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THE NEWSLETTER OF ACTIVE MOTIFOCTOBER 2015 | VOLUME 16 NUMBER 2

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.

4

NEW: Improve the Success of RNA
Applications with Subcellular Isolationof High-Quality RNA

5

NEW: Service for Quantitation of Histone
Post-translational Modifications in
Multiplex

6

NEW: Quantitative ELISA for the
Detection of Global 5-hmC

7

NEW: Determine the Genome-wide
Distribution of 5-Hydroxymethylcytosine

8

Tools to Enable Insight into theTranscriptional Programs of
Neurogenesis

10 Perform ChIP Without Protein-specific
Antibodies using Tag-ChIP-IT®
11 Enhance the Reproducibility of Any
Probe Sonicator with the EpiShear™
Cooled Sonication Platform

There is increasing evidence that several neurodevelopmental,
neurodegenerative and psychiatric disorders are, in part, caused
by aberrant epigenetic modifications. As a result, researchersin 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
are found in neurons
These modifications include classicalepigenetic marks such as DNA
methylation and histone modifications.
Other epigenetic mechanisms in playinclude 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) and a
growing body of evidence linking
epigenetic mechanisms to neuraldevelopment, plasticity and CNS
disorders, such as schizophrenia anddrug addiction. More than a dozen
neurological syndromes so far have been
linked to mutations in single genesthat encode DNA methyltransferase
and histone modifying enzymes. This
list includes neurological disordersthat appear in early childhood (Rettsyndrome) 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, 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 toenable scientists to link specificstates of chromatin to individual
gene loci in a cell to understand how
genes are regulated• Global and locus-specific methods toenable researchers to analyze shifts
in DNA methylation status of repetitiveelements or at specific promoters• Bisulfite conversion of methylated
DNA prior to sequencing to allow
detection of methylated cytosines“What Roles do Epigenetic Mechanisms Play in
Neural Development and Diseases of the Nervous System?”2

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Epigenetics of the Nervous System

Using the right tools for ChIP
ChIP is a technically challenging
method. Researchers who are notexperts in ChIP techniques are best
served using well-validated and reliable
kits to perform these assays. Active
Motif has developed a number of kits
and accessory reagents tailored to aid
researchers with their ChIP experiments.
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, visit us atwww.activemotif.com/chip.

“How do MicroRNAs
Regulate Gene Expressionin Neurons?”
MicroRNAs in neural function
MicroRNAs (miRNAs) are regulators ofgene expression that are implicated innearly all biological processes, including
neural development and dysfunction.
miRNAs regulate neural specification
and also contribute to neurodegenerativeand psychiatric conditions such as
Fragile X syndrome and drug addiction.
While miRNAs are abundant in neurons,
their biological roles are still emerging.
Active Motif’s LightSwitch™ portfolio of
products includes over 12,000 3’UTR
constructs, miRNA mimics and inhibitors,
and custom services to ensure you
have all the functional genomics
tools at your disposal for the study ofmiRNA function. To learn more about
LightSwitch™ miRNA products, visit us
at www.activemotif.com/lightswitch.
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 tissues. This has led to speculation that 5-hmC may have a uniquebiological role in the CNS. Also, recent
studies have shown a correlation
between DNA methylation levels andsales@activemotif.com

behavioral disorders such as anxiety
and depression. In addition, mutations
in genes involved in DNA methylation(e.g. MeCP2) are linked to neurologicaldiseases. However, the mechanism bywhich DNA methylation exerts theseeffects is not fully understood.
Tools for DNA methylation analysis
Active Motif offers a number of products
specific for this area of research, including
kits and antibodies that enrich for DNA
fragments that contain 5-mC and 5-hmC.
Our DNA methylation related antibodies
have been validated for various applications including ChIP, methylated DNA
immunoprecipitation (MeDIP) and immunofluorescence. For complete details
on all our DNA methylation products,
visit www.activemotif.com/dnamt.
Epigenetic techniques require
validated antibodies
One of the greatest challenges for
epigenetics research has been thelack of available antibodies that showproper specificity and that have beenvalidated for use in techniques suchas ChIP and ChIP-Seq. Histones havemultiple post-translational modifications
along the N-terminal tail portion thatare involved in gene regulation anddisease. Antibodies need to be able toclearly differentiate between these

