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THE NEWSLETTER OF ACTIVE MOTIFOCTOBER 2014 | VOLUME 15 NUMBER 2

Special Neuroepigenetics Edition

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

New to Neuroepigenetics?
We Can Help.

IN THIS ISSUE2

New to Neuroepigenetics?
We Can Help.

45-hmC and the Brain

5

Tools for Neuroepigenetic Research

6

NEW: Quantify Global Changes in
DNA Methylation

7

NEW: ChIP-Bisulfite-Sequencing for
Mapping Allele-Specific Methylationof Protein/DNA Binding Sequences

8

NEW: High-Resolution ChIP Analysis
of Transcription Factors

10 NEW: Nucleosome Substrates forMore Biologically Relevant
Screening Assays
11 NEW: Epigenetic Services: A Novel
ChIP-Seq Spike-in Strategy
12 NEW: Validate the Specific mRNA
Targets of miRNAs
13 miRNAs in Neural Development &Neurological Diseases
14 Tools to Streamline Epigenetic Drug
Research
15 NEW: Small Molecules to Modulate
Epigenetic Processes

There is increasing evidence that several neurodevelopmental,
neurodegenerative and psychiatric disorders are, in part,caused by aberrant epigenetic modifications. As a result,researchers in neurogenetics are now beginning to investigate
the mechanisms underlying epigenetic regulation in neuronal
gene expression and how this impacts normal brain function.
The transition into epigenetics research does not have to be
intimidating because there are an increasing number of tools
available to help researchers obtain their desired results.

Various epigenetic modifications arefound in neurons
These modifications include classical epigenetic marks such as DNA
methylation and histone modifications. Other epigenetic mechanisms inplay include histone subunit exchange,
cytosine methylation variants andnon-coding RNAs. In recent years,
there has been a rapid increase in thenumber of publications suggesting arole for epigenetics in regulating theCentral Nervous System (CNS) anda growing body of evidence linking
epigenetic mechanisms to neuralplasticity and CNS disorders, such as
schizophrenia and drug addiction (Table 1). More than a dozen neurologicalsyndromes so far have been linked tomutations in single genes that encode
DNA methyltransferase and histone
modifying enzymes. This list includes
neurological disorders that appear inearly childhood (Rett syndrome) or
later in life, such as Hereditary Sensory and Autonomic Neuropathy Type
1 (HSAN1).

The epigenetic tool kit
The development of methods such as
chromatin immunoprecipitation (ChIP)
and DNA methylation enrichment techniques, along with Next-Generation Sequencing tools, is allowing researchers
to get a better picture of the genomewide changes associated with neurological and other diseases. There are
a number of techniques that are key toepigenetic analysis. These include:• Antibodies to discriminate between
the multiple post-translational
modifications on the histone tails,
and to detect methylated DNA• ChIP and ChIP-Seq techniques to enable scientists to link specific statesof chromatin to individual gene lociin a cell to understand how genes
are regulated• Enrichment methods for isolation of
methylated DNA variants (5-methylcytosine, or 5-mC, and 5-hydroxymethylcytosine, or 5-hmC)• Bisulfite conversion of methylated
DNA prior to sequencing to allow
detection of methylated cytosines“What Roles do Epigenetic Mechanisms Play inComplex Human Diseases of the Nervous System?”2

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Using the right tools for ChIP
ChIP is a technically challenging
method. Numerous factors can cause
it to fail, so researchers who are notexperts in the techniques are best
served using well-validated and reliable kits to perform these assays.
Active Motif has developed a numberof kits and accessory reagents tailoredto help the researcher with their ChIP
experiments. We provide all the critical components needed in a single kit,along with easy to follow instructions.
These kits and associated antibodies
have been used in hundreds of labs
and cited in over 1,000 papers in peerreviewed journals. For the full list of
kits and reagents for ChIP, please visit
us at www.activemotif.com/chip.

“How is DNA Methylation
Actively Regulatedin Neurons?”

What is the role of DNA methylationin brain function?
DNA methylation of genes is foundin all human tissues, including thebrain. There are several methylation
variants, and one of these, 5-hmC, ishighly expressed in the brain relative
to other parts of the body. This has led
to speculation that 5-hmC may havea unique biological role in the CNS.
Also, recent studies have shown acorrelation between DNA methylationlevels and mental health problems,such as anxiety and depression. In
addition, mutations in genes involved
in DNA methylation (e.g. MeCP2) arelinked to neurological diseases. However, the mechanism by which DNA
methylation exerts these effects is not
fully understood.

enrich for DNA fragments that contain
5-mC and 5-hmC. Many of our DNA
methylation antibodies have been validated for use in ChIP, methylated DNA
immunoprecipitation (MeDIP) and/or
immunofluorescence. For complete
details on all of our DNA methylation
products, please visit us atwww.activemotif.com/dnamt.

