepted marker for global DNA methylation.

cell lines. One clone represents one single SST1 element.

www.activemotif.com

Products to Enable Cancer Research

4

10
8
6
4
2
0

K9me3 K27me3

IgG

K9me3 K27me3

Caco-2IgG

LS174

may ultimately affect chromosomal
integrity. In addition to ChIP-IT FFPE
products, we also offer the ChIP-IT
PBMC Kit to perform ChIP on difficultto-lyse peripheral blood mononuclear
cells (PBMCs), including lymphocytes (Tcells, B cells & NK cells) and monocytes.
For more complete information on
ChIP-IT products for primary cells,
visit us at www.activemotif.com/chip.

For more information, please visit
www.activemotif.com/chip-bis-seq.

Data was provided courtesy of Dr. Johanna Samuelsson
and Dr. Manuel Perucho. Samuelsson et al., in preparation.

Validate results obtained from cell
models in primary tissues
Cell model systems often do not reflect
true biology. Thus, it is important toconfirm results in primary tissues.
The Active Motif ChIP-IT® FFPE
Chromatin Preparation Kit and ChIP-IT®
FFPE Kit were used on colon primary
FFPE samples and confirmed the shift
from high levels of DNA methylationand H3K9me3 in normal tissues to
SST1 hypomethylation and increased
levels of H3K27me3 in tumor cells(Figure 6).These findings suggestthat demethylation of SST1 elements
may be a marker of an epigenetic
reprogramming event associated withchanges in chromatin structure that

900
800
700
600
500
400
300
200
100
0

H3K9me3

GAPDH

1.0

Methylation Level

0.8

SST1

0.6

0.4

0.2

0.0
Input K27me3 Input K27me3

Caco-2

LS174

Figure 5: SST1 elements associated to H3K27me3 arehighly demethylated.

Binding Events Detected/1000 Cells

Fold Enrichment Relative Input

12

Fold Enrichment Relative Input

Heterogeneity of SST1 methylationlevels was observed in the LS174 T
colon cancer cell line employed to
study the putative effects of SST1
demethylation on chromatin structure.
Therefore, Active Motif’s ChIP-Bis-Seq
Kit, which offers a method to directly
assess DNA methylation patterns
associated with chromatin modifications(or chromatin-associated factors), wasutilized to test whether SST1 H3K27me3
actually co-occurs in association withdemethylated SST1 elements. LS174
and Caco-2 chromatin was immuno-

precipitated with an anti-H3K27me3
antibody followed by bisulfite treatmentof isolated DNA. The SST1 methylation
status of H3K27me3-associated DNA
was then determined by bisulfite sequencing (Figure 5). The ChIP-Bis-Seq
results confirmed that the H3K27me3associated SST1 elements are actuallydemethylated.

Binding Events Detected/1000 Cells

Assess chromatin featuresassociated with DNA methylationin normal & diseased cells
Investigation into the chromatinchanges that occur during loss of
methylation at SST1 repeat elements
in colon and ovarian cancer cell lines
revealed that, while the cell lines withretained SST1 methylation (Figure3) exhibit enrichment of Histone H3lysine 9 trimethylation (H3K9me3),
SST1 demethylation is associated with
Histone H3 lysine 27 trimethylation(H3K27me3), an epigenetic repressive
mark of facultative heterochromatin(Figure 4).

NormalTumor

120

H3K27me3

100
80
60
40
20
0

GAPDH

SST1

3

Figure 6: ChIP analysis of histone modifications associated with SST1 repetitive elements in colon cancer FFPE primary2tissue samples confirms the shift from high levels of SST1 methylation and H3K9me3 enrichment in the normal tissue(purple) to SST1 demethylation, a reduction of H3K9me3 and increase in H3K27me3 levels in the tumor (copper).

1
0

K9me3 K27me3

IgG

K9me3 K27me3

A2780 ADR

IgG

OV-90

Figure 4: SST1 methylation correlates positively with
H3K9me3 levels and negatively with H3K27me3.
ChIP analysis corresponding to data sets in Figure
3 show demethylation of SST1 is associated withincreased H3K27 trimethylation and a reduction in
H3K9me3. Fold change calculated relative to input.

sales@activemotif.com

Product

FormatCatalog No.