multiple modifications. It is a major
challenge to develop antibodies with
sufficient specificity that will also perform
in demanding techniques such as ChIP.
Active Motif has established a validation
program for its antibodies that includes
ChIP-Seq testing, ChIP validation, as
well as a test on the specificity of ourhistone 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.
Let us do the work for you Active Motif Epigenetic Services
Generating high quality, interpretabledata from ChIP experiments can bechallenging as it requires optimized
protocols for various cell types andknowledge of cell type-specific bindingsites. Add in the technical and bioinformatics challenges associated withgenerating whole-genome data sets,
and ChIP-Seq may literally be beyond
your reach. Our Epigenetic Services
team provides a wide variety of ChIP
services. This makes it possible foryou to utilize our expertise and researchtools without having to be an expert
in the techniques yourself. To find out
more, or to get a quote, please go towww.activemotif.com/services.

GLOBAL EPIGENETIC SHIFTS DURING NEUROGENESIS
Self-renewal
NPCs

Fate
Specification

Differentiation

Maturation

5-mC level
5-hmC level
H3/H4 acetylation
H3K4me2
H3K27me3ncRNAsProliferation miRNAs

Neurogenesis miRNAs

3

RNA Isolation

NEW

Improve the Success of RNA Applications with
Subcellular Isolation of High-Quality RNA
RNA molecules exist at different levels of maturation and processing within a cell at any
given time. Such heterogeneity can bias downstream analysis and data interpretation. Unlike
other commercial RNA isolation kits, Active Motif’s RNA Subcellular Isolation Kit enablesisolation of RNA from specific subcellular fractions, greatly improving the accuracy of gene
expression profiling by reverse transcription quantitative PCR (RT-qPCR) or RNA-Seq.
Additionally, subcellular isolation can be used to localize long non-coding RNAs (lncRNAs)
to their site of action which can provide important insights into their functional role.

• Separates nuclear &cytoplasmic RNA withoutcross-contamination• Works with cell or tissue samples• Spin columns included for
RNA purification• Isolates RNA sizes 75-7000 nt• Purified RNA validated for use
in RT-qPCR and RNA-Seq

Minimize sample heterogeneity withcellular fractionation
Traditional methods for RNA analysis
often utilize total RNA which consistsof a mixture of intronic RNA originatingfrom immature transcripts in the nucleus,
mature RNA from the cytoplasm anda variety of non-coding RNAs. Suchheterogeneity can bias downstream
analysis and data interpretation by
reducing the sensitivity of detection
for low abundance transcripts and theability to study RNA processing
dynamics. Total RNA also lacks
information regarding RNA localization.
To overcome these challenges, Active
Motif has developed the RNA Subcellular
Isolation Kit to efficiently isolate separate
nuclear and cytoplasmic fractions of

4

RNA for downstream analysis. Thismethod can be used to isolate both longand short RNA sequences from cells
or tissue without cross-contaminationor the use of phenolic compounds.
How does it work?
Lysis Buffer is added to cell or tissue
samples. The lysate is then separated
by centrifugation, with the supernatantcontaining cytoplasmic RNA and the pellet
containing nuclear RNA. A guanidinebased buffer and ethanol are added toeach RNA fraction before loading onto
individual spin columns. Following
a wash step to remove proteins, theRNA fractions are eluted and quantified.
The RNA Subcellular Isolation Kit canbe used in combination with Active
Motif’s new DNase I Treatment Kit toremove any genomic DNA contaminationfrom the purified RNA sample.
What’s in the box?
Each kit contains enough reagents toperform either 15 cytoplasmic and
15 nuclear RNA isolations, or 30
whole-cell RNA isolations.
For more information, please visit us
at www.activemotif.com/rna-iso.

Subcellular RNA Expression Analysis
NuclearCytoplasmic
100

Percent of Total RNA

RNA Subcellular Isolation Kit

Figure 1: Analysis of subcellular RNA fractions by
RT-qPCR provides insight into RNA localization.
Nuclear, cytoplasmic and total RNA were isolated fromHeLa cells using the RNA Subcellular Isolation Kit.