Epigenetic techniques require
validated antibodies
One of the greatest challenges for epigenetic-based research has been thelack of antibodies that show proper
specificity and that have been validated for use in techniques such as ChIP
and ChIP-Seq. Histones have multiple
post-translational modifications along
the so-called tail portion of the proteinthat appear to be involved in gene
regulation and disease. Antibodies
need to be able to clearly differentiate between these multiple small
modifications. It is a major challenge
to develop antibodies with sufficient
specificity that will also perform indemanding techniques such as ChIP.
Active Motif has established a validation program for its antibodies toqualify them for their intended use. This
includes ChIP-Seq testing, ChIP validation, as well as a test on the specificity
of our histone antibodies with a unique
Modified™ Histone Peptide Array(Catalog No. 13001). For the full list of
our epigenetics antibodies please visit
www.activemotif.com/abs.

H
is
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ifi
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DN atio
n
A
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et
hy
la
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on tionRN codA ingEpigenetics of the Nervous System

Learning and memory

√

√

√

Addiction√√√

Autism spectrum

?

?

?

Epilepsy

√

√

√

PTSD

√

√

Alzheimer's disease√√

Stress√√Schizophrenia√√

Bipolar disorder√√

Adult neurogenesis

√

√

√
√

Table 1: Examples of neural processes and disorders
associated with epigenetic modifications.

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 yourreach. That’s why our Epigenetic Services team provides a wide variety of
ChIP services. This makes 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
to www.activemotif.com/services.

Tools for DNA methylation analysis
Active Motif offers a number of products specific for this area of research,
including kits and antibodies thatsales@activemotif.com

3

5-Hydroxymethylcytosine (5-hmC)

5-hmC and the Brain
Epigenetic regulation is an important part of human brain development. It links
our environment and our genome and influences learning and memory processes
by what many now refer to as behavioral epigenetics, or neuroepigenetics.
Neurogenesis is said to continue in certain parts of the brain throughout adultlife. However, it may be neuroepigenetic changes that hold the key to understanding disease-related risk factors associated with our neural development
and the aging process. One of the most studied epigenetic modifications, DNA
methylation, is at the center of research involving brain diseases, major
psychosis and addictions.

DNA methylation
DNA methylation functions to regulate
gene transcription in normal cells.
Aberrant DNA methylation is responsible for repression of genes throughpromoter hypermethylation, which is
implicated in tumorigenesis, and theubiquitous hypomethylation common
in early carcinogenesis. Abnormal
DNA methylation is also linked tovarious neurological diseases.

5-hmC and the brain
Ten-eleven translocation (TET)
enzymes catalyze 5-methylcytosine(5-mC) conversion to 5-hydroxymethylcytosine (5-hmC). 5-hmC is
considered the sixth base in mammalian DNA because of its increasing functional significance apartfrom an intermediary in the demethylation process. While 5-hmC isenriched in many active genes, it appears more prominent in the brain.
5-hmC is approximately 10 times
more abundant in brain cells than instem cells, being highly enriched in
the cerebellum and hippocampus. It
has been shown that, in areas of enriched 5-hmC, there is loss of 5-mC
and vice versa, suggesting uniquefunctions in neural development andmaintenance. Interrogation of 5-hmC
genome patterns in the brain has revealed important interdependencies,
one being the correlation between

45-hmC and methyl CpG binding
protein 2 (MeCP2), a member of the
MBD family that is important forneuronal maturation. While 5-hmC
has limited binding affinity to most
MBD proteins, in the case of MeCP2,
both 5-mC and 5-hmC can bind itwith high affinity (Figure 1) raising thequestion of how each affects neuronalprocesses. While MeCP2’s repressive properties are important fornormal brain function, it has recently
been shown that mutations in MeCP2
proteins are associated with certain

diseases. In the case of Rett syndrome, there is a strong correlation
between 5-hmC and MeCP2 binding
when these mutations are present.
As researchers continue to study
5-hmC’s contribution to disease andto normal brain function, Active Motif
offers highly characterized and validated 5-hmC antibodies for a numberof important applications, including
hMeDIP-Seq, IF and IHC. For more
information please visit us atwww.activemotif.com/dnamethabs.

Figure 1: 5-hmC antibody (Catalog No. 39769) shown in confocal microscopy.
The homogeneously (euchromatic) distributed MeCP2 co-localized with 5-hmC (top, arrowhead), but the aggregated (heterochromatic) MeCP2 dissociated with 5-hmC in more mature neurons (bottom, arrowhead). It is shown that MeCP2 are mostly distributed
at DAPI-dense regions with heterochromatin where 5-hmC is usually absent during neuronal maturation (legend edited for content).
Copyright © 2014 Chen, Damayanti, Irudayaraj, Dunn and Zhou (2014) Front Gent. 5:46 doi: 10.3389/fgene.2014.00046. This is an
open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY)

Product

FormatCatalog No.

5-Hydroxymethylcytosine (5-hmC) antibody (pAb)

100 µl39769

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Products for 5-hmC ResearchTools for Neuroepigenetic Research
OH

NH2

N

O

N
R

C

5-hm

5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification thatis known to play a major role in the Central Nervous System (CNS) in
the regulation of development, neuroplasticity and disease. Due to its
importance in neuroepigenetic research, tools to accurately detect andquantify 5-hmC are highly warranted. Active Motif offers a comprehensive portfolio of products specific for 5-hmC, including antibodies, kitsfor 5-hmC enrichment, assay-ready enzymes and custom services toaid in this area of research.

