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THE NEWSLETTER OF ACTIVE MOTIFNEUROEPIGENETICS EDITIONOCTOBER 2016 | VOLUME 17 NUMBER 2

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

ACTIVE MOTIF – Advancing
Neuroepigenetics Research

IN THIS ISSUE2The Role of Epigenetic Modulation in
Brain Disorders

6

NEW: High Quality ChIP-Seq Data fromas Few as 5,000 Cells

7

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

8

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

9

NEW: Easily Isolate Intact Histones
for PTM Analysis

10 NEW: RRBS Service for Genome-scale
DNA Methylation Profiling
11 NEW: Is DNA Methylation a Biomarker
in Your FFPE Sample?
12 NEW: Simple High-throughput Screen
to Evaluate Changes in Global DNA
Methylation
13 NEW: Sortase 2.0 – A Versatile
Platform for Rapid, Controlled,
Site-specific Protein Modification
14 A Genome-wide 3’UTR Collection for
High-throughput Functional Screeningof miRNA Targets

Epigenetic modifications including histone modifications, DNA
methylation, and non-coding RNAs influence chromatin structure
and, ultimately, gene regulation. Epigenetic modifications are heritablebut, unlike genetic modifications, are dynamic and responsive toexternal stimuli such as stress, toxins and learning. Neuroepigenetics is a growing area of research focused on the study of
epigenetics in various neural processes, including development
and plasticity, behavior, stress, aging, and brain disorders. Below
are two publication summaries illustrating how Active Motif, theleading provider of innovative epigenetic research tools, is aiding
scientists every day in advancing their neuroepigenetics research.
THE ROLE OF EPIGENETIC
MODULATION IN BRAIN DISORDERS
There is increasing evidence thatseveral neurodevelopmental,
neurodegenerative and psychiatricdisorders are, in part, caused by aberrant
epigenetic modifications. As a result,researchers in neurogenetics are now
beginning to investigate the mechanisms underlying epigenetic regulationin neuronal gene expression and how
this impacts normal brain function.
Recently, Active Motif collaborated in
two separate published studies where
loss-of-function mutations of chromatin
modifiers Wiz and Kdm5C were used as
mouse models to study how disrupting
the function of epigenetic modifiers
contributes to brain disorders. These
recent publications in eLife by thelaboratory of Dr. Emma Whitelaw at
La Trobe University in Melbourne,
Australia and in Cell Reports by thelaboratory of Dr. Yang Shi at Harvard
Medical School, respectively, aresummarized below.
Identification of a role for the epigenetic
modifier Wiz in neural function
This following article is a summary of the research published in
eLIfe.: Isbel et al., 2016;5:e15082. DOI: 10.7554/eLife.15082.

To identify novel modifiers of epigenetic
reprogramming, the laboratory of

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Dr. Emma Whitelaw performed amutagenesis mouse screen in which they
identified the gene Widely interspaced
zinc finger motifs, or Wiz, as a modulatorof gene expression. Although little is
known about Wiz, it is highly expressedin both the embryonic and adult brain
and is known to mediate protein-protein
and protein-DNA interactions of theG9a/GLP histone methyltransferase
complex which catalyzes methylationof H3K9me1 and H3K9me2, marks thatare associated with transcriptional
repression. G9a is also associated withtranscriptional activation independentof its histone methyltransferase activity,and it has been suggested that Wiz
may also be involved in G9a-mediatedtranscriptional activation.
Reduced levels of Wiz are associatedwith gene silencing
In the mutagenesis screen, haploinsufficiency for Wiz (WizMD30/+) resulted
in silencing of both the targeted GFP
transgene reporter under the controlof erythroid promoter/enhancer
sequences as well as an independent
locus, the Agouti viable yellow (Avy)allele, known to be sensitive to levelsof epigenetic modifiers, lending further
evidence that Wiz is involved in regulationof G9a-mediated transcriptional