80

Purified RNA was then subjected to RT-qPCR. Each
subcellular fraction was plotted as a percentage of total

60

RNA. The data shows localization of lncRNAs, MALAT1,

40

NEAT1 and TUG1, that is consistent with published
RNA-FISH data. Ribosomal protein (S14) and mRNA

20

(BIRC5) are both shown to localize to the cytoplasm, as
0

MALAT1

NEAT1

TUG1

S14

BIRC5

expected and consistent with previously reported results.

Product

FormatCatalog No.

RNA Subcellular Isolation Kit

30 rxns

25501

DNase I Treatment Kit

30 rxns

25502

www.activemotif.com

Histone PTM Quantitation Service

NEW

Service for Quantitation of Histone Post-translational
Modifications in Multiplex
Active Motif’s Histone PTM Quantitation Service gives you access to ground-breaking
technology that enables high-throughput multiplexed screening of histone modifications.
The service uses Luminex® xMAP® magnetic bead-based multiplexing technology tosimultaneously measure multiple histone modification targets in a single reaction. This
first-of-its-kind multiplex epigenetic assay generates more data using less sample
material than traditional Western blot, mass spectrometry or ELISA methods andenables you to analyze both specific and off-target effects in your sample simultaneously.

HISTONE PTM QUANTITATION SERVICES
Simply submit frozen cell pellets and we will:

1 Prepare cell lysates
2 Perform Luminex
histone quantitation®

3 Perform normalization
to Histone H3

How does it work?
The service utilizes the same Luminex
technology as our Histone H3 PTM
Multiplex Assay to enable multiplexing
and quantitation of histone PTM
targets (Figure 1). The end-to-end,
customizable service includes sample
preparation, assay design andexperimental execution, data analysis
and support. Simply submit yoursamples and our expert scientists
will consult with you to formulate an
appropriate experimental design.

Service Features:• Minimal cell requirements(250K cells are sufficient toquantify 12 histone modifications)• Detects ≥ 25% difference
between samples• Controls include total H3 andsamples containing known PTMchanges
For more complete information, visit
www.activemotif.com/services-luminex.

4 Perform analysis anddelivery of data

Simultaneously screen specific andoff-target effects in the same sample
Profiling variances in histone
post-translational modification (PTM)
levels in clinical or compound-treatedsamples using traditional Western blot
or ELISA techniques requires large
numbers of cells from samples that may
already be in scarce supply. Traditional
assays are also limited by low throughput
and the inability to simultaneously
analyze specific and off-target effects.
Our Histone PTM Quantitation Service
provides a quantitative and highly
sensitive method to enable screeningof variances in histone modificationlevels in multiplex across clinical orcompound-treated samples.

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AVAILABLE TARGETS

Streptavidinphycoerythrin

SA-PE
in

ot

Bi

Biotinylated
Histone H3 Ab
H3Histonecontaining PTM

in

in

ot

ot

Bi

Bi

H3

H3

Ac

SA-PE

SA-PE

P

Me3

PTM Abconjugated beadH3 pan-ac

H3K56ac

H3K4me3

H3S10ph

H3K9ac

H3T11ph

H3K9me1

H3K27ac

H3K9me2

H3K27me2

H3K9me3

H3K27me3

Figure 1: Schematic of histone PTM multiplexing technology.
Histone-specific modifications in the N-terminus are captured using
antibodies conjugated to fluorescent-labeled magnetic beads. Eachbead emits a unique fluorescent signal to enable detection of multiple
targets within the same well. A biotinylated H3 C-terminal antibody
is used to capture histones. Streptavidin-phycoerythrin is thenadded to bind biotin and produce a signal. A Luminex® instrument
is used to read signals and decipher both the bead identity and the

To inquire about servicesorto receive a quote, contact

sales@activemotif.com

number of binding events pertaining to each histone modification.

55-Hydroxymethylation

NEW

Quantitative ELISA for the Detection of Global 5-hmC
Hydroxymethylation is known to play a role in transcriptional regulation and embryonicdevelopment. Research has indicated it may also serve as a prognostic indicator incertain cancers and neurodegenerative disorders. To better understand the influence ofchanges in global 5-hydroxymethylcytosine (5-hmC) levels on normal and diseased
states, Active Motif has developed the Global 5-hmC Quantification Kit. Obtain the same
quantitative capabilities as mass spectrometry, but with higher throughput and lower
sample requirements.