5-hmC is highly abundant in the CNS
where it regulates various processes,
including development, aging andneuroplasticity. High 5-hmC levels inthe developing brain are associatedwith maintenance of active transcription. In adult neurons, alternating DNA
methylation and demethylation are
observed in response to neuronal ac­
tivity that suggests 5-hmC is involvedin cognitive function and developmentof psychiatric disorders such as stressinduced PTSD. Also, aberrant 5-hmC
distribution is linked to the etiopathology of various neurodegenerative
disorders, including Alzheimer’s andHuntington’s disease.

Assays for 5-hmC DNA enrichment
To analyze 5-hmC at the resolutionof individual genomic loci by PCR or
sequencing, Active Motif offers two
methods for hydroxymethylated DNA
enrichment. Both methods utilize
magnetic beads for faster processing.
Our hMeDIP Kit is an antibody-based
technique that utilizes a highly specific 5-hmC antibody to immunoprecipitate DNA fragments containing
5-hmC from the rest of the genomic
DNA. The assay is optimized towork with both single-stranded anddouble-stranded DNA.
Alternatively, the Hydroxymethyl

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Collector™ Kit utilizes a β-Glucosyltransferase enzyme to label 5-hmC
residues within double-stranded
DNA fragments with a modified
glucose moiety to enable distinction
from other cytosine modifications
during enrichment. This sensitivetechnique can enrich DNA fragments containing as little as two
5-hmC residues.

Assay-ready enzymes to study 5-hmC
Little is known about how 5-hmC
functions as a transcriptional regulator or as an intermediary of DNA
demethylation. Functional studies
often involve assays to study theseprocesses in vitro. For this purpose,
Active Motif provides several enzymes
for 5-hmC analysis to readily incorporate into your assays.
To study the process of DNA demethylation, Active Motif offers Recombinant Tet1 protein that catalyzes theconversion of 5-mC to 5-hmC andother oxidative cytosine variants.

Active Motif also offers the
β-Glucosyltransferase enzyme tochemically label 5-hmC with a glucosyl moiety and the PvuRts1I restriction
endonuclease that specifically cleaves
hydroxymethylated DNA. These enzymes distinguish 5-hmC from othercytosine variants and are valuable
tools for analysis of hydroxymethylated DNA patterns in the genome. For
more information, please visit
www.activemotif.com/dnametenzymes.

Methylation variant profiling services
Active Motif’s Custom Epigenetic
Services include genome-widehydroxymethylcytosine (hmC), formylcytosine (fC) and carboxylcytosine(caC) profiling. The services use
antibodies that specifically recognize
each modification for DNA immunoprecipitation to generate genomewide enrichment profiles. For more
complete information, please visit
www.activemotif.com/meth-variant-svc.

Product

FormatCatalog No.

hMeDIP

10 rxns

55010

Hydroxymethyl Collector™

25 rxns

55013

Recombinant Tet1 protein, active

25 µg

31363

β-Glucosyltransferase enzyme

500 Units

55012

PvuRts1I restriction enzyme

50 Units

55011

5

Global DNA Methylation Assay

NEW

Quantify Global Changes in DNA Methylation
Because global hypomethylation is a hallmark of human cancer and disease,simpler methods than HPLC or bisulfite sequencing are warranted for analyzing
genome-wide DNA methylation to correlate variances with factors such as treatment conditions or clinical outcome. 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 tooffer better specificity and reproducibility than other methods that utilize nonspecific passive adsorption.
LINE-1 & global DNA methylation
The Global DNA Methylation – LINE-1
Kit is uniquely designed to quantitate
methylation in Long Interspersed
Nucleotide Element 1 (LINE-1) repeats in human genomic DNA. LINE-1
methylation serves as a surrogate
readout for global DNA methylationlevels. The ELISA-based assay provides a highly specific and sensitive
high-throughput method for screening relative changes in 5-mC levels
across samples to correlate with variables, such as treatment conditions,environmental and social factors andclinical prognosis (Figure 1).

How does it work?
In the Global DNA Methylation –LINE-1 Assay, the genomic DNA ofinterest is enzymatically fragmented
and hybridized to a biotinylated probe
containing a human LINE-1 consensus sequence. Hybridized DNA is thenimmobilized onto a 96-well streptavidin-coated plate. Any unbound DNA
fragments are then washed away

Global DNA Methylation – LINE-1
2,5

OD 450nm

2
1,5
1
0,5

HeLa

HeLa + Aza

STD 75

STD 100

STD 50

STD 30STD 20

STD 0

STD 100
BLANKDNA methylation occurs in the context
of CpG dinucleotides that are mainly
found within repetitive elements. It
is largely understood that the global
hypomethylation observed in cancers
is primarily due to hypomethylation
at these elements. Not surprisingly,
aberrant LINE-1 methylation is also
linked to the onset and poor prognosisof cancer. Assessing the methylation
status of repetitive elements such as
LINE-1 has emerged in the literature
as a comparable method of ascertaining global methylation levels to
supplant HPLC and other current
methods used to measure total 5-mC
as a percentage of total cytosine.