Global DNA Methylation - LINE-1 Kit

1 x 96 rxns

55017

ChIP-Bis-Seq Kit

10 libraries

53048

®

ChIP-IT FFPE Chromatin Preparation Kit

5 rxns

53030

ChIP-IT FFPE Kit

16 rxns

53045

ChIP-IT PBMC Kit

16 rxns

53042

®

®

11

lncRNA Promoter Reporter Assays

NEW

Study Regulation of lncRNA Promoters
It has been estimated that while only 1% of the mammalian genome codes for protein,
70% to 90% is transcribed at some point during development. The vast majority ofthis transcriptome is thought to be long noncoding RNAs (lncRNAs). Previously
thought of as simply “junk DNA”, the relatively recent revelations that lncRNA regulation
is distinct from protein-coding genes and that they are important regulators of bothtranscription and chromatin organization has elevated their status to that of “dark matter”,
reflecting that their mechanisms of action are still poorly understood. Active Motif’s
LightSwitch™ Assay System offers a simple solution for unraveling lncRNA regulation.

lncRNA promoter regulation
As compared to protein-coding genes,
lncRNA genes appear to be regulated
in a more condition-specific manner.
Evidence suggests the possible
existence of distinct modes of regulation
for the two different gene groups,
making functional assays imperative.
The LightSwitch Assay System wasdesigned to make it fast & easy to
study promoter regulation. The backbone of the LightSwitch System is theLightSwitch Promoter Reporter vector.
After a promoter of interest is cloned
upstream of RenSP in this vector, it
is transfected into live cells. The cells
can be stimulated, if desired, and thepromoter activity can then be determined simply by assaying for the luminescence signal, which is measured
quickly and accurately as light output(Figures 1 & 2).
% signal relative toto ACTB promoter control

140

DMSO

120

Paclitaxel

100
80
60
40
20
0

MALAT1

FIRRE

TUG1

GAS5

Figure 1: Measuring lncRNA promoter activity.
The promoters of 4 different lncRNA genes were cloned
into the LightSwitch Promoter Reporter vectors andtransfected into MCF-7 cells. Following stimulation withDMSO or paclitaxel, luciferase assays were performed
and the data normalized using an ACTB positive control.

12

Select pre-made lncRNA promoter
constructs, or clone your own
For your convenience, we havecloned a number of widely studied
lncRNA promoters, including FIRRE,
GAS5, HOTAIR, MALAT1, MEG3 and
TUG1, into the LightSwitch Promoter
Reporter vector. These are available
as sequence-verified, transfectionready reporter constructs, so you
can begin assays to study how thesepromoters are regulated immediately,
without the need to clone, sequenceor prepare the DNA. Alternatively, you
can clone your own sequence into theempty reporter vector or utilize our
economical Custom Cloning Services.

Optimized luciferase and assay
reagents for the best results possible
All LightSwitch vectors utilize an optimized Renilla luciferase gene (RenSP)
that has been engineered for maximum
brightness and minimal backgroundwhen reacted with the proprietary
substrate found in LightSwitch
Luciferase Assay Kits. In addition, theLightSwitch reagents were developed
to enable one-step addition directly tocultured cells, eliminating the needfor a separate lysis step. Collectively,
these improvements ensure that
LightSwitch delivers superior resultswith simple and robust protocols. For
more information, please visit us atwww.activemotif.com/ls-lncrna.

LightSwitch™ lncRNA Promoter Assays

PromoterPre-cloned intoa Reporter
VectorlncRNA of interest
RenSP
Luciferase

LightSwitch™
Promoter
Reporter
1. Transfectvector.

2. Treat withcompounds,
pathogens,or altered
growthconditions.

Light signal

3. Measure luciferase activity to determine
the functional effects of treatments
and/or sequence variations.
Figure 2: LightSwitch lncRNA Promoter Reporter Assays.
To study a lncRNA promoter’s regulation, it is cloned intoa LightSwitch Promoter Reporter vector and transfected.
The cells are treated, if desired, and then assayed. The
luciferase produced oxidizes the assay substrate in areaction that also produces light. The amount of light is
proportional to the activity of the cloned lncRNA promoter.

www.activemotif.com

Tools to Study miRNA & 3´UTR Interactions

Dysregulation of miRNAs in Cancer
MicroRNAs (miRNAs) regulate many different biological processes, including development,
differentiation, proliferation and apoptosis. Expression levels of specific miRNAs havebeen observed to either be upregulated or downregulated in a variety of cancers. An
estimated 50% of miRNA genes are located at fragile sites in the genome that are deleted,amplified or translocated in human cancer, making miRNAs useful as biomarkers to
identify patients at risk of developing a cancer, as well as predicting those that may
respond to therapy. For a list of miRNAs implicated in specific cancer types, visit
www.activemotif.com/mirna-cancer.