0.9
0.8
0.7

% 5-hmC

Simplified global 5-hmC quantification
Gene-specific analysis techniques do
not provide information about the
global levels of DNA hydroxymethylation
within a genome. Chromatographic
methods followed by mass spectrometry
are commonly used for global DNA
methylation analysis. Although they arehighly quantitative and reproducible,
these methods are labor-intensive,
require special equipment and expertiseand large amounts of high quality
DNA. Active Motif’s new Global 5-hmC
Quantification Kit enables you to obtainthe same quantitative results using
less sample material and in a higher
throughput and easier-to-use format
than mass spectrometry (Figure 1).

0.6
0.5

Global 5-hmC ELISA
Mass Spectrometry

0.4
0.3
0.2
0.1
0
mouse
mouse
cerebellum heartmouse
kidney

mouse
liver

mouse
lung

human
brain

human humankidneyliver

Figure 1: The Global 5-hmC ELISA generates comparable 5-hmC quantification to mass spectrometry using 10-fold less sample material.
The Global 5-hmC ELISA Kit was used to determine the % 5-hmC in genomic DNA isolated from mouse and human tissues. Genomic
DNA was tested in the range of 20 ng/well for human brain to up to 50 ng/well for all other DNA samples. The %5-hmC was calculated foreach sample using the included DNA standards. The data was compared to mass spectrometry data obtained from 500 ng of the same

How does it work?
Genomic DNA of interest is enzymaticallydigested and heat denatured before
Global 5-hmC ELISA features• Direct detection assay is
specific for 5-hmC DNA• Requires only 20-50 ng per wellof starting DNA• Includes DNA standards forquantification of 5-hmC levels

DNA samples. Results show the Global 5-hmC ELISA provides equivalent quantification using only a fraction of the starting material.

addition to coated 96-well plates.
Unbound DNA fragments are thenwashed away. A 5-hydroxymethylcytosine
antibody and a secondary antibody
conjugated to horseradish peroxidase(HRP) are used for detection of thehydroxymethylated fragments. The
colorimetric readout is then easily quantified by spectrophotometry. Results
are compared to the included DNA
standards to extrapolate the percent
5-hmC in the DNA sample (Figure 1).

What’s in the box?
Each kit contains an optimized protocol
and all the reagents necessary to perform
DNA fragmentation, plate coating,capture and colorimetric detection.
DNA standards with known levels of
5-hmC are included for standard curve
generation and sample quantification.
For more information, please visit
www.activemotif.com/global-hmc.

• Detects as little as 0.02% 5-hmC• Higher throughput quantification
than mass spectrometry

6

Product
Global 5-hmC Quantification Kit

FormatCatalog No.

1 x 96 rxns

55018

www.activemotif.com

Hydroxymethyl Collector™-Seq

NEW

Determine the Genome-wide Distribution of
5-Hydroxymethylcytosine
Because 5-hydroxymethylcytosine (5-hmC) is known to play a major role in the Central
Nervous System (CNS) in the regulation of development, neuroplasticity and disease,it has gained prominence in neuroepigenetic research. Therefore, tools to accurately
detect and quantify 5-hmC are highly warranted. A recent study from the laboratory ofFrancois Fuks utilized Active Motif’s Hydroxymethyl Collector™-Seq assay to investigate
the impact of oxidative stress on TET-mediated hydroxymethylation. Their results show,
for the first time, that oxidative assaults deeply modify the hydroxymethylome.

Profile changes in 5-hmC
5-hmC is highly abundant in the CNS
where it regulates various processes.
High 5-hmC levels in the developing
brain are associated with maintenanceof active transcription. In adult neurons,
alternating DNA methylation anddemethylation are observed in responseto neuronal activity that suggests 5-hmC
is involved in cognitive function anddevelopment of psychiatric disorders
such as stress-induced PTSD. Also,
aberrant 5-hmC distribution is linked
to the etiopathology of various neurodegenerative disorders, including
Alzheimer’s and Huntington’s disease.
Active Motif’s new Hydroxymethyl
Collector™-Seq Kit is designed specifically to determine the genome-wide
localization of 5-hmC in your samples(Figure 1).
Genome-wide 5-hmC pattern changes
in response to oxidative stressIn a recent study, Delatte et al. investigated the impact of oxidative stress on
TET-mediated hydroxymethylation. To
determine the genome-wide 5-hmC
distribution in neuroblastoma cells that
had been subjected to oxidative stress,
they utilized Active Motif’s Hydroxymethyl
Collector™-Seq assay (hmC-Seq).
Interestingly, the cells subjected tooxidative stress displayed a significant
global decrease in 5-hmC as well as
local differential hmC patterns at genes