DNA (100 ng/well)Figure 1: Global DNA Methylation Assay results showing
a decrease in 5-methylcytosine levels resulting from

What’s in the box?
The Global DNA Methylation – LINE-1
Kit contains an optimized protocol andall the necessary reagents to perform
DNA fragmentation, hybridization,
capture and colorimetric detectionof 5-mC. For added convenience,
methylated and non-methylated DNA
standards containing known levels ofLINE-1 methylation are included in
the kit that can be utilized to generatea standard curve for determination of% 5-mC values of your samples.

treatment of HeLa cells with 5-azacytidine (Aza), a DNA
methyltransferase inhibitor.

Product
Global DNA Methylation - LINE-1 Kit

6

and a 5-Methylcytosine antibody anda secondary antibody conjugated tohorseradish peroxidase (HRP) areused for detection of methylated fragments. The colorimetric readout isthen easily quantified by spectrophotometry. This LINE-1 targeted hybridization approach produces lower
background and enables more specificand reproducible capture of methylated genomic DNA fragments thanmethods using passive adsorption. To
learn more, please visit
www.activemotif.com/gdm.

FormatCatalog No.

1 x 96 rxns

55017

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ChIP-Bisulfite-Sequencing

NEW

ChIP-Bisulfite-Sequencing for Mapping Allele-Specific
Methylation of Protein/DNA Binding Sequences
Active Motif’s new ChIP-Bis-Seq Kit offers a method to directly interrogate boththe chromatin and DNA methylation profile on the same sample. The kit combines our highly validated high sensitivity chromatin immunoprecipitation (ChIP)
protocol to enrich for the sites of chromatin associations or modifications ofinterest, followed by a bisulfite conversion and sequencing (Bis-Seq) procedure
to generate single base-pair resolution methylation profiles at these sites.

What is ChIP-Bis-Seq?
ChIP-Bisulfite-Sequencing (ChIP-BisSeq) enables direct interrogation ofboth the chromatin and DNA methylation profile on the same sample within
a single experiment. ChIP-Bis-Seq
combines a ChIP procedure for targetspecific enrichment of protein-boundor modified chromatin with bisulfite
conversion and sequencing (Bis-Seq)
to provide a DNA methylation profile of
ChIP-enriched chromatin sequences.
This approach is ideally suited forgenome-wide studies of the interplay
between DNA methylation and chromatin features, such as in assessing
allele-specific DNA methylation variances (e.g. imprinting, X-inactivation),or in evaluating global gene regulatory
effects.

How does it work?
The ChIP-Bis-Seq Kit consists of threemodules. The first is Active Motif’s
ChIP-IT® High Sensitivity Kit which isused to immunoprecipitate chromatin
associated with a protein or modification of interest in order to reduce therepresentation to targeted sites of
the genome. The second kit module
contains methylated adapters andenzymes to enable preparation of a
Next-Generation Sequencing (NGS)
library from the ChIP DNA. By using
methylated adapters, the cytosines
within the adapter sequence will

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remain unchanged during bisulfite
conversion and retain their original sequence. The third kit module
includes reagents to perform bisulfite
conversion on the adapter-ligatedDNA. The converted library is PCRamplified, size selected and submitted
for NGS.

Save time and money
Active Motif saves you time and effort
by providing an optimized, validated
start-to-finish solution at a lower
price than purchasing components
separately. Check out the list ofincluded items in the box below.

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

What’s in the ChIP-Bis-Seq Kit?
• ChIP-IT® High Sensitivity Kit –includes lysis buffers, proteaseinhibitors, protein G agarose
beads, IP buffers and DNA
purification reagents to preparechromatin from cells or tissues
for 16 ChIP reactions• Library Preparation Reagents –includes end polishing enzymes
and methylated adapters compatible with Illumina sequencing
platforms to prepare 10 NGS
libraries from your ChIP DNA

Figure 1: DNA gel for size selection of the PCR amplified
ChIP-Bisulfite-Sequencing library.

Product
ChIP-Bis-Seq Kit• Bisulfite Conversion –includes sodium bisulfite conversion reagents with specialized DNA purification columns
suitable for use with converted,fragmented ChIP-enriched DNA

FormatCatalog No.

10 libraries

53048

7

ChIP-exonuclease Assay

NEW

High-Resolution ChIP Analysis of Transcription Factors
Active Motif’s new ChIP-exo Kit* provides high-resolution genome-wide mapping of transcription factor binding sites. The ChIP-exo method utilizes exonuclease-mediated digestion of DNA during the immunoprecipitation procedure
to eliminate non-targeted DNA and increase binding resolution to within 20-95
base pair sequencing reads. ChIP-exo significantly improves the accuracy ofidentification of protein/DNA binding motifs and is ideal for discovery-based
studies or evaluation of mutation and SNP effects.

DNA and reduce fragments to the siteof transcription factor binding prior todeep sequencing. By removing the excess unbound DNA, background signal
is reduced so that fewer sequencing
reads map to non-specific genomic
regions. This also enables identification of weak peaks allowing detection of transcription factors that are
inefficiently bound to the genome thatwould be indistinguishable from noisewith traditional ChIP-Seq.

What is ChIP-exo?
ChIP-exonuclease (ChIP-exo), a
technology that was developed in thelaboratory of B. Franklin Pugh, is a
modified ChIP-Seq approach that
includes an exonuclease digestion
step during chromatin immunoprecipitation to excise non-cross-linked

A high-resolution method for genomewide mapping of protein binding sites
offers many advantages. It improves
identification of transcription factor
consensus sequences that will aid in
the discovery of new binding motifs(Figure 1). Additionally, studies ofbinding specificities at high resolution
assist in deducing the impact of mutations and SNPs on transcriptional
regulation and disease.

Figure 1: Identification ofunique p53 binding sites.
In addition to reducing thesize of the identified p53
binding site (red triangle)
as compared to ChIP-Seq
results, Active Motif’s ChIPexo method identifies several
other unique binding sites
not revealed by ChIP-Seq (redHow does the assay work?
Cells are fixed with formaldehyde tocross-link protein/DNA binding interactions. Cells are then lysed and chromatin is fragmented by sonication. An
antibody directed against the protein
of interest is conjugated to protein G
magnetic beads for immunoprecipitation of the DNA of interest. With thechromatin still bound by the beads,
the DNA is end-polished and P7-exo
adapters are ligated onto the blunt
ends. The nicked DNA is repaired andthen digested by lambda and RecJF
exonucleases to excise DNA in a 5´ to
3´ direction, trimming DNA to the siteof the cross-linking and selectively
eliminating the P7 adapter at the 5´end. Following cross-link reversal andelution from the beads, the DNA is
made double-stranded by P7 primer
extension, and a P5-exo adapter isadded to the exonuclease-treatedends.
The DNA library is PCR amplified andsize selected before it is subjected
to high-throughput sequencing. The
sequence of the DNA is mapped backto the reference genome to determine
the binding locations of the protein ofinterest. The 5´ ends of the DNA fragments on the forward strand indicate
the left border of a protein/DNA interaction, while the 5´ ends of the DNA

circles).

8

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ChIP-exonuclease Assay

ChIP-exo Flow Chart

ChIP-exo advantages• 20-95 bp resolution of binding sites ascompared to 300 bp for ChIP-Seq• Provides high-resolution genome-wide
mapping of transcription factors• Exonuclease digestion of non-specific DNA
reduces non-specific background• Improves detection of inefficiently-boundtranscription factors• On-bead enzymatic reactions streamline
sample processing

fragments on the reverse strand indicate the right border of a protein/DNA
interaction. These borders demarcate
the precise site of protein/DNA crosslinking, providing high-resolution(20-95 base pairs) identification of
genomic binding sites (Figure 2).

What’s in the box?
ChIP-exo Kits contain lysis buffers, protease inhibitors, protein G
magnetic beads, exonuclease andpolymerase enzymes and reaction
buffers, dNTPs, sequencing adapters, indexing primers and DNA
purification and size selection beads
sufficient to prepare chromatin andperform 12 immunoprecipitation andsequencing library reactions. The kit
includes a streamlined protocol thathas been adapted for use on Illumina
sequencing platforms.
For more complete information on the
ChIP-exo Kit, please visit us atwww.activemotif.com/chipexo.

*Technology is covered under U.S. Patent No. 8367334 b2.

Figure 2: Schematic of the ChIP-exo Kit workflow following chromatin preparation and capture
with antibody-conjugated beads.

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Product

FormatCatalog No.

ChIP-exo Kit

12 rxns53043

9

Nucleosomes

NEW

Nucleosome Substrates for More Biologically
Relevant Screening Assays
Whether you are performing drug discovery screening, analyzing enzyme kinetics
or monitoring changes in histone modifications, substrate selection is critical.
Nucleosomes offer the advantage of providing a more biologically relevant substrate when compared to histone proteins alone or synthetic peptides. To best
mimic cellular biology with your in vitro assay, use Active Motif’s Nucleosome
Preparation Kit to prepare nucleosomes from your samples, or utilize one of our
pre-assembled Recombinant Nucleosomes.

Prepare your own nucleosomes
Active Motif’s new Nucleosome
Preparation Kit contains lysis buffers,
protease inhibitors, digestion buffer,
enzymatic shearing cocktail and an
optimized protocol to isolate intact
nucleosomes from your samples (Figure 1). Simply adjust your enzymatic
digestion time to modify the amountof mononucleosomes or oligonucleosomes you extract. Use yourisolated nucleosomes as substrates in
the analysis of histone post-translational modifications, enzyme kinetics
or inhibitor screening studies.