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ADVANCES IN NEUROEPIGENETICS RESEARCH

activation. Furthermore, a reduction inlevels of G9a/GLP are associated with
Kleefstra syndrome, a neurologicaldisorder that manifests in a range ofsymptoms including anxiety. Becauseof the high levels of Wiz in the brain,
the researchers postulated that
reduced levels of Wiz might also haveneurological effects. Utilizing a suite ofmolecular and bioinformatics tools, theresearchers demonstrate that Wiz
binds active promoters and they
provide evidence that suggests Wiz
functions as a transcriptional activatorin neural tissue. Furthermore, theyshow that Wiz mutant mice present withbehavioral abnormalities, revealing ananxiety-like phenotype similar to what
is observed with reduced G9a/GLP levels.
Wiz binds promoters and shows highconsensus overlap with CTCF
In order to determine where Wiz wasbound within the genome with respect
to active and inactive genes, cerebellumsof adult Wiz+/+ (WT, wild-type) mice werecollected and submitted to Active Motiffor chromatin immunoprecipitation
followed by sequencing (ChIP-Seq)
services. ChIP-Seq analysis confirmedthat Wiz binds across the genome atpromoters and showed strong enrichA

B

Figure 1: Wiz binds across the genome and at promoter
elements. (A) Pie graph depicting the percentage of Wiz
peaks that overlap with the promoter (up to 2 kb from aTSS) and genic sequence of active or silenced genes,
according to RNA-Seq data mapped to Ensembl genes
annotations. (B) Wiz ChIP-Seq occupancy over all
Ensembl gene bodies is shown as deep sequencing readdensity along the transcription unit, including 2 kb up anddownstream of the transcriptional start and stop site.

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Figure 2: Wiz binding consensus shows a high degreeof overlap with CTCF. (A) The six most enriched motifs
in Wiz ChIP-Seq peaks (n = 39,781) and their distribution
inside peaks are shown. (B) The most significant of thesematches the CTCF-binding site consensus sequence(JASPAR CORE database - MA0139.1). (C) Read density

causes reduced expression of anumber of genes genome-wide.
However, differential expression isobserved mainly within gene clusters,in particular within the protocadherinb (mainly expressed in neurons) andcadherin 11-like clusters, and adjacent
to telomeres, showing significant
overlap with embryonic and adultexpression patterns (Figure 3A).
Telomeres are well-known heterochromatic regions, and protocadherin
clusters exist as large tandem arrays
important for gene silencing during
heterochromatization, suggesting a rolefor Wiz in chromosome looping. Analysis
of ChIP-Seq read density at the TSSof the differentially regulated genes in
the WizMD30/+ mice showed enrichmentof Wiz at the TSS of genes withdecreased expression, but not at thegenes with increased expression,
further lending evidence that Wiz
acts as a transcriptional activator
rather than repressor (Figure 3B).

for Wiz and the Encode CTCF and H3K4me3 ChIP-Seq
datasets were calculated across a 4 kb region centeredon the ~40,000 Wiz ChIP-Seq peaks. Input sequencing
from the Wiz ChIP-Seq experiment is also shown. (D) Venn
diagram showing the overlap for Wiz, CTCF and H3K4me3
ChIP-Seq peaks.

ment at the transcription start site(TSS) (Figure 1).
Motif analysis revealed that one of thecommon Wiz motifs showed correlation
with the CTCF binding motifs (Figure 2A
and 2B). A comparison of Wiz ChIP-Seq
data with existing ENCODE ChIP-Seq
data sets for CTCF and H3K4me3, amarker of active transcription, verified
strong correlation with Wiz ChIP-Seq
peaks, with almost all CTCF peaks
overlapping with Wiz peaks (Figure 2C
and 2D).
Wiz is associated with transcriptional
activation in neural tissue
To identify sites within the genome
where Wiz haploinsufficiency wascausing differential gene expression,
RNA-Seq was performed using brainsfrom both E13.5 and adult WizMD30/+ andWiz+/+ male and female mice. The
results show that Wiz haploinsufficiency