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important for a protective response tooxidative stress, in agreement with previous mass spectra and dot blot (5-hmC
antibody, Catalog No. 39769) results.
Further, hmC-Seq in mice lacking theglutathione peroxidases 1 and 2 confirmed
and extended the above findings to show
restructuring of the hydroxymethylome
in response to oxidative stress in vivo.
Further, Delatte et al. observed an unexpected high proportion of differentiallyhydroxymethylated microRNA (miRNA)
coding sequences. The miRNAs displaying the most robust change in 5-hmC
were those with target mRNAs of toxico-

genomic pathways involved in oxidative
stress. In summary, these results
suggests that TET1 may play a role inprotecting cells against oxidative stress,
and the resulting changes in 5-hmC
pattern occur, not only in DNA regionsthat code for proteins, but also miRNAs.
In addition to Hydroxymethyl Collector™Seq, Active Motif offers a comprehensive
portfolio of 5-hmC products, including
antibodies, kits for 5-hmC enrichment,
assay-ready enzymes and custom services
to aid in this area of research.
This article is a summary of the research published in
Sci Rep.: Delatte et al., 2015.

Input

Brain

KidneyLiver

Figure 1: 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.

Hydroxymethyl Collector™-Seq

25 rxns

55019

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

100 μl39769

7

Tools to Enable Neuroepigenetics ResearchTools to Enable Insight into the Transcriptional
Programs of Neurogenesis

5

Mouse
Human

4
3
2
1
0
200 0

-400

-800

bits

2

The role of NFIB in neural progenitor
cell differentiation
To determine the underlying mechanismof NFIB-mediated neural progenitor
cell differentiation, the laboratory of
Dr. Richards performed a microarray
screen to analyze differential expression
in the hippocampus of NFIB-/- mice.
Interestingly, targets involved in the

8

-1600

-2000

1
0

TTGGCT GGCGCCAAGCCAAQUERY MOTIFTTGGC NNNNN GC C AATTGGC GGGAC C C C AA
TTG T T CTTTT GC C AA
Human (112): TT AGC ATAAA GC C AA(-845): TTGGC GGGAC GC C AA
Query Motif:

Mouse (-294):(-228):

Figure 1: An in silico screen for predicted NFI-bindingsites identified conserved potential NFI-binding sites
within the proximal promoter of the mouse EZH2 and theFI
EZ conH tr
EZ 2 -2 ol
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EZ 2 -2 4H 9
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human EZH2 gene.

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NFI EZH2 EZH2
control -294 -228
anti-NFIB

+

+

+

SS

For more detailed information,visit
www.jneurosci.org/content/34/8/2921.

-1200

Distance from TSS (bases)

1
2
3
4
5
6
7
8
9
10
11
12
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NFIB-mediated repression of EZH2
regulates cortical development
The chromatin-modifying protein
Enhancer of zeste homolog 2 (EZH2)
has recently emerged as a central
player in promoting progenitor cell
self-renewal during cortical development. However, the mechanism bywhich EZH2 itself is regulated remains
unclear. Furthermore, the nuclear
factor I (NFI) family of transcription
factors has been implicated in driving
neural progenitor cell differentiation.
Thus, researchers from the laboratoryof Dr. Linda Richards at the Queensland
Brain Institute set out to determine
the role of transcription factor nuclear
factor IB (NFIB) in this process. Utilizing
a suite of molecular and bioinformatics
tools, they identified the transcriptional
repression of EZH2 by NFIB to be an
important component of the processof neural progenitor cell differentiation
during cortical development.