miRNA-mediated regulation
by RISC complexes
miRNAs act as post-transcriptional
regulators of gene expression bybinding to the 3´ Untranslated Region(3´UTR) of messenger RNA transcripts (mRNAs). The interaction of amiRNA with a 3´UTR results in either
the repression of translation or the
degradation of the transcript.
The targeting of a given miRNA to a
specific mRNA is mediated throughthe formation of an RNA Induced
Silencing Complex (RISC). While RISCs
can contain a combination of different
RNA-binding proteins, at a minimuma RISC is comprised of an Argonaute
protein (Ago) and a miRNA. The Agoprotein binds the miRNA in a mannerthat enables the miRNA-loaded RISC
complex to base-pair with a mRNAtranscript (Figure 1).

Validate miRNA/mRNAinteractions using Ago IP
Active Motif’s miRNA Target IP Kit
utilizes protein G-coupled magnetic
beads and a pan-Ago antibody thatrecognizes Ago1, Ago2 and Ago3
proteins to immunoprecipitate miRNA/
mRNA complexes that have associatedwith Ago1, Ago2 or Ago3. Following
their immunoprecipitation as part of
the RISC complex, the mRNA targets
can be purified and then amplified by
RT-PCR using gene-specific primers.

Translational Repression by a miRNA-directed, RNA Induced Silencing Complex (RISC)

Ribosome
5´
mRNA

ORF

3´UTR
Ago

AAA-3´
miRNA

RISC Complex
Figure 1: The miRNA contained in a RISC complex enables precise silencing of specific mRNA transcripts.
The key components in a RISC complex are an Argonaute protein (Ago) and a miRNA. The Ago protein binds the miRNA, positioning
it in a conformation that enables the RISC to base-pair in a Watson-Crick manner with a mRNA transcript. This leads to either
inhibition of translation (shown) or increased degradation of the targeted transcript.

sales@activemotif.com

By comparing cells transfected with orwithout a miRNA mimic, one can validate the mRNA targets of a particular
miRNA based on over-expressionof that miRNA. It is also possible toprofile the immunoprecipitated mRNA
molecules using conventional
expression microarrays or RNA-Seq.
For more information, please visit us
at www.activemotif.com/mirna-ip.

Study how sequence variation
alters miRNA-3´UTR interactions
Single Nucleotide Polymorphisms(SNPs) in an mRNA’s 3´UTR have beenshown to play a role in the miRNAdysregulation seen in cancers. Active
Motif’s LightSwitch™ System is ideal
for studying miRNA-3´UTR interactions and for assessing the functional
impact of variations in the sequenceof the target 3´UTR. The system
includes a collection of over 12,000
human 3´UTRs that can be purchased
as ready-to-transfect LightSwitch
luciferase reporter vectors. These
constructs can easily be mutagenized
to determine how sequence variations
impact function. Combined with ourlarge collections of miRNA Mimics &Inhibitors, you have everything needed
to study miRNA-3´UTR interactions,validate miRNA targets, and measure
the functional impact of miRNAs on agene-by-gene basis. For information,
visit www.activemotif.com/ls-3utr.

13

Epigenetics in Drug Discovery

Enzymes & Substrates for Drug Discovery
Recombinant proteins are a critical component of epigenetic drug discovery
programs. Robust enzymatic assays are critical for screening, structural andmechanistic studies that guide chemical design of epigenetic modulators. To
achieve robust enzymatic assays requires high quality epigenetic recombinant
proteins. Active Motif’s protein biochemistry team has produced hundreds ofhistone and nucleosome substrates and highly potent enzymes to many of the
hot drug targets, such as NSD2, Dot1L, LSD1, KDM5A, KDM5B, KDM6A, as well as PRC2 andMLL complexes, that are ready to incorporate into your assay design.