Looking for ready-to-use
nucleosome substrates?
Active Motif’s Recombinant Nucleosomes save you time and effort byproviding pre-assembled humannucleosomes. Each recombinantnucleosome is comprised of octamersof unmodified core histone proteins(H2A, H2B, H3 and H4) bound by DNA.
These intact nucleosomes are ideal
substrates for in vitro studies. Nucleosomes are available to study both Histone H3.1 and Histone H3.3 variants.
Additionally, choose from unlabeled
or biotin-labeled nucleosomes to give1

Nucleosome Preparation Kit

2

3

4

2

3

4

NSD2-SET

To illustrate the usefulness of nucleosome substrates, recombinantnucleosomes were compared withhistone octamers in an activity assay
using SET domain-containing histone
methyltransferase enzymes NSD1-SET
and NSD2-SET (Figure 2). The results
show there is greater methyltransferase activity when using recombinantnucleosomes as a substrate as compared to octamers alone.

5

NSD1-SET
1

you flexibility in your experimental
design.

5

1. No enzyme
2. No substrate
3. Histone Octamers
4. Recombinant Nucleosomes A
5. Recombinant Nucleosomes B

Figure 2: Histone methyltransferase activity assay comparing recombinant nucleosomes and histone octamers as substrates.

Product

FormatCatalog No.

Nucleosome Preparation Kit

20 rxns

53504

Recombinant Nucleosomes (H3.1)

20 μg

31466

Figure 1: DNA analysis of HeLa (A2, B2) and MCF-7

Recombinant Nucleosomes (H3.1) - biotinylated

20 μg

31467

(C2, D2) cells enzymatically digested into mono- andRecombinant Nucleosomes (H3.3)

20 μg

31468

Recombinant Nucleosomes (H3.3) - biotinylated

20 μg

31469

oligonucleosomes using Active Motif’s Nucleosome
Preparation Kit.

10

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Epigenetic Services

NEW

Epigenetic Services:
A Novel ChIP-Seq Spike-in Strategy
A persistent problem with chromatin immunoprecipitation is that traditional
ChIP-Seq protocols are not always able to detect global changes in histone modifications caused by treatment of samples with epigenetic inhibitors. Active Motif’s
Epigenetic Services team has a solution. We have created and validated a ChIP-Seq
spike-in normalization strategy that is able to reveal these differences. This method
is now available as part of our end-to-end ChIP-Seq Service.

Problem:• ChIP-Seq does not always detect
inhibitor-induced global changes
in histone modifications
Solution:• ChIP-Seq Normalization to a
spiked-in control

How does it work?
ChIP-Seq reactions: A standard ChIPSeq reaction is set up using experimental chromatin (e.g. human) and an
antibody of interest (e.g. H3K27me3
antibody). However, in addition,
Drosophila melanogaster chromatin isadded, or “spiked-in”, to each reaction
as a minor fraction of total chromatin. An antibody that recognizes the
Drosophila-specific histone variant,H2Av, is also added to the reaction.
The H2Av antibody provides a mechanism to reliably pull down a small
fraction of Drosophila chromatin.
Following ChIP, immunoprecipitated
DNA sequences are analyzed by NextGeneration Sequencing (NGS).

Normalization: Following NGS, sequence tags are aligned to the experimental reference genome (e.g. human)
and the Drosophila genome. Differences in Drosophila tag counts areequalized across samples. Human tag
counts are then normalized using thesame ratio used to equalize Drosophila
tag counts.

Results: Biases that are introduced
during Next-Generation library amplification and sequencing also occur
in the Drosophila spike-in chromatin.
Normalization using our spike-in
strategy eliminates these biases toenable ChIP-Seq analysis to reveal
any significant biological changes inyour samples (Figure 1).

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

For more information, or to request a quote, please go to www.activemotif.com/services-normalize.

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11

MicroRNA Target Validation

NEW

Validate the Specific mRNA Targets of miRNAs
MicroRNAs (miRNAs) regulate many important cellular processes, including
development, differentiation, proliferation and apoptosis. Because changes inmiRNA expression levels have been found in a variety of cancers, as well as in
cardiac disease and neurological disorders, miRNA-mediated regulation is an
important topic in biomedical research. Active Motif’s new miRNA Target IP Kit
was designed to identify the physical interactions of miRNAs with endogenous
mRNA transcripts in order to validate the binding targets of specific miRNAs.

The targeting of a miRNA to a specific mRNA is mediated through theformation of an RNA Induced Silencing Complex (RISC). While RISCs cancontain 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 (Diagram 1).

Validate miRNA/mRNAinteractions using Ago IP
Active Motif’s new 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 associated with 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

By comparing cells transfected with orwithout a miRNA mimic, one can validate the mRNA targets of a particular
miRNA based on over-expression ofthat miRNA (Figure 1). It is also possible to profile the immunoprecipitatedmRNA molecules using conventional
expression microarrays or RNA-Seq.
For more information, please visit us
at www.activemotif.com/mirna-ip.

14

miR-122
Non-targeting
Mock tfxn

12

Fold Enrichment

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). In mammalian cells,
the interaction of a miRNA with a3´UTR usually results in repression oftranslation.

10
8
6
4
2

AAA-3´
miRNA

0

G6PC3

SLC1A5GAPDH

Figure 1: miR-122 targets G6PC3 and SLC1A5.
The miRNA Target IP Kit was used on samples of HT1080
cells that were transfected with a miR-122 mimic or a
non-targeting miRNA control for 8 hours, or that were

RISC Complex
Diagram 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.