-log10(p-value)

The transcriptional programs of neural progenitor cells change dynamically during
neurogenesis. While most research has focused on identifying changes in these gene
regulatory networks and the underlying mechanism causing them, it is equally important
to determine the actual functional impact of these changes or events. In a recent study
performed by the laboratory of Dr. Linda Richards at the Queensland Brain Institute inAustralia, numerous tools from the Active Motif product line were utilized to investigate
and functionally validate the epigenetic and transcriptional mechanisms regulating neuralprogenitor cell self-renewal during cortical development.

*

*

FP

FP

Figure 2: EMSAs performed with mouse brain nuclear
extracts and probes for (left) NFI control (lanes 1-2) andthe -294 (lanes 3-4) or the -228 (lanes 5-6) consensus
sites. (Right) NFI control (lane 1) and the -294 (lane 2),
mutated -294 sequence (lane 3), -228 (lane 4), or mutated-228 (lane 5) consensus sites.

modulation of chromatin structure
were highly enriched in the NFIB-/compared to wild-type mice, suggestingthat NFIB may influence chromatin
structure during development. Furthermore, an in silico screen searchingfor the high-affinity NFI-binding site
(TTGGC(N5)GCCAA) in the 2 kb region
upstream of the transcription start site(TSS) of all known Polycomb Repressive
Complex (PRC) genes identified EZH2
as having two putative NFI-binding
motifs in its basal promoter (Figure 1).
To determine whether NFIB is able
to regulate the transcription of EZH2,
researchers first investigated whether
NFIB could actually bind to thesepredicted sites within the EZH2
promoter. Using EMSA, they determinedthat a protein from nuclear extracts
derived from embryonic cortical tissue
bound to one of the two predicted NFIbinding sites (-228). Supershiftingof the -228 probe with an anti-NFIB
antibody indicated that this protein
complex contained NFIB (Figure 2,left). EMSA experiments using aprobe in which the -228 site had been
mutated did not exhibit the same bandas the wild-type -228 probe, indicatingthat NFIB could no longer bind thissequence (Figure 2, right).
To learn about Active Motif’s Gelshift™
Chemiluminescent EMSA Assay, visit
www.activemotif.com/gelshift.

www.activemotif.com

Tools to Enable Neuroepigenetics Research

NFIB represses EZH2 expression inprimary cortical cells
To address whether the observed
upregulation of EZH2 led to increased
repression of its downstream targets,
such as the INK4A locus, ChIP was
EZH2 Prom

2.0
1.5
1.0

NFIB pCAGIG+ EZH2 mut Prom

EZH2 mut
Prom

EZH2 Prom

NFIB pCAGIG0

NFIB pCAGIG+ EZH2 Prom

0.5

Figure 4: LightSwitch Dual Assay reporter assay
performed in Neuro2A cells. NFIB expression constructalone did not elicit luciferase activity (NFIB pCAGIG),
whereas the EZH2 promoter luciferase constructdisplayed high levels of luciferase. Cotransfection of the
NFIB expression and EZH2 promoter luciferase constructs
yielded significantly reduced levels of luciferase. Mutating
the NFI-binding site at -228 in the EZH2 promoter abolished
NFIB-mediated repression of luciferase activity.
ns: Not Significant. *p<0.05, ANOVA.

Dr. Richards at the Queensland Brain Institute in Australia,
published in J Neurosci.: Piper et al., 2014.

NFIB +/+NFIB -/-

4

*

2

*
0
-2
-4

*
*

-6

Ink4C

Ink4D

*
Ink4B

performed against H3K27me3 using
Active Motif’s Histone H3K27me3
antibody (Catalog No. 39155).
H3K27me3 levels were significantly
enriched at exons 1 and 2 of the INK4A
locus in NFIB-/- cortical tissue, consistent
with the elevated levels of EZH2 inthese mice. Further, the proximal
promoter regions of the INK4B (p15,
CDKN2B), INK4C (p18, CDKN2C), and
INK4D (p19, CDKN2D) loci carriedsignificantly reduced levels of H3K27me3(Figure 5). Collectively, these data showthat NFIB represses EZH2 expressionin cortical cells in vitro and that theelevation of EZH2 expression in NFIB-/mice promotes epigenetic changes to
key downstream target genes in vivo.

Figure 5: ChIP-IT Express performed using Histone
H3K27me3 antibody (Catalog No. 39155) on chromatin
isolated from NFIB+/+ and NFIB -/- cortices.

For more complete information on all
available ChIP validated antibodies,
visit www.activemotif.com/chipabs.
Product

NFIB-/- mice exhibited significantly elevated levels of
H3K27me3 at exons 1 and 2 of the INK4A locus, but
significantly reduced levels at the INK4B, INK4C, and INK4D
loci. *p = 0.05, Wilcoxon rank-sum test.

FormatCatalog No.

LightSwitch Custom Cloning

1 GoClone

32051

LightSwitch Custom Mutagenesis

1 GoClone

32052

LightSwitch Dual Assay Kit

100 assays

32035

LightSwitch Luciferase Assay Kit

100 assays

32031

LightSwitch ACTB Promoter Control

5 µg

32003

Gelshift Chemiluminescent EMSA

100 rxns

37341

ChIP-IT Express Kit

25 rxns

53008

™

Input

™

anti-IgG™
™

Figure 3: ChIP performed on tissue from mouse wild-type
cortex with antibodies against NFIB (Catalog No. 39091)
or IgG control (Catalog No. 53011). PCR was performed
with primers encompassing the predicted NFI-binding

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This article is a summary of the research performed by

Neg Conanti-NFIB

site within the EZH2 promoter.

Conclusion
Using analysis of NFIB-/- mice and asuite of molecular and bioinformatics
techniques, the laboratory of Dr. Richards
were able to show that EZH2 is atarget for transcriptional repression
by NFIB, suggesting that the deficits inneural progenitor cell differentiation
evident within NFIB-/- mice may bedue, at least in part, to the loss of
NFIB-mediated repression of thisepigenetic regulator. This study is justone example of how the Active Motifsuite of gene regulation products andservices can accelerate your functional
genomics research.

Ink4A exon 2

For more information on LightSwitch™
products & services, please visit us atwww.activemotif.com/lightswitch.

2.5

Ink4A exon 1

To determine whether NFIB could
functionally regulate EZH2 promoterdriven gene expression, the researchers
performed luciferase assays using theLightSwitch™ Dual Assay Kit. A 461
base pair region of the mouse EZH2
promoter containing the putative NFI
motif at -228 was cloned upstreamof the luciferase gene. As depicted in
Figure 4, NFIB is able to repress EZH2
promoter-driven transcription in vitro.
Further, this repression was abolishedwhen the NFI-binding site at -228 in
the EZH2 promoter was mutated.

ns

3.0

Relative H3K27me3
occupation/input(- Ct)To learn more about Active Motif
ChIP-IT Express Kits, please visit
www.activemotif.com/chipit.

*

3.5

Relative luciferase
Activity (Rluc/Cluc)

NFIB directly regulates EZH2transcriptional activity
To confirm the binding of NFIB to the-228 site of the EZH2 basal promoter,chromatin immunoprecipitation (ChIP)
was performed using Active Motif’s
ChIP-IT® Express Kit on wild-type
embryonic cortical samples. ChIP
confirmed the enrichment of NFIB at
the EZH2 promoter region containing
the predicted NFI-binding site (Figure 3).

™

™

®

9

Tag-ChIP

Perform ChIP Without Protein-specific Antibodies
using Tag-ChIP-IT®
Can’t find a suitable antibody for chromatin immunoprecipitation (ChIP)? Don’t limit yourChIP experiments based on antibody availability. Active Motif’s new Tag-ChIP-IT
system enables you to create a fusion protein expressing Active Motif’s uniqueAM-tag sequence that offers several advantages over FLAG, GFP and HA tags for
ChIP. Simply clone your protein of interest into the provided vector and our systemprovides everything else you need to analyze the AM-tag fusion protein by ChIP.

No ChIP-validated antibody for yourprotein of interest? No problem!
ChIP analysis is often hindered by thelack of available antibodies capable ofrecognizing fixed protein targets, orthe inability of antibodies to distinguishbetween protein isoforms. Active Motif
has developed the Tag-ChIP-IT Kit toenable ChIP without the use of a targetspecific antibody. Tag-ChIP-IT utilizes
a unique AM-tag that, unlike traditional
FLAG, GFP or HA tags, is specifically
optimized for ChIP. Furthermore, we havedesigned a high specificity antibody tothe tag to optimize pull-down efficiency
during immunoprecipitation (IP).