Available Products• Histone modifying enzymes - HMTs / HDMs,
HATs / HDACs

Writers, Readers and Erasers
Writers (Histone methyltransferases)

Erasers (Histone demethylases)
JMJC family

LSD family

LSD1/KDM1A
LSD2/KDM1B• Binding domains - Bromodomains,
SET domains• Substrates - Unmodified & modified
histones, histone octamers & nucleosomes
JASC1/KDM4C• DNA modifying enzymes - DNMTs, TET

SETD4
SETD3

• Transcription Factors - NFkB, AP-1,
p53, and more...
14
Readers (Bromodomains)

Bulk sizes available
Call us at 877-222-9543 to inquire about
custom or bulk orders.

BAZ2B

Fold Enrichment

BRPF1

Assay-ready ‘Writers’, ‘Readers’ & ‘Erasers’
There is no need to waste valuable time andresources generating proteins for screening,
drug discovery and target validation programs.
With Active Motif’s comprehensive portfolioof purified assay-ready recombinant proteins– including methyltransferases and demethylases, acetyltransferases and deacetylases,
as well as binding domains – you are sure tofind what you need to develop more efficient
epigenetic assays.

miR-122
Non-targeting
Mock tfxn

12
10
8
6
4
BRWD1 (1)

2
0

G6PC3

SLC1A5GAPDH

The epigenetic state of the cell is controlled by the activity of three major classes of epigenetic proteins:the “Writers” (enzymes that deposit modifications), the “Erasers” (enzymes that remove modifications)and the “Readers” (proteins that recognize and bind epigenetic modifications). Recent drug development
strategies that target these enzymes have proved highly successful, resulting in many FDA approved
cancer drugs, with more promising leads on the horizon.

To learn more, go to www.activemotif.com/proteins or download a copy of our Tools for Drug Discovery product brochure.

14

www.activemotif.com

Epigenetic Services

NEW

Epigenetic Services: RIME for Identification ofTranscriptional Networks Associated with Disease
Active Motif’s newest addition to it’s suite of epigenetic and gene regulation
focused services, RIME (Rapid Immunoprecipitation Mass Spectrometry ofEndogenous Proteins), sheds light on the complex process of gene regulation
by enabling the capture and identification of interactomes, or the associatedprotein networks, of an endogenous protein of interest. The RIME service
includes immunoprecipitation of a target protein from cross-linked cell
extracts followed by ChIP-Seq, mass spectrometry and data analysis.

Identify interactomes regulating
gene expression
Gene regulation is complex but tends
to be oversimplified by researchers
focused on one particular transcription
factor (TF) in any given cellular model.
In reality, differential gene expression
is greatly influenced by cofactors andother protein interactions with chromatin.
For example, gene activation by the
Estrogen Receptor is thought to bemodulated through ligand-induced
conformational changes in the receptor
that mediate cofactor recruitment.
Additionally, ChIP-Seq data sets often
show transcription factor binding at
the same promoters in multiple cells
although they display differentially expressed target genes, suggesting protein networks are responsible for suchdifferential gene activation. RIME sorts
out this complexity by providing a means
to identify the protein interactions thatare important for gene regulation.

RIME Service Workflow for Identification of Endogenous Interactomes

Cross-link cells

Sonicate

Identify bindingsites by ChIP-Seq
Immunoprecipitate target
Identify interacting
proteins by mass
spectrometry

Why RIME?
Immunoprecipitation (IP) followed bymass spectrometry has traditionallybeen the approach used to identify
proteins interactions with a target ofinterest. RIME is an adaptation of the
ChIP-Seq method that, in addition
to enabling the mapping of
RIME advantages
transcription factor binding sites,
enables the identification unknown• Identify transcriptional cofactors
interacting proteins. RIME differsfrom traditional IP approaches• Identify co-occurance of TFs bound to
because the starting material has
DNA at sites adjacent to your target TF
been cross-linked, which offers the• Identify important targets foradvantage of 1) enabling capture
ChIP-Seq studiesof low affinity interactions which
are lost in IP/mass spectrometry,• Detect low affinity interacting proteins
2) allowing more stringent wash

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Isolate nuclei

POLR2A Coverage
500 µg chromatin starting material: 48% coverage

50 µg chromatin starting material: 43% coverage

Target protein detection in the mass spectrometry data
is a measure of success in a RIME experiment.

conditions resulting in less nonspecific interactions and better
preservation of protein complexes, and3) stabilizing protein/DNA interactions
to enable the capture of proteins thatare bound at adjacent sites in the DNA
and are independent of protein/protein
interactions.
To learn more, please visit us atwww.activemotif.com/rime.

15

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Enabling Epigenetics Research