motif vations

THE NEWSLETTER OF ACTIVE MOTIFAPRIL 2017 | VOLUME 18 NUMBER 1

special focus on cancer epigenetics

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

ACTIVE MOTIF – The ComplicatedPicture of Epigenetics in Cancer

IN THIS ISSUE
2The Complicated Picture of Epigenetics in Cancer

4

NEW: High Quality ChIP-Seq Data fromas Few as 1,000 Cells

5

NEW: NGS DNA Library Preparation
with Molecular Identifiers Eliminates
Bias from PCR Duplicates

6

NEW: Is DNA Methylation a Biomarker
in Your FFPE Sample?

7

Obtain Whole-Genome Profiles from
Clinical Samples with ChIP IT® FFPE?

8

NEW: Reveal Histone Modification
Patterns in Clinical Samples with Active Motif’s FFPE ChIP-Seq Services

9

NEW: Epigenomic Profiling of Tumors

10 NEW: AbFlex™ Recombinant
Antibodies
12 NEW: A Genome-wide 3’UTR Collection for High-Throughput Functional
Screening of miRNA Targets
14 ChIP Normalization Reagents
Reduce Effects of Technical Variation and Reveal Subtle Biological
Changes
15 NEW: Assay-Ready RecombinantProteins, Substrates, & Inhibitors

The efforts of The Cancer Genome Atlas Project (TCGA), as well as
those of the ENCODE and other consortia, have revealed a great
deal of underappreciated biology by examining the combined genetics and epigenetics of patient tumors and cultured cells. These
efforts yielded a large collection of new SNPs and translocations
associated with misregulation of the tumor genome. Several clinical trials are underway, and the field of epigenetics has moved
from an area of research to more of an applied tool.

EPIGENETIC DRUGS & GENETICS
OF EPIGENETICS
According to records available atwww.clinicaltrials.gov, 340 studies
are listed that contain the keyword“epigenetics” (completed, active orenrolling trials). These translational
studies are diverse in the aspectof epigenetics explored and thediseases being investigated. In drug
discovery, the families of deacetylases and DNA methyltransferases
were the first to be targeted, withresulting drugs such as 5-azacytidine (DNMTi) and Vorinostat (HDACi)
approved in 2004 and 2006 respectively for myelodysplastic syndromeand cutaneous T-cell lymphoma.
While the DNMT protein family isrelatively small (including DNMT1,
3a, and 3b), the pleitropic systemic
effects of these compounds resulted in toxicity challenges. For
the HDACi compounds, specificallytargeting individual HDACs (10 family members) was not a possibility at
the time, and direct targets of theirindividual biological actions werelargely unknown.
Genetic mutations, including SNPs
and translocations, that impact epigenetic proteins are now implicated in2

a wide spectrum of cancer types. As
such, some of these proteins are now
considered therapeutic targets for
drug discovery (see table). This tablelists five therapeutic targets that havebeen studied over the last decade.
Epigenetic proteins remain challenging targets for drug discovery for multiple reasons. These proteins typicallyfunction in multiprotein complexes,
and their enzymology is just now being elucidated with crystallographic
studies. Collectively, activities of these
epigenetic modifiers contribute to the“chromatin state” of a cell. Chromatinstate can be determined by measuring certain histone modifications. It
indicates regulatory regions within our
genome, and whether a gene is active
or repressed.
Chromatin interrogation tools - a
protein toolbox
Exploration of this interesting
and complex biology has
centered around chromatin
immunoprecipitation (ChIP) using
antibodies specific to various
histone modifications. These
post-translational modifications(e.g. acetylation and methylation)
result from the activities of
epigenetic “writers” (HATs andwww.activemotif.com

ADVANCES IN CANCER EPIGENETICS RESEARCH

KMTs) and “erasers” (HDACs andKDMs). Early in the discovery
process, biochemical assays were
established using synthetic peptides
as substrates for these enzymes inin vitro enzymatic assays. However,
not all enzymes were active forthese peptides, and the enzyme
activity in vitro did not always match
reports of what was observed incells or animal experiments. In
some cases, enzymes could not
be shown to recognize peptides as
substrates, and were thought to beinactive due to missing components
in multiprotein complexes. A
need for more “native” substrates
emerged, meaning fully assembled
nucleosomes containing both
DNA and 8 polypeptides from thehistones H2A, H2B, H3, and H4.
When we consider what we now
know about the targets in the table,
challenges remain. We now appreciate the BET inhibitor target BRD4
contains protein recognition domains
for 3 distinct modifications on a native nucleosome. It requires threemodifications (H3K9ac, H3S10ph,and H4K16ac) to be engaged by its
bromodomains before it can stabilize
transcriptional elongation, and thiscombinatorial mechanism was not
obvious from peptide binding studies.
On the other hand, EZH2 and its protein partners have multiple allostericmechanisms that impose regulation on whether H3K27 is methylated, and to what extent. The drug
discovery research field requires
protein substrates that more closely
resemble what these enzymes utilize
in the cell, and often the enzymes
being studied must be co-expressedwith other proteins to be sufficiently
active. Hence, recombinant naked
and modified nucleosomes are allpart of the toolbox now. This protein
complexity is difficult to recapitulate
in a screening environment, andit remains somewhat of a barrier
to progress in drug discovery. BET
inhibitors, targeting bromodomain
containing proteins, are being widely

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applied in clinical trials. Yet we do
not understand fully the biology ofthe various members of this 8 member protein family, and in certain
cases, unexpected findings havebeen observed in clinical trials.
The future
Our toolbox will evolve to accommodate the needs of these applied researchers. Both in DNA methylation
assays, which can be diagnostic or
predictive for disease progression,as well as in trying to therapeuticallytarget the complex machines thatmake up the core of cellular epigenetic machinery. Today, in addition to thehistone modification antibodies thatapplied researchers need for assay
development, we offer full length andmodified histones, recombinant andmodified nucleosomes (see page 15).
We will continue to develop products
and services to help facilitate progress
towards epitherapeutic development.

E P I G E N E T I C C A N C E R TA R G E T S
TA R G E T

SUMMARY

P300(EP300)Lysine acetyltransferase associated with several blood andsolid tumor types. Multiple mutations including nonsense,
missense, amplification, deletion, and translocation. Promiscuous activity on histone and non histone lysines, e.g. H3K14,
H3K18, H3K23, H4K5, H4K8, H4K12, and H4K16.

EZH2(KMT6)

Lysine methyltransferase, enzymatic component of PRC2
complex, targeted in myelomas and overexpressed in a varietyof tumor types. Exhibits both loss and gain of function phenotypes. Specific for H3.1 and H3.3 K27, mono, di, and tri
methylation.

DOT1L(KMT4)

Lysine methyltransferase, DOT1L has minimal somatic mutations but interacts with MLL translocation and fusion proteins.
These fusion proteins are associated with various leukemias.
Specific for H3K79 mono, di, and tri methylation.

LSD1(KMD1A)

Lysine demethylase, overexpressed in prostate cancer. Genetic lesions in non-cancer contexts, it is targeted in MLL
rearranged leukemias. Non-competitive inhibitors that disrupt
it’s important protein-protein interactions offer a therapeutic
opportunity in certain genetic contexts. Demethylates H3K4
mono and di methylation, H3K9 mono and di methylation, andother non-histone proteins.

BRD4

Bromodomain, translocation and fusion partner of NUT in
midline carcinoma. Both BRD3 and BRD4 have fusion partners
that promote tumor growth in this disease. BRD4 dependency
has been shown in certain leukemias. Many applications for
BET inhibition are being evaluated in numerous cancer andnon-cancer disease related studies. Its recognition of acetyllysines is promiscuous depending on assay, may recognize
H3K9ac, H3S10ph, and H4K16ac simultaneously.

To learn more about our active enzymes, histone substrates, and detection
antibodies are available at www.activemotif.com/drugdiscovery

3

TOOLS FOR ChIP-SEQ

NEWHigh Quality ChIP-Seq Data from as Few as 1,000 Cells
ChIP-Seq analysis typically requires millions of cells per immunoprecipitation (IP) reactionin order to obtain meaningful information about global changes across a large population ofcells. This requirement is often not feasible for researchers working with primary cells,
FACS sorted cells, or other rare sample types. To enable studies of the complex proteinDNA interactions from limited sample material, improvements to the traditional ChIP-Seq
protocol are needed. Active Motif’s Low Cell ChIP-Seq Kit addresses this need by providing acomplete, optimized low cell ChIP-Seq workflow that enables generation of genome-widebinding profiles from as few as 1,000 cells.

Expect more from your low cell ChIP
Active Motif’s Low Cell ChIP-Seq Kit isdesigned for highly sensitive chromatin immunoprecipitation from limited
amounts of sample material. Active Motif
has utilized its extensive expertise in
ChIP-Seq to optimize chromatin preparation and immunoprecipitation procedures to lower the input requirements
for detection of histone and transcription
factor protein-DNA binding interactions.
Chromatin can be prepared from as few
as 1,000 cells or small tissue biopsies.
Figure 1: Low Cell ChIP Seq works wtih 1,000 cells. The Low Cell ChIP-Seq Kit was used to immunoprecipitate both robust and

Low Cell ChIP-Seq not only reduces
sample input requirements, but also
resolves the issues often associatedwith low-cell ChIP, including poor
signal-to-noise, inefficient library
amplification, and high duplication
rates. Low background Protein G
agarose beads and blockers are used to
Low Cell Number ChIP-Seq Kit• Reproducible ChIP-Seq datafrom as little as 1,000 cells• Works with histones andtranscription factor targets• Includes reagents to prepare ahigh complexity NGS library for
use with Illumina® platforms• Multiplex up to 16 samples on
the same sequencing flow cell4

low abundance proteins and both active and repressive histone modifications using the input cell number listed in the y-axis.
Nice peaks are observed above background from as little as 1,000 cells for histone marks H3K4me3, H3K4me1 and H3K27me3.
Lower abundance proteins BRD4 and insulator protein CTCF show nice ChIP-Seq signal with 10,000 cells. Results shown
represent a collection of individual Low Cell ChIP-Seq reactions from several cell types. Data has been reproducibly generated
across multiple experiments.

minimize non-specific binding during
the IP, while filtration columns provide
a fast, easy, and consistent solution
for wash steps. Illumina-compatible
sequencing libraries can be generated
from as little as 10 pg ChIP DNA.
Additionally, molecular identifiers(MIDs) are added with the P5 adapter
during library preparation to distinguish
PCR duplicates from fragmentation
duplicates, thereby increasing thenumber of unique alignments.

What’s in the box?
Each kit contains enough reagentsto perform 16 Low Cell ChIP-Seq
reactions. This includes reagentsfor chromatin preparation, immunoprecipitation, purification, as well as
Active Motif’s Next Gen DNA Library Kit
and Next Gen Indexing Kit (see page 5)
to make 16 unique NGS libraries foruse on Illumina platforms.
To learn more on Low Cell ChIP-Seq,
visit www.activemotif.com/chip-lowcell.

Product

FormatCatalog No.

Low Cell ChIP-Seq Kit

16 rxns

53084

www.activemotif.com

TOOLS FOR ChIP-SEQNEWNGS DNA Library Preparation with Molecular
Identifiers Eliminates Bias from PCR Duplicates
Improve the quality of your Next-generation sequencing (NGS) data sets by including
molecular identifiers (MIDs) into your DNA library preparation. Active Motif’s Next
Gen DNA Library Kit includes MIDs as part of the P5 adapter for accurate de-duplicationof PCR replicates from single read sequencing. The ability to distinguish and selectively
eliminate PCR duplicates from fragmentation duplicates improves the quality of databy increasing the number of unique alignments. The Next Gen DNA Library Kit can beused to prepare high complexity NGS libraries from double-stranded genomic DNA,ChIP DNA, FFPE DNA, or cell-free DNA (cfDNA).

High complexity NGS DNA Libraries
The Next Gen DNA Library Kit* isdesigned to generate high complexity
DNA libraries for Next-generation
sequencing for use with Illumina®
platforms (Figure 1). Libraries canbe generated from as little as 10 pg
dsDNA, or from as low as 100 ng
DNA if preparing PCR-free libraries.
The library kit is used in combination
with the Next Gen Indexing Kit, whichcontains 16 unique index adaptersthat may be used to multiplex different
samples for co-sequencing on thesame flow cell.

Molecular identifiers (MIDs)
The Next Gen DNA Library Kit offers
the advantage of including molecular
identifiers during library generation.
The MID is a 9 base random N
sequence that is added with the P5
adapter (Figure 2). Addition of the
MID is strand-specific to enableaccurate de-duplication from single
read sequencing by distinguishing
PCR duplicates from fragmentation
duplicates. This increases thenumber of unique alignments
resulting in more robust data.

Figure 1: Next Gen DNA Library Kit outperforms the

Figure 2: Schematic of a completed MID tagged andindexed DNA library molecule.

What’s in the box?
The Next Gen DNA Library Kit contains
the reagents needed to repair both 5’
and 3’ termini and sequentially attach
Illumina adapter sequences
Fragmented dsDNA
to the ends offragmented ds10 min
Repair I
DNA. The Next
+ SPRIGen Indexing
Kit contains
20 minRepair II+ PEG/NaCl
16 uniqueindices. Indices
15 minLigation I
may be used
+ PEG/NaCl
individually or
10 minLigation II
in combination+ PEG/NaCl (x2)
to multiplex
samples within Optional PCR varies+ SPRI
the samesequencing
reaction.
Library

competition. Active Motif’s Low Cell ChIP-Seq protocol
was used to prepare chromatin from GM12878 cells
using the number of cell equivalents listed. ChIP-Seq

Product

FormatCatalog No.

libraries were prepared using either the Next Gen DNA

Next Gen DNA Library Kit

16 rxns

53216

Next Gen Indexing Kit (16 indices)

64 rxns

53264

Library Kit or a competitor kit. Results show strong
peaks that match the ENCODE data sets for the Next
Gen Libraries, even if starting from low cell numbers.

sales@activemotif.com*The Next Gen DNA Library and Indexing Kits are Powered by Swift Biosciences.

5

TOOLS FOR FFPE ANALYSISNEWIs DNA Methylation a Biomarker in Your FFPE Sample?
Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource forretrospective research on clinical samples because information about treatments
and outcomes is often available. Data obtained from FFPE collections can be used to
identify biomarkers for disease research. Active Motif’s FFPE Bisulfite Conversion Kit
enables you to obtain DNA methylation profiles at single base-pair resolution fromFFPE core samples or tissue sections. Bisulfite converted DNA can be analyzed to
identify locus-specific or genome-wide DNA methylation patterns.

DNA methylation as a biomarker
FFPE samples are highly valuable for
their ability to relate clinical outcomesto disease states and epigenetic profiles.
A common biomarker observed in
the cancer epigenome is altered DNA
methylation patterns including global
DNA hypomethylation and promoterspecific hypermethylation silencing
tumor suppressor genes. Aberrant
methylation profiles correlate withmultiple developmental diseases andcancers. By analyzing DNA methylation profiles in FFPE tissues, a better
understanding of disease progression
and treatments can be obtained.
Me

Me

Me

Bisulfite Conversion

Me

GATCGAUGATCGGAGUGTAGGTAUGACGTTMeMePCR AmplificationMeGATCGATGATCGGAGTGTAGGTATGACGTT
Figure 1: Schematic of bisulfite conversion. The
bisulfite conversion reaction only modifies unmethylated
cytosine residues. Methylated cytosines are unchanged.

Benefits of bisulfite conversion
Bisulfite conversion followed by DNA
sequencing has long been considered
the “gold standard” in site-specific
DNA methylation analysis as itprovides single base-pair resolutionof DNA methylation. The conversion
reaction occurs as a 3-step deamination of cytosine residues into uracil. As
only unmethylated cytosine residues

6

Prepare sections
5-20 µm thickness

Removal of paraffinwith xylene & ethanol

Reversal of cross-links& protease digestion

On-column
DNA isolation

On-column desulfonation& purification

DNA conversion
5 hours at 50°C

RNase digestion& DNA purification

Downstream applications:
Conversion-specific PCR
DNA cloning & sequencing
Methyl-seq analysis

Figure 2: Flow chart of FFPE Bisulfite Conversion Kit.

are susceptible to bisulfite conversion,
the original methylation state of the
DNA can be determined.

MeGATCGACGATCGGAGCGTAGGTACGACGTTMeFFPE Tissue Blocks

FFPE challenges
FFPE samples are often stored forlong periods of time under less than
optimal conditions and the harshchemicals needed to preserve tissue
structure and prevent putrefaction
often lead to highly fragmented anddegraded DNA, making it challenging
to obtain usable data. Active Motif’s
FFPE Bisulfite Conversion Kit hasaddressed this challenge by providing
optimized reagents throughout theworkflow to improve the recovery ofhigh quality DNA, minimize degradation,and provide greater than 99% bisulfite
conversion efficiency (Figure 2).

What’s in the box?
Active Motif’s FFPE Bisulfite Conversion
Kit contains enough reagents toperform 40 DNA isolations from up to
four 20 µm FFPE tissue sections or
35 mg of unsectioned, core samples
per reaction. Bisulfite conversion
reagents are included for 40 samples,with input DNA requirements ranging
from 5 pg - 2 µg per reaction. A positive control conversion-specific PCRprimer pair that is specific for bisulfite-converted human or mouse DNA
is also included in the kit. Because
the primer pair only produces a PCR
product if conversion has occurred,
you can confirm the procedure worked
before starting downstream sequencing(Figure 3). To learn more, visit www.
activemotif.com/bis-conv-ffpe.

Marker5 pg

500 ng5 pg

100 pg

5 pg

50 pg

200 bp –Human kidney
FFPE sample

Human stomach
FFPE sample

Mouse kidney
FFPE sample

Figure 3: Bisulfite conversion from as low as 5 pg DNA.
Active Motif’s FFPE Bisulfite Conversion Kit was used toisolate genomic DNA from human kidney, human stomach, and mouse kidney FFPE tissue sections. Bisulfite
conversion was performed using the input quantities
listed. Results show a 220 bp conversion-specific PCRamplicon that is present only in bisulfite converted DNA.

Product

FormatCatalog No.

FFPE Bisulfite Conversion Kit

40 rxns55021

www.activemotif.com

TOOLS FOR FFPE ANALYSIS

Obtain Whole-Genome Profiles from ClinicalSamples with ChIP-IT FFPE
Active Motif has utilized its years of experience and expertise to develop the first
chromatin immunoprecipitation (ChIP) kit designed specifically for use with formalinfixed, paraffin-embedded (FFPE) samples. The ChIP-IT® FFPE Chromatin Preparation
Kit can extract high-quality chromatin from FFPE slides or tissue blocks, while theChIP-IT® FFPE kit is sensitive enough to enrich histone and transcription factor targets with minimal background signal. To learn more about ChIP-IT FFPE, please visit
us at www.activemotif.com/ffpechip.

Active Motif’s ChIP-IT FFPE Kit is
the first ChIP Kit available that can
extract high-quality chromatin fromextremely limited starting material,
while producing minimal backgroundsignal in ChIP, thereby enabling specific detection of the target protein ofinterest. The assay is sensitive enoughto detect histone and transcription
factor targets using qPCR or Nextgeneration sequencing (NGS).

What’s in the box?
ChIP-IT FFPE Chromatin Preparation
Kit provides specially formulated
reagents and protocol guidelines toextract chromatin from histologicalslides or tissue sections. While thequality of the extracted chromatin isdependent upon sample fixation andstorage conditions, we have successfully extracted ChIP-grade chromatinfrom normal and tumor human colon
FFPE blocks that were stored for morethan 10 years under less than ideal
conditions (Figure 1).

1200
1000

logical sections of a human colon tumor and matchednormal colon for use in H3K4me3 ChIP-Seq. A subset of

ChIP-IT FFPE advantages

the genome-wide profile shows nearly equal H3K4me3• Use with slides or tissue blocks

H3K4me3 is present at the promoter of the PTK7 gene,• Sensitive assay detects histone
and transcription factor targets

the tumor sample.

• Positive controls are included tovalidate results at each step

Product• Compatible with downstream
NGS and qPCR analysis

occupancy at SRF and CUL9 promoters. However,a gene known to be up-regulated in colon cancer, only in

ChIP using YY1 Antibody
Normal
Tumor

800
600
400
200
0

Figure 1: ChIP-Seq on normal and tumor FFPE samples. Chromatin was extracted from 10-year-old histo-

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ChIP-IT FFPE includes our highly
sensitive immunoprecipitation andDNA purification reagents, blockers, protease inhibitors and protein G
agarose beads. Positive controls, suchas a Histone H3K4me3 ChIP-Seqvalidated antibody and human PCRprimer sets, are included to confirm
success at each step of the process.
For downstream applications such as
NGS, the ChIP-IT qPCR Analysis Kit isrecommended to evaluate the qualityof the ChIP-enriched DNA prior tosequencing.

Binding events detected / 1000 cells

First of its kind assay
FFPE tissue serves as the “goldstandard” for the preservation ofpathology samples, and represents anopportunity to study clinical outcomesof disease and treatment conditions.
Traditionally, FFPE samples have notbeen useful in chromatin immunoprecipitation due to the limited size of thesamples and the fact that the formalin
fixation process often causes degradation and loss of antigenicity.

Human Neg Primer Set 1

Human Pos Primer Set ATG4B

Figure 2: qPCR analysis of normal and tumor colon
samples assayed using ChIP-IT FFPE. Chromatin was
extracted from five 20 µm sections of matched normal
and tumor human colon FFPE blocks and assayed with
the ChIP-IT FFPE kit using an antibody for transcription
factor YY1. The quality of the ChIP DNA was validatedwith the ChIP-IT qPCR Analysis Kit. qPCR results show
the sensitivity and specificity of ChIP-IT FFPE.

FormatCatalog No.

ChIP-IT® FFPE Chromatin Preparation Kit

5 rxns

53030

ChIP-IT® FFPE

16 rxns

53045

ChIP-IT® qPCR Analysis Kit

60 rxns

53029

7

TOOLS FOR FFPE ANALYSIS

NEW

Reveal Histone Modification Patterns in ClinicalSamples with Active Motif’s FFPE ChIP-Seq Service
Formalin-fixed, paraffin-embedded (FFPE) tissue blocks and histology slides are avaluable resource for retrospective research on clinical samples. In the field of epigenetics, the benefits of this archived material have not been fully realized becauseof the difficulty these sample types present for use in conventional epigenetic techniques such as chromatin immunoprecipitation (ChIP). Challenges arise due to thelimited size of FFPE materials and the harsh conditions under which these samples
have been prepared. Active Motif’s new FFPE ChIP-Seq Service gives you access tobreakthrough technology from our Epigenetic Services lab that now enables the generation of genome-wide histone modification profiles from FFPE samples.
Accessing epigenetic informationfrom archived FFPE samples
The ability to study FFPE samples
provides researchers with an opportunity
to link FFPE data to disease, diagnosis, and biomarker discovery. Thereis increasing interest in utilizingthese clinical samples for studies in
research areas, such as epigenetics
where aberrant histone and DNA
modifications have been linked tohuman pathologies, including neurological, immune, and neoplastic
disorders. ChIP is widely used in
epigenetics research to determine global
histone modification patterns. In somediseases, such as cancer, enzymes that
regulate histone modification deposition
and removal have been shown to be
mutated. Research into cancer therapies
aimed at restoring normal histonelandscapes through small molecule
inhibition are now underway and raise
the possibility that histone modification
occupancy profiles can inform treatments or predict patient outcome.

Active Motif’s new FFPE ChIP-Seq
Service performs the work for you!
A major challenge arises whenattempting to extract epigenetic information from clinical samples. FFPE
tissue is typically limited in size, lacks
consistency in preparation, and often
displays degradation and loss of antigenicity due to harsh fixation conditions
or prolonged storage, presenting
an obstacle for performing sensitive
epigenetic techniques such as ChIP.
These factors can increase the difficultyof chromatin preparation and often

require for optimization. Active Motif’s
new FFPE ChIP-Seq Service takes
the burden of optimization out of yourhands and makes it possible to profile
histone modification binding
patterns or perform biomarker
identification studies in FFPE patient
samples with ease. Simply send us
your FFPE blocks or sections and wewill do the rest!
To learn more about our end-to-end
FFPE ChIP-Seq Service, visit us awww.activemotif.com/services-ffpechip.

Sample types:• FFPE blocks, sections, or curls
Targets:• H3K4me1, H3K4me2, H3K4me3• H3K9ac, H3K14ac• H3K27ac, H3K27me3• H3K36me3• CTCF, and more...

8

Figure 1: ChIP-seq from an FFPE preserved colon tumor. 10µm sections were taken from a FFPE preserved colon tumor
followed by chromatin extraction and ChIP-Seq. Each 10µm section yielded enough chromatin for a single histone PTM ChIPSeq experiment. The seven histone modification ChIP-Seq data sets presented above show the expected genomic localization
patterns.

www.activemotif.com

TOOLS FOR FFPE ANALYSIS

NEW

Epigenomic Profiling of Tumors
Epigenetic landscapes, like other molecular profiles such as gene expression, can be used to
stratify patients into different therapeutic groups. As such, epigenomic biomarker development
will be important to identify patients that will respond favorably to a given treatment. In recent
years, researchers have begun to explore epigenetic modifications as biomarkers, an approachthat has been met with success as related to DNA methylation. Histone modifications, on theother hand, have not been widely used or widely accepted as biomarkers, in part due to thechallenges associated with the methods used to reliably measure histone modification patterns
in patient samples. Advances in ChIP and ChIP-Seq at Active Motif now enable tumor profilingand biomarker discovery from tumor biopsies.

Epigenetic biomarkers in
medulloblastoma
Medulloblastoma is a highly
malignant pediatric brain tumor
that accounts for 20% of all childhood brain tumors. These tumors
are classified into four biologicallyand clinically distinct subgroups.
Treatment choice is based on themolecular subgroup and treatment
options include: surgery, radiation, and chemotherapy. Molecularsubgroups can be identified using
genome-wide profiles of H3K27ac
as illustrated in a recent publication in Cell Reports: Lin et al. Active
medulloblastoma enhancers reveal
subgroup-specific cellular origins(2016) (Figure 1).
Fresh frozen biopsies are well suited
for ChIP-Seq since molecular interactions are preserved during freezing
and are maintained during storage.
Frozen samples can immediately flow
into the preparative step of formaldehyde fixation needed for ChIP. However, the vast majority of tumor samples
are stored as FFPE blocks, not frozen

samples. FFPE samples are morechallenging due to extensive crosslinking which hampers chromatin extraction. Recent advances at Active Motif
now enable the routine generation ofhigh quality ChIP-Seq data sets fromlimited amounts of FFPE preserved
tumors (Figure 2).
Active Motif provides end-to-end FFPE
ChIP-Seq services as well as tumor profiling on frozen biopsies. To learn more
about our custom services, or to get aquote, please visit us at www.activemotif.
com/epigenetic-services-info.

Figure 1: H3K27ac profiles characterize medulloblastoma subgroups. ChIP-Seq was performed at
Active Motif using frozen biopsies from each of the four

Figure 2: H3K27ac ChIP-Seq comparison between fresh/frozen and FFPE preserved tumors. ChIP was

medulloblastoma subgroups: WNT (n=3), SHH (n=5),

performed using chromatin from two human medulloblastoma biopsies. Biopsies were provided as both fresh/

Group 3 (n=9) and Group 4 (n=11). Profiles from all

frozen (green) and FFPE (blue). ChIP-Seq data sets were similar with correlation coefficients of 0.889 and 0.826

samples within a group were overlayed for visualization.

between FFPE and fresh/frozen samples. Tumor-specific H3K27ac occupancy was detectable in the FFPE and

Subgroup-specific H3K27ac patterns were identified.

fresh/frozen data sets (highlighted in red).

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9

RECOMBINANT ANTIBODIESNEW

AbFlex™ Recombinant Antibodies
Batch to batch variability and poor characterization of antibodies is a concern within the
research community and has led to calls for a better approach to antibody development. To
address these concerns Active Motif has developed AbFlex™ recombinant antibodies using
defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex™
antibody contains tag sequences allowing attachment of a variety of labels directly to theantibody.

The problem with antibody variability
A 2008 study found fewer than halfof 6,000 routinely used commercial
antibodies recognized their specifictargets exclusively (Berglund et. al.,2008). In 2011, looking explicitly athistone modification state specificantibodies, another group found thatamong 246 antibodies, one-quarter
failed tests for specificity (Egelhofer,
et. al. 2011). Additionally, lot-to-lot
variability in the commercial products
often plague scientists who want toreproduce results from within theirown lab or others. These and otherconcerns were summarized in two
Nature comments articles written by
two researchers with over 100 cosignatories. These articles expressedthat new standards were needed tocircumvent several issues involved ingenerating these reagents in animals(Bradbury, et.al. Nature 2015). These
new standards included a call for all
antibodies to be derived from definedsequences, and for those sequences
to be available to the researchers.

flexible conjugation options. AbFlex™
recombinant antibodies are derived
from high value parent antibodies(monoclonals or recombinants froma variety of species), and they havebeen engineered to contain a tripartite “tag” of functional molecularfeatures (Figures 1 & 2).

Figure 2: SDS-PAGE analysis of AbFlex™ antibodies
compared to a standard monoclonal. AbFlex™ antibodies
were heavy and light chains are indicated for the various
antibodies.

Figure 1: Schematic diagram of an AbFlex recombi™

Introducing AbFlex™ recombinant
antibodies
In response to these requests, Active
Motif has created a new type of antibody tool that will enable reproducible results across antibody batches.
Active Motif’s AbFlex™ recombinant
antibodies, address the need forrecombinant antibodies and includes
value-added features for easy and

The tripartite tag specifies the following features on the carboxyl terminus of the heavy chain molecules:• Avidin Tag for site directed,
efficient biotinylation• Sortase Tag, a LPETG motif
for site directed conjugation
via an optimized transpeptidase• 6X-His Tag for standard affinity purification by nickel-based
chromatographynant antibody. Heavy and light chains are shown withthe cloned variable regions indicated in copper. Antibodies are expressed and purified with the molecular tagson the carboxyl terminus of the heavy chain as shown.

Molecularly cloned variable regions
are combined with various heavy
and light chain frameworks (e.g.
murine IgG2a) to reconstitute thebinding capabilities.

Designed for flexibility
These engineered, AbFlex™ recombinant antibodies, are expressedfrom mammalian cells to ensure
lot-to-lot uniformity, performance,and standardized presentation.
When combined with the Sortase labeling* system, the molecular tags
are compatible with the following
tri-glycine dependent conjugation
options:• Biotin*US 2014/0057317 A1

10

www.activemotif.com

RECOMBINANT ANTIBODIES

•

•
•
•

TEV Biotin (for protease digestion to release from biotinstreptavidin matrices)
Horseradish Peroxidase (HRP)
Alkaline Phosphatase (AP)ATTO488, 535, 550, 647N

Quality is key
To ensure quality of AbFlex™ antibodies, each conversion is re-validated in applications that the parent
antibody was validated in (Figures3-5). Examples of applications
testing (depending on the parent
antibody’s product claims) include:• Western blot
• ChIP or ChIP-Seq• Plate or beadbased ELISA• Immunofluorescence (with orwithout secondary
amplification)

include two options for enzymatic addition of biotin or TEV Biotin in molar
ratios of 1 label to 1 heavy chain fora maximum possible labeling of 2 labels per conjugation. Biotinylation issite directed and near the C-terminus,
thus enabling consistent orientation in
streptavidin bead or plate applications
so that the antigen recognition site
is uniformly presented on surfaces.
This advantage, as well as the ability
to prevent over-labeling, eliminates
the need to re-validate an antibody’s
performance post labeling. This isparticularly advantageous, as chemical labeling can often interfere with agiven antibody’s ability to recognize its
antigen specifically post labeling. The
enzymatic ligation is performed under

mild conditions, and there is no riskof labeling conjugates on the antigen
recognition portion of the antibody.
The only restriction on coupling via
Sortase requires the antibody to havethe LPXTG is the antibody recognition
sequence, and the molecule of interest requires a poly-glycine for conjugation purposes. AbFlex™ custom
services is also available for customers interested in converting their own
monoclonals into the AbFlex™ framework.
To learn more about our custom
services, or to get a quote contact
us at 877-222-9543 or visit us atwww.activemotif.com/services.

Figure 3: AbFlex™ Histone H3K9ac
recombinant antibody tested by Western blot. HeLa
nuclear extract (20 µg per lane) probed with AbFlex™
Histone H3K9ac antibody (2 µg/ml dilution).Lane 1: untreated cells. Lane 2: cells treated with HDAC inhibitor
sodium butyrate.

Figure 5: AbFlex™ Histone H3K9ac recombinant antibody tested by ChIP-Seq. ChIP was performed using
the ChIP-IT® High Sensitivity Kit (Cat. No. 53040) with 30 µg of chromatin from PC9 cells with 4 µg ofantibody. ChIP DNA was sequenced on the Illumina® HiSeq and 22 million sequence tags were mapped to
identify histone H3K9ac binding sites. The image shows binding across a region of chromosome 1.

AbFlex™ Recombinant Antibodies••
Figure 4: AbFlex™ Histone H3K9ac recombinant antibody tested by immunofluorescence. HeLa cell stained•

Recombinant full length antibodies with heavy and light chains (e.g.
mouse IgG2a)Three functional tags for purification, biotinylation, and protein ligation(6X-His Tag, AvidinTag, and Sortase Tag)
Consistent, site directed-labelingwith 2 µg/mL of AbFlex™ Histone H3K9ac antibody
followed by anti-mouse-IgG-488.

Functionality of the molecular features—The AbFlex™ advantage
New AbFlex™ antibodies will be added
routinely, and currently include antibodies to histone modifications, tags,
transcription factors, and epigenetic
readers, writers, and erasers. The
advantages offered by the tag features

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Product

FormatCatalog No.

AbFlex Histone H3K9ac antibody (rAb)

100 µg

91103

AbFlex Histone H4K20me3 antibody (rAb)

100 µg

91107

AbFlex Cas9 antibody (rAb)

100 µg

91123

AbFlex His Tag antibody (rAb)

100 µg

91183

AbFlex JMJD2A Antibody (rAb)

100 µg

91143

AbFlex EED antibody (rAb)

100 µg

91135

™
™
™
™
™
™

11

miRNA TARGET IDENTIFICATIONNEWA Genome-wide 3’UTR Collection for High-Throughput
Functional Screening of miRNA Targets
Active Motif’s LightSwitch™ Luciferase Assay System includes a genome-wide collectionof 12,000 3’UTR reporters to enable researchers to better understand microRNA(miRNA) and 3’UTR interactions. In a recent study, the laboratory of Dr. Settlemanperformed a functional screen of 879 human miRNAs which identified miR-371-3p as
a potent suppressor of drug tolerance. Using the LightSwitch system, they identified
PRDX6 (peroxiredoxin 6) as a key target of miR-371-3p in establishing drug tolerance,suggesting that co-targeting of PRDX6 or modulating miR-371-3p expression together
with targeted cancer therapies may delay or prevent acquired drug resistance.

luciferase
promoter

3’UTR

3’UTR
Reporter Vector

miR-371-3p mimicco-transfector
non-targeting control

RISC
complex

HybridLUC-3UTR
Transcript

Figure 1: Experimental design for miR-122 functional
screen using the LightSwitch System.

122

Log2 ratio (targeting/non-targeting miRNA)

0

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

-2

-4

-6

PIGK
PRDX6
PLCB4
ATP6V1E1
GDE1
PLD6
HMOX1
STX12
TOMM20
SLC37A2
ZNF213
GLT25D1
ZNF576
FAM73BWNT7B
REM2GABBR2SRXN1FGFR1CXorf23
WDR33
miR-371-3p_3’UTR
Empty_3’UTR
GAPDH_3’UTR
R04_3’UTR

LightSwitch™ 3’UTR Reporter
Collection and Assay System
MicroRNAs are important regulatorsof gene expression involved in bothnormal and pathological processes.
Accumulating evidence suggests arole for miRNAs in epigeneticallyregulating various phenotypicstates in cancer cells. miRNAs canimpact genetic programs throughpost-transcriptional silencing oftarget genes either by promoting
degradation of target messenger
RNAs (mRNAs) or by inhibiting theirtranslation. miRNAs have been
implicated in the regulation of various
aspects of cancer biology, including

Figure 2: Identification of relevant miR-371-3p targets using the LightSwitch luciferase reporter assay and 3’UTR target
reporters. Downregulation of RenSP luciferase by miR-371-3p upon erotinib treatment from 3’UTR LightSwitch reporters
corresponding to candidate miR-371-3p target genes *p<0.05.

drug resistance, cancer cell stemness,
epithelial-to-mesenchymal transition,and metastasis (Sahu et al., 2016).
To enable researchers to leverage 3’
untranslated region (3’UTR) reporter
assays in their cancer research ona high-throughput scale, we havecreated a genome-wide collectionof 12,000 human 3’UTR luciferase
reporters in the highlyoptimized
LightSwitch Luciferase Assay System.
The Active Motif LightSwitch assay
is ideal for performing miRNAtarget validation and assessing thefunctional impact of miRNA-3’UTR
interactions. Combined with ourlarge collection of miRNA mimics andinhibitors, you have everything needed

to study miRNA-3’UTR interactions,validate miRNA targets, and to measure
RNA stability and the functional
impact of miRNAs on a gene-by-genebasis (Figure 1).
To learn more, please visit
www.activemotif.com/ls-3utr.
*Functional screening implicatesmiR-371-3p and PRDX6 in reversible
tolerance to cancer drugs
In a recent study, Dr. Nisebita Sahu
explores the potential role for miRNAs in transient drug tolerance.
Acquired resistance to cancer drug
therapies almost always occurs inadvanced-stage patients, even following

www.activemotif.com

miRNA TARGET IDENTIFICATION

1 kb

0.5 kbRLU (%)

150

100

PRDX6 3`UTR WT

PLCB4 3`UTR WTPRDX6 3`UTR mutant

PLCB4 3`UTR mutant

Erlotinib + siNTC
Erlotinib + miR-371-3p

Media + siNTCMedia + miR-371-3p

*

*

NS

*

*

*

50
0

1.5 kb

0.5 kb2 kb

1 kb

TTTTGGCACTAACATTTTTGTAACTAACAT

150
*

*

100

NS

TTTGGGCACTATATT
TTTGGTAACTATATT

Erlotinib + siNTC
Erlotinib + miR-371-3p

Media + siNTCMedia + miR-371-3p

*

*

2 kb
3`UTR

*

0

STX12 3`UTR WT
STX12 3`UTR mutant

Media + siNTCMedia + miR-371-3p

150

*

50

1.5 kbmiR-371 predicted site

miR-371 predicted site

RLU (%)TTTTGGCACTAACATTTTTGTAACTAACAT

1 kb

3`UTR

3`UTR
miR-371 predicted site

RLU (%)

0.5 kb

*

NS NS

100

Erlotinib + siNTC
Erlotinib + miR-371-3p

NS NS

*

*

50
0

Figure 3: Mutational validation of relevant miR-371-3p targets using the LightSwitch system. Mutations of seed sequences of the putative miR-371-3p recognition element in target 3’UTRs prevented
inhibition by miR-371-3.

a significant response to treatment. In
addition to mutational mechanisms,
various non-mutational resistancemechanisms have now been recognized.
To investigate whether miRNAs are
required to engage or maintain the
drug-tolerant state, Sahu et al. functionally screened 879 human miRNAs
and identified miR-371-3p as a potent
suppressor of drug tolerance.
Identification of miR-371-3p targets
Next, candidate gene targets ofmiR-371-3p were identified using theTargetScan prediction algorithm. The
KDM5A and IGF1R genes, which encode
proteins previously found to regulate
drug-tolerant persisters (DTPs), did
not display any (3’UTRs) that could be
potentially targeted by miR-371. Direct
regulation by miR-371-3p of the top 70
predicted targets was assessed using
the Active Motif LightSwitch Luciferase Reporter Assay System following
Erlotinib treatment in the presenceof miR-371-3p. In addition, KDM3B,
which displayed partial sequencehomology in the 3’UTR with miR-371,
was explored using the 3’UTR luciferase reporter assay. However, miR-371
was unable to target the 3’UTR of
KDM3B, suggesting that miR-371 does
not regulate DTPs via KDM3B. Significantly, 21 genes displayed reduced

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luciferase activity upon Erlotinib treatment, implicating these genes as bonafide targets of miR-371-3p (Figure 2).
To further validate the selective
downregulation of specific genetargets by miR-371-3p, the single
putative miR-371-3p recognitionsite within PRDX6, PLCb4, and STX12
3’UTR sequences was mutated, which
abolished the ability of miR-371-3p toinhibit luciferase reporter expression.
These results confirm miR-371-3p as
a direct regulator of these genes andsuggest that a single recognition element
is sufficient for their regulation by
miR-371-3p (Figure 3).

Sahu et al. to reveal the miR-371-3p
target gene PRDX6 as a key regulatorof the reversible drug tolerance thatfrequently emerges within heterogeneous cancer cell populations. In
conclusion, the findings presented
by the Settleman laboratory in thisNature Communications report reveal
a major regulatory role for miRNAs
in the emergence of reversible drug
tolerance.
*This article is a summary of the research performed by Dr.
Settleman at the Discovery Oncology department at Genentech,
Inc,. published in Nature Communications: Sahu, N. et al.
Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs. Nat. Commun.

The LightSwitch Assay System infunctional validation of miRNA targets
Taken together, The Active Motif
LightSwitch™ miRNA Target Validation
and Custom Mutagenesis Services
were key components in enabling
Product

7:12351. DOI: 10.1038/ncomms12351 (2016).

To learn more about LightSwitch
products and services, visit us atwww.activemotif.com/lightswitch.

FormatCatalog No.

LightSwitch 3’UTR GoClone

5 µg

32011

LightSwitch Custom Cloning

5 µg

32051

LightSwitch Custom Mutagenesis

5 µg

32052

™
™
™

LightSwitch miRNA Mimics & Inhibitors™

LightSwitch™ Luciferase Assay Kit
LightSwitch Synthetic miRNA Target™

LightSwitch miRNA Target Validation Service™

5 nmol29000

100 assays

32031

5 µg

32003

Custom32055

13

ChIP NORMALIZATIONChIP Normalization Reagents Reduce Effects ofTechnical Variation and Reveal Biological Changes
ChIP is a multi-step process in which variations caused by sample loss during immunoprecipitation and library preparation, uneven sequencing read depths, or hand-to-handdifferences make it difficult to compare data sets. 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 and sample processing bias. Additionally, the normalization strategy can be used for detecting conditional
effects, such as those induced by compound treatments or mutants.

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 biological changes in yoursamples (Figure 1). ChIP normalization
can easily be implemented simply byintegration of our Spike-in reagents into
your standard ChIP protocol.
A standard ChIP reaction is set up using
experimental chromatin (e.g. human)
and an antibody of interest. In addi-

Figure 1: ChIP-Seq Spike-in Normalization Strategy reveals changes in H3K27me3 levels following treatment with an EZH2
inhibitor. 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 ofExperimental
Chromatin

Spike-in
Chromatin
Spike-in
Antibody

Antibody ofinterest

Chromatin Immunoprecipitation

Sequencing

Map to experimental
genomeNormalize sample
tag counts by same ratio

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

Figure 2: ChIP-Seq Normalization Workflow.

14

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

tion, Drosophila melanogaster Spike-in
Chromatin is added, or “spiked-in”, toeach 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
Spike-in Antibody provides a mechanism
to reliably pull down a small fraction of
Drosophila chromatin that is consistentacross all samples (Figure 2).

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 tothe sample genome.
To learn more about ChIP normalization, visit www.activemotif.com/spikein

Product

FormatCatalog No.

Spike-in Chromatin

15 rxns

53083

Spike-in Antibody

50 µg

61686

Drosophila Positive Control Primer Set Pbgs

96 rxns

71037

Drosophila Negative Control Primer Set 1

96 rxns71028

Drosophila Negative Control Primer Set 3

96 rxns

71038

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ASSAY SUBSTRATESNEWAssay-Ready Recombinant Proteins, Substrates, andInhibitors
There is no need to waste valuable time and resources generating proteins and substrates for screening, drug discovery, and target validation programs. Active Motif
continues to add to its collection of over 300 purified, assay-ready recombinant proteinsand physiologically-relevant assay substrates for use in enzyme activity assays, inhibitor screens, and binding studies. We also offer a collection of activator and inhibitor
compounds to modulate the activity of proteins that regulate DNA methylation, histones, and binding proteins.

Assay-Ready Proteins

Assay-Ready Substrates

Epigenetic Activators & Inhibitors• Histone modifying enzymes
HATs, HDACs, HMTs, & HDMs

• Lysine Demethylase &Methyltransferase• Binding domains
Bromodomains, & SET domains• Arginine Methyltransferase• Histone Acetyltransferase &Deacetylase• DNA modifying enzymes
DNMTs & TET• Sirtuins• Transcription factors
NFkB, AP-1, p53, and more...

• DNA Methylation• Bromodomain• Bulk sizes available
Product

FormatCatalog No.

NEW: Recombinant Histone H2A.X

100 µg

31251

Recombinant Histone H3K4me3

50 µg

31210

Recombinant Histone H3K9ac

25 µg

31253

Recombinant Histone Octamer (H3.1)

100 µg31470

Recombinant Histone Octamer (H3.1) - biotinylated

50 µg

31471

Recombinant Mononucleosomes (H3.1) - biotinylated

20 µg

31467

31468) or 2 µg Histone Octamer (H3.3) (Catalog No.

Recombinant Mononucleosomes (H3.3) - biotinylated

20 µg

31469

31472) at the concentrations shown above in reaction

Recombinant Nucleosomes (H3.1)

20 µg

31466

Recombinant Nucleosomes (H3.3)

20 µg

31468

NEW: Recombinant AGO1 protein

20 µg

31522

NEW: Recombinant HDAC3 / NCOR2 Complex

20 µg

31526

Figure 1: Recombinant NSD2 (E1099K) activity assay.
Recombinant NSD2 (E1099K)was incubated with either
2 µg Recombinant Nucleosomes (H3.3) (Catalog No.

buffer for 3 hours at room temperature. Activity was detected by Western blot for H3K36me2. Results show thesubstrate preference of NSD2 (E1099K) for nucleosomes
over octamers.

A few of the available proteins, substrates, and epigenetic modulators
are shown in the table to the right.

NEW: Recombinant NSD2 (E1099K) protein

20 µg

31546

NEW: Recombinant SIRT1 (193-741) protein

100 µg31533

NEW: SGC707 (PRMT3 inhibitor)

5 mg

14131

To learn more, visit us atwww.activemotif.com/proteins andwww.activemotif.com/smallmol

BML-278 (SIRT1 activator)

5 mg

14025

5´-Azacytidine (DNMT inhibitor)

50 mg

14103

sales@activemotif.com

15

NORTH AMERICAJAPAN

EUROPE

CHINA

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Direct: 760 431 1263
Fax: 760 431 1351
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Direct: +86 21 2092 6090
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Customer Service:orders@activemotif.com

Enabling Epigenetics Research