12

mock transfected. Following IP using the Ago1/2/3 antibody or Negative Control IgG included in the kit, qRT-PCR
was performed on the samples using primers for G6PC3and SLC1A5, which are known targets of miR-122, and for
GAPDH, a common housekeeping gene that is not known
to be targeted by miR-122.

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Post-transcriptional Regulation by microRNAs

miRNAs in Neural Development &Neurological Diseases
MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that act as
post-transcriptional regulators in many different biological processes. Observedchanges in expression levels of specific miRNAs, both upregulated and down,
demonstrate that they are involved not just in the development and function of thebrain, but also in the occurrence and progression of many neurological diseases.
Consequently, miRNAs are proving to be useful as novel biomarkers in studies ofischemia & reperfusion, traumatic brain injury, as well as in disorders, such as
epilepsy and Alzheimer’s, Parkinson’s & Huntington’s diseases.

Post-transcriptional regulators
miRNAs regulate gene expression
by binding to the 3´UTRs of mRNAtranscripts. This interaction results ineither inhibition of translation or transcript degradation. Most miRNAs
target multiple mRNAs, and it has
been shown that a single miRNA canregulate multiple signaling pathways
simultaneously. In addition, individualmRNAs frequently contain bindingsites for more than one miRNA. This
creates a complex feedback networkthat regulates and fine-tunes theexpression levels of individual genes,
as well as entire pathways, in a tissue
and cell-state dependent manner.

miRNAs in neurological disorders
A review article by Wang et al. (Biomed.
Rep. (2014) 2(5): 611-619.) provides
an overview of research articles
that study the role miRNAs play in a
number of neurological disorders. It
highlights papers that show how thealtered expression of specific miRNAs
can play a neuroprotective role bymodulating the expression of genes
involved in diseases of the central
nervous system. For example, Harraz
et al. (PNAS USA (2012) 109: 1896218967.) shows miR‑223 overexpression
can decrease levels of both GluR2 andNR2B, which protects the brain fromneuronal cell death during stroke.

Study the impact of miRNAs
Active Motif’s LightSwitch™ System
is ideal for assessing the functional
impact of miRNA-3´UTR interactions.
It includes a collection of over 12,000
human 3´UTRs that can be purchased
as ready-to-transfect LightSwitch
luciferase reporter vectors. Combinedwith our large collections of miRNA
Mimics & Inhibitors, you have everything needed to study miRNA-3´UTRinteractions, validate miRNA targets,and to measure RNA stability and thefunctional impact of miRNAs on agene-by-gene basis (see below). For
more information, please visit us atwww.activemotif.com/ls-3utr.

Studying miRNA-3´UTR Interactions with the LightSwitch™ Luciferase Assay System

Gene of interest
RenSP
Luciferase

3´UTR
Pre-cloned intoa Reporter
VectorLightSwitch™
3´UTR
Reporter
1. Choose a 3´UTR of interest fromour collection of over 12,000
ready-to-transfect constructs.

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miRNA Mimic

Light signal

RISC
complex

Hybrid mRNA
2. Co-transfect reporter
construct with miRNA
mimics or inhibitors.

3. Measure luciferase activity to determine
the effect on translation of miRNA binding
to the hybrid luciferase-3´UTR mRNA.

13

Epigenetics in Drug Discovery

Tools to Streamline Epigenetic Drug Research
Drug discovery and development programs focused on epigenetics have attracted
much attention and investment in recent years. This is driven by the potential todevelop novel therapeutic strategies that can reverse epigenetic and transcriptional
abnormalities. Active Motif is a leading provider of innovative technologies for
epigenetics and gene regulation research. Our assays, reagents and services canbe easily integrated into your research to streamline the drug discovery workflow.
Biologically relevant assay substrates
The outcome of your in vitro biochemical assays is vastly
improved when the system mimics actual cellular biology. To
provide you the best choice of substrate for use in your assay,
Active Motif offers over 60 different recombinant full-length
modified H3 and H4 histones that are engineered using patented technology to have structural and functional similarity
to their native counterparts. These can be used as standalone substrates or assembled along with recombinant H2A
and H2B to generate nucleosomes and oligonucleosomes.
By choosing the most biologically relevant substrate, you canensure the most accurate results.
To analyze epigenetic modifications or protein interactions
in a chromatin context, Active Motif’s Chromatin Assembly
Kit provides an end-to-end solution for designing your own
chromatin. The technique involves an ATP-dependent process
to produce a chromatin substrate that mimics native in vivo
chromatin structure. The assembled chromatin is ready to use
in downstream assays, such as in vitro transcription assays,
chromatin immunoprecipitation and enzyme activity assays.

Which Histone Substrate is Right for Your Assay?

Choosing the correct histone substrate for assay development is key to achieving the best

Assay-ready proteins, enzymes and domains
There is no need to waste valuable time and resources generating proteins for assay development. With Active Motif’scomprehensive portfolio of over 300 purified assay-readyrecombinant proteins – including transcription factors, histones,
DNA- and histone-modifying enzymes, and bromodomains –you are sure to find what you need to develop more efficient
assays for your drug discovery and development program.

Assays for enzymatic activity
Active Motif offers a variety of robust, sensitive, highthroughput assays for quick and easy screening and profiling of cellular changes in the activity of histone modifying
enzymes. These include our HAT Assay Kit to measure theactivity of histone acetylases, the HDAC Assay Kits to analyze
histone deacetylase activity and our Histone Demethylase
Assay to analyze the lysine demethylase activity of LSD1.

results. Opinion leaders in epigenetics recognize the power of reconstituting recombinant
chromatin for creating biologically relevant substrates.

For more details, go to www.activemotif.com/info to download a copy of our Tools for Drug Discovery product brochure.

14

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Inhibitors & Activators

NEW

Small Molecules to Modulate Epigenetic Processes
Active Motif has an expanding collection of small molecule compounds (activators and inhibitors) thattarget the activity of proteins that regulate epigenetic changes in DNA methylation, chromatin remodeling and histones. These include targets to DNA Methyltransferases (DNMTs), Histone Acetyltransferases(HATs), Histone Deacetylases (HDACs), Histone Methyltransferases (HMTs), Histone Demethylases(HDMs), and the BET family bromodomains. As epigenetic aberrations are implicated in many
diseases, these modulators are important tools for use in lead generation and assay development.
Description

TargetFormatCat. No.

HISTONE DEACETYLASE
Apicidin

HDAC inhibitor

1 mg
5 mg

14041
14040

BML-210HDAC inhibitor

5 mg
25 mg

14049
14048

CUDC-101

HDAC inhibitor

5 mg
25 mg

HPA (Hexyl-4-pentynoic acid)

HDAC inhibitor

Description

TargetFormatCat. No.

DNA methyltransferase inhibitor

50 mg
250 mg

14103
14102

Decitabine(5-Aza-2´-deoxycytidine)

DNA methyltransferase inhibitor

10 mg
50 mg

14101
14100

14061
14060

RG108

DNA methyltransferase inhibitor

5 mg
25 mg

14105
14104

10 mg
50 mg

14035
14034

Description

TargetFormatCat. No.

5 mg
25 mg

14053
14052

METHYLATION
5-Azacytidine

MS-275HDAC inhibitor

5 mg
25 mg

14043
14042

HISTONE ACETYLTRANSFERASE
HAT inhibitor
C646

Panobinostat

HDAC inhibitor

5 mg
25 mg

14045
14044

CTPB

HAT activator

Phenylbutyrate Na

HDAC inhibitor

1g

14033

5 mg
25 mg

14065
14064

Romidepsin

HDAC inhibitor

1 mg

14083

Garcinol

HAT inhibitor

Trichostatin A

HDAC inhibitor

1 mg
5 mg

14039
14038

10 mg
50 mg

14077
14076

Description

TargetFormatCat. No.

Tubastatin A

HDAC inhibitor

1 mg
5 mg

14085
14084

SIRTUIN
AK-7

SIRT2 inhibitor

Valproic acid

HDAC inhibitor

5g

14021

5 mg
25 mg

14055
14054

Vorinostat (SAHA)HDAC inhibitor

10 mg
50 mg

14027
14026

BML-278

SIRT1 activator

5 mg
25 mg

14025
14024

TargetFormatCat. No.

EX-527

SIRT1 inhibitor

5 mg
25 mg

14029
14028

LYSINE METHYLTRANSFERASE & LYSINE DEMETHYLASEHMT inhibitor
5 mg
BIX-01294
25 mg

Piceatannol

SIRT1 activator

14073
14072

5 mg
25 mg

14037
14036

Resveratrol

SIRT1 activator

Chaetocin

HMT inhibitor

200 µg14051

100 mg
500 mg

14023
14022

Daminozide

HDM inhibitor

50 mg
250 mg

14059
14058

SirtAct

SIRT1 activator

5 mg
25 mg

14081
14080

DMOG

HDM inhibitor

10 mg
50 mg

14063
14062

Sirtinol

SIRT2 inhibitor

5 mg
25 mg

14075
14074

GSK-J1 (cell impermeable)

HDM inhibitor

5 mg
25 mg

14069
14068

Splitomycin

Sir2p inhibitor

5 mg
25 mg

14087
14086

GSK-J4 (cell permeable)

HDM inhibitor

5 mg
25 mg

14071
14070

Description

TargetFormatCat. No.

IOX1

HDM inhibitor

5 mg
25 mg

14057
14056

BROMODOMAIN
JQ1 (racemic)

ML-324

HDM inhibitor

5 mg
25 mg

14079
14078

Bromodomain
inhibitor

5 mg
25 mg

14067
14066

Description

TargetFormatCat. No.

Tranylcypromine hemisulfate

HDM inhibitor

50 mg
250 mg

14047
14046

NAD biosynthesis
inhibitor

5 mg
25 mg

14031
14030

Description

OTHER
FK-866 HCl

For an up-to-date list of available activators & inhibitors, please visit www.activemotif.com/smallmol.

sales@activemotif.com

15

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SAN DIEGO, CA

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Enabling Epigenetics Research