Tag-ChIP-IT Identifies Estrogen Receptor (ER) Binding Motifs

The AM-tag is engineered to have minimal cross-reactivity with mammaliansamples. Additionally, in contrast tolarger GFP and FLAG tags, the smaller
footprint of the AM-tag reduces obstruction of the DNA binding domain. The
AM-tag design also maximizes exposure
during the IP reaction to increase theenrichment efficiency of low abundance

transcription factors for more reliable
and consistent ChIP results (Figure 1).

Tag-ChIP-IT advantages• Ideal for targets lacking
ChIP-qualified antibodies

Input
Endogenous ER

ER Motif Identified

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. Results show 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 expression ofyour tagged protein, the Tag-ChIP-IT Kit

What’s in the box?
The Tag-ChIP-IT Kit contains all thebuffers and reagents necessary for ChIP
analysis using the AM-tag. For cloning,
the pAM_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

• Distinguishes between isoforms

pAM_1C_JunD Vector

50 μg

53044

• Improved sensitivity & specificity
over FLAG, GFP and HA tags

AM-Tag antibody

100 μg

61677

FuGENE HD Transfection Reagent

0.2 ml32042• Specifically designed for ChIP

10

Product

can be used to isolate chromatin andperform immunoprecipitations using
AM-Tag antibody specific for the AM-tag.

®

®

www.activemotif.com

EpiShear™ Sonication Products

Enhance the Reproducibility of Any Probe Sonicatorwith the EpiShear™ Cooled Sonication Platform
The most critical factor of sonication is reproducibility. The EpiShear Cooled
Sonication Platform enhances sample-to-sample reproducibility by enabling you toprecisely position the probe at the same depth in the sample every sonication. Also
included is a Tube Cooler that ensures that the sample stays cold throughout the entire
sonication, eliminating the need to move samples to an ice bucket between pulses.
Use with any programmable sonicator like our EpiShear™ Probe Sonicator. Simply set
your parameters, press START, then walk away.

Achieve better consistency whenshearing chromatin or DNA
Ensure sample-to-sample reproducibility from shearing when preparing
samples for ChIP. Once you determine
the optimal settings for sonication,
recreate them every time with the
EpiShear Cooled Sonication Platform.
How does it work?
The EpiShear Cooled Sonication
platform is designed to be used with
Active Motif’s Tube Coolers, which areavailable in sizes that fit microfuge,
15 ml and 50 ml tubes. The Tube

when it is in the Cooler. Once theappropriate adjustments have been
made to ensure proper probe height
within the tube, you can record anduse the same setting for all future
experiments to ensure consistency
between samples. Each unit is suppliedin a sound enclosure to reduce thenoise of sonication. To learn more
and for ordering information, visit
www.activemotif.com/platform.

Coolers keep the sample cold duringsonication to save time spent moving
samples to an ice bath to prevent
overheating.
The durable stainless steel andaluminum platform includes a handcrank, a height counter and a verticalalignment tool. The supplied verticalalignment tool is used to determine
the depth of the probe inside the tube

Sample 1

The EpiShear™ Cooled Sonication Platform

SampleAverage

Range (bp)1

325 bp55-10882322 bp46-9883

320 bp54-1012

Sample 2
Figure 1: Bioanalyzer data confirms reproducibility.
Three different samples of sheared chromatin were prepared from HeLa
cells using the EpiShear Probe Sonicator (1/8” probe) and the ChIP-IT®

Sample 3

Express Kit. Each sample was sonicated using the identical settings on
the Cooled Sonication Platform. Analysis performed using an Agilent
2100 Bioanalyzer shows that all 3 samples were sheared to near
identical average sizes and comparable size ranges. (The 15 bp and1500 bp peaks in the graphs above are DNA markers that correspondto the green and purple bars present in the gels.)

Product

FormatCatalog No.

EpiShear Cooled Sonication Platform, 1.5 ml

1 platform

53080

EpiShear™ Cooled Sonication Platform, 15 ml

1 platform

53081

EpiShear™ Cooled Sonication Platform, 30 ml

1 platform

53082

110V

53051

230V

53052

™

EpiShear™ Probe Sonicatorsales@activemotif.com

11

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Enabling Epigenetics Research