Wiz haploinsufficiency in mice resultsin phenotypic alterations in behavior
To characterize any changes in behavior
resulting from Wiz haploinsufficiency,
A

B

Figure 3: Wiz binding at the protocadherin β locus in
the adult cerebellum. (A) Genome browser screenshotsof the protocadherin β locus and the HS5-1bL enhancer(*indicated) located ~400 Kb downstream showing Wiz
binding. (B) Shown is Wiz ChIP-Seq read density in adult
cerebellum as a heatplot for the 29 genes that decreased
and the 53 genes that increased in expression in the cerebellum of WizMD30/+ mice. Also shown is CTCF ChIP-Seq
read density. ChIP-Seq density was calculated for the 1
Kb surrounding the TSS of each gene showing that thosewith decreased expression showed high enrichment for Wiz
and some for CTCF, whereas upregulated genes showed
little enrichment for Wiz and no enrichment for CTCF.

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ADVANCES IN NEUROEPIGENETICS RESEARCH

the researchers conducted behavioral
studies on cohorts of WizMD30/+ and WT
male mice. Their findings show
various abnormalities in behavioral
activity exhibited by the WizMD30/+ mutant
vs. WT mice, including increased
displays of anxiety-like behavior.
Kdm5c-knockout mouse model ofX-linked intellectual disability (XLID)This article is a summary of the research published in Cell
Reports.: Iwase et al., A Mouse Model of X-linked Intellectual
Disability Associated with Impaired Removal of Histone Methylation (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.091.

Intellectual disability (ID), which manifests in limited adaptive behavior andintellectual functioning, is a prevalentneurological disorder affecting 1-2%of the total population. Several genetic
variations have been associatedwith different forms of ID, including
mutations in the X-linked (XL) KDM5C
histone H3K4 di- and tri-methyl(H3K4me2/3)-specific demethylase
gene which account for close to 3% of
all XLID cases. KDM5C is ubiquitously
expressed, although at higher levels
in brain and skeletal muscle, and has
been implicated in various otherneurological disorders, including
autism spectrum disorder, Huntington’sdisease and cerebral palsy, suggesting
it has a critical role in the developmentof the central nervous system.
Mouse models have been instrumental
in investigating brain disorders caused
by mutations, including those ofchromatin modifiers such as Crebbp
and Ehmt1 methyltransferases.
However, there have never been any
mouse models of mutations of ‘erasers’of methylation to investigate theircontribution to brain function. To test
the hypothesis that loss-of-function of
Kdm5c is causal to XLID, researchers
from the laboratory of Dr. Yang Shi at
Harvard created a Kdm5c-knockout(KO) as a mouse model of XLID andcollaborated with Active Motif’s
Epigenetic Services team to gain abetter understanding of the role ofregulation of histone modifications innormal and diseased brains.

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Figure 4: Altered transcription profiles in Kdm5c-KO brain tissues. RNA-Seq data were obtained from the amygdala (AMY) andthe frontal cortex (FC) of three adult mice for each genotype. (A) Differentially expressed (DE) genes between WT and Kdm5c-KO
brain. AMY and FC show a modest overlap. (B) KO/WT ratio of expression levels of DE genes. (C) LR path analysis of misregulated GO
categories followed by clustering. The odds ratio for enrichment is shown. Yellow and blue indicate upregulation and downregulationin KO compared with WT, respectively. Four major classes (I to IV) represent differential alteration patterns between AMY and FC.

Kdm5c-KO mice exhibit abnormal social,
adaptive and cognitive behaviors
To determine whether Kdm5c-KO micedisplayed abnormalities observed inhuman XLID patients, the investigators
performed a series of tests to assess
the physical and behavioral variations
between Kdm5c-KO (-/y, KO) andwild-type (+/y, WT) male littermates.
Physically, the KO mice were reducedin size and body weight, also observed
in XLID patients, but were otherwise
in good health and fertile. The KO
mice exhibited increased aggressive
behavior, anxiety and hyperactivity, andimpairments in cognitive and spatial
learning and memory, which are also
observed in XLID patients. In addition,
KO mice displayed higher, althoughmarginally non-significant, testosterone levels. Furthermore, KO micedisplayed a lower interest in social
interaction which is consistent with
autistic behaviors that manifest in someindividuals with KDM5C mutations.

Kdm5c loss affects both cellular phenotypes and gene expression in the brain
Abnormal dendritic arborization andspine morphology have been implicatedas a cellular basis for ID. Previous in
vitro studies have shown that Kdm5c
is required for dendritic arborizationin rat neurons in culture. However,no in vivo evidence exists to show aconnection between Kdm5c and brainarchitecture. Therefore, the researchers examined whether the behavioral
alterations observed in Kdm5c-KO
mice is due to alterations in brain
morphology. Their analysis revealed
reduced dendritic arborization in thebasolateral amygdala (BLA), an area
critical for perception and emotional
control, including fear and aggression.
They also observed reduced spine
density, abnormalities which have beenpreviously linked to anxiety and cognitive
defects, in the Kdm5c-KO vs. WT mice.
In the cortex, a slight reduction in spine
density was observed and dendritic
arborization was found to be unaffected.

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ADVANCES IN NEUROEPIGENETICS RESEARCH

To determine the genome-wide impactof Kdm5c loss on the brain, the researchers performed RNA-Seq analysisof the amygdala (AMY) and frontal
cortex (FC) transcriptomes of adultmice. They found a comparable numberof differentially regulated genes inboth brain areas, with a larger numberof upregulated genes in KO vs. WT
mice, consistent with the function of
Kdm5c in removal of the H3K4me2/3
active chromatin mark (Figure 4A
and 4B). Differential gene analysis
showed that differentially regulated
genes in the AMY and FC regions areassociated with neural development
and differentiation, synaptic pathways
and behavior (Figure 4C). Interestingly,
they also observed a statisticallysignificant enrichment of misregulated
androgen-responsive genes in the
AMY and FC, consistent with higher
testosterone levels observed in brainsof Kdm5c-KO mice. Taken together, thedata suggest these cellular alterations
caused by Kdm5c loss-of-function may

potentially contribute to the behavioral
phenotypes observed in XLID.
Molecular analysis of Kdm5c regulationof neuronal function
The researchers performed integratedfunctional genomic analysis of corticalneurons to investigate gene regulation
by Kdm5c in a relatively homogenous
cell population. ChIP-Seq analysis of
Kdm5c data sets generated by Active
Motif revealed that Kdm5c is mainly
enriched at promoter and enhancer
functional elements (Figure 5A). 93%
of Kdm5c peaks were found within
DNase hypersensitivity sites, a marker
of open chromatin (Figure 5B). At
promoters, a pronounced Kdm5c peak
was observed at the transcription
start site, and peaks often coincidewith CpG islands, indicating that Kdm5c
modulated transcription of active
genes (Figure 5B and C). CpG islands
often correlate with high H3K4me3
and mRNA levels, and Kdm5c-boundgenes were found to coincide with

Figure 5: Kdm5c Is recruited to CpG promoters with high H3K4me3 in post-mitotic neurons and represses transcription.
ChIP-Seq and RNA-Seq were performed on two biological replicates of WT and Kdm5c-KO neurons. (A) Enrichment of Kdm5c peaks
in promoter and enhancer regions (left) compared with genomic fractions (right). (B) UCSC browser shot of Kdm5c, H3K4me3, andH3K4me1 across the Kdm5c-bound locus Slc29a1 (gray, WT; red, KO). RNA-Seq in WT neurons is shown in red; CpG island position isindicated in green. (C) Most Kdm5c-bound promoters harbor CpG islands (left), which represents strong enrichment over the genomic
average (right). (D) Western blot showing global H3K4me levels are not altered in KO neurons. (E) WT neurons were used to divide
genes into four groups according to expression level. Kdm5c-bound promoters with low expression levels show significantly higher
H3K4me3 levels than unbound genes with the same expression levels. (F and G) Expression and H3K4me3 changes in KO neurons. WTneurons were used to divide genes into three groups according to expression level. Low-expressed Kdm5c-target genes showed themost noticeable increase in expression and H3K4me3 in KO neurons. (H) 206 differentially expressed (DE) genes in Kdm5c-KO neurons
identified by RNA-Seq. (I) Expression level fold change of unchanged and DE genes in KO neurons compared with WT. (J) Overlap of
Kdm5c-bound promoters with DE genes from KO/WT. (K) Upregulated and downregulated genes showed significantly higher or lower
H3K4me3 levels in Kdm5c-KO neurons, respectively.

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increased expression and H3K4me3
levels, suggesting CpG islands andH3K4me3 serve as primary signatures
for recruitment of Kdm5c (Figure 5D).
Because the enzymatic function of
Kdm5c is to demethylate the H3K4me2/3
active mark, it was postulated that
Kdm5c functions as a transcriptional
repressor at H3K4me3 promoters.
Combined RNA-Seq and ChIP-Seq
analysis of WT and KO mice showedthat Kdm5c is not a global regulatorof H3K4 methylation, but rather acts
on a subset of actively transcribedpromoters (Figure 5E). Combined
RNA-Seq and H3K4me1 and H3K4me3
ChIP-Seq data from WT and KO mice
showed that low, but not middle orhigh expressed Kdm5c targets wereupregulated in knockout mice andshowed increased H3K4me3 levels at
promoters. Since this was only observedin low expressing genes, the researchersconcluded that, although Kdm5cfunctions as a transcriptional repressor,
it mainly acts to fine-tune expression of
more sensitive low-expressing targets.
RNA-Seq analysis demonstrated
significant overlap in the differentiallyregulated genes identified using cultured
neurons vs. KO mouse models forKdm5c. In both cases, the changes
observed were modest and upregulationand downregulation of expressionin KO mice correlated with increaseor decrease in H3K4me3 levels at
promoters, respectively. Although thechanges were modest, the researchersconclude that the additive effects ofthese changes collectively contributeto the observed behavioral and cellularabnormalities characteristic of ID.
Let us do the work for you Active Motif Epigenetic Services
Active Motif’s Epigenetic Services
team provides a wide variety of
ChIP-Seq and RNA-Seq services tomake it possible for you to utilize
our expertise and research tools
without having to be an expert in
the techniques yourself. To learn
more about our custom services,or to get a quote, please visit us atwww.activemotif.com/services.

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TOOLS FOR ChIP-SEQ

NEWHigh Quality ChIP-Seq Data from as Few as 5,000 Cells
ChIP-Seq analysis typically requires millions of cells per immunoprecipitation (IP)reaction in order to obtain meaningful information about global changes across a large
population of cells. This is often unattainable for researchers working with primary cells
or other challenging sample types. To enable studies of the complexities of protein-DNA
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 byproviding a complete, optimized low cell ChIP-Seq workflow that enables generation of
genome-wide binding profiles from as few as 5,000 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 cell or tissue material. Active
Motif has utilized its extensive expertisein ChIP-Seq to optimize chromatin
preparation and immunoprecipitation
procedures to lower the input requirements for detection of histone andtranscription factor protein-DNA binding
interactions. Chromatin can be preparedfrom as few as 5,000 cells or small tissue
biopsies.

Figure 1: The Low Cell ChIP-Seq Kit was used to immunoprecipitate 10,000 MDA-MB-468 cells with low abundance BRD4,
or 5,000 GM12878 cells with either active histone mark H3K27ac or repressive histone mark H3K27me3. Following ChIP,

Low Cell ChIP-Seq not only reduces
sample input requirements, but also
resolves the issues often associated with
low-cell ChIP, including poor signal-tonoise, inefficient library amplification andhigh duplication rates. Low background

Low Cell Number ChIP-Seq Kit• Reproducible ChIP-Seq datafrom as little as 5,000 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 cell

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Illumina-compatible sequencing libraries were prepared using the included Next Gen DNA Library and Next Gen Indexing Kits.
Samples were sequenced on the NextSeq 500. Results show high quality ChIP-Seq peaks can be obtained using as little as 5,000
cells for both active and repressive histone modifications and as low as 10,000 cells for low abundance BRD4.

Protein G agarose beads and blockers
are used to minimize non-specificbinding during the IP, while filtration
columns provide a fast, easy and consistent solution for wash steps. Illuminacompatible sequencing libraries can begenerated 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 7)
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

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TOOLS FOR CHIP-SEQNEWNGS DNA Library Preparation with Molecular
Identifiers Eliminates Bias from PCR Duplicates
Improve the quality of your Next-generation sequencing 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. Libraries can be generated
from as little as 10 pg double-stranded
DNA, or from as low as 100 ng DNA if
preparing PCR-free libraries (Figure 1).
The library kit is used in combination
with the Next Gen Indexing Kit, whichcontains 16 unique index adapters that
may be used to multiplex different samples together during cluster generation
for co-sequencing on the same 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 sequencethat is added with the P5 adapter(Figure 2). Addition of the MID is
strand-specific to enable accurate
de-duplication from single readsequencing by distinguishing PCR
duplicates from fragmentation
duplicates. This increases the numberof unique alignments from sequencing
for more accurate data analysis.

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 to the
Fragmented dsDNA
ends of frag10 min
Repair I
mented dsDNA.
+ SPRIThe Next Gen
20 minIndexing Kit
Repair II+ PEG/NaClcontains 16
unique indices.
15 minLigation I+ PEG/NaCl
Indices may be
used individually
10 minLigation II
or in combination + PEG/NaCl (x2)
to multiplex
varies
Optional PCR+ SPRI
samples together
within the same
sequencing
Library
reaction.

Figure 1: 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.

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TOOLS FOR ChIP-SEQ

NEW

Reveal Histone Modification Patterns in ClinicalSamples with Active Motif’s FFPE ChIP-Seq Service
FFPE tissue blocks and histology slides are a valuable resource for retrospective
research on clinical samples. In the field of epigenetics, the benefits of this archived
material has not been fully realized because of the difficulty these sample types
present for use in conventional epigenetic techniques such as chromatin immunoprecipitation (ChIP) due to their limited size and the harsh conditions under which thesesamples have been prepared. Active Motif’s new FFPE ChIP-Seq Service gives youaccess to breakthrough 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. There isincreasing interest in utilizing theseclinical samples for studies in various
research areas, such as epigenetics
where aberrant histone and DNA
modifications have been linked tovarious human 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

lead to the need for optimization. Active
Motif’s new FFPE ChIP-Seq Service
takes the burden of optimization out ofyour hands and now 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 or end-to-end
FFPE ChIP-Seq Service, visit us atwww.activemotif.com/services-ffpechip.

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

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Figure 1: ChIP was performed using chromatin from two human glioblastoma biopsies and an antibody against H3K27ac.
Biopsies were provided as both FFPE prepared samples (purple) and fresh/frozen samples (copper). FFPE and fresh/frozen
ChIP-Seq data sets were similar. Tumor-specific H3K27ac occupancy was detectable in the FFPE and fresh/frozen data sets(highlighted in red).

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HISTONE ISOLATION

NEW

Easily Isolate Intact Histones for PTM Analysis
Histone post-translational modifications (PTMs) play a crucial role in the regulation oftranscription and chromosome packaging. Epigenetic modifications such as histone
methylation, acetylation, and phosphorylation help to recruit chromatin remodelingproteins that assist in unraveling the chromatin to allow access to the basaltranscriptional machinery, or deposit repressive modifications which lead tochromatin condensation and gene silencing. Don’t risk losing your histone PTMs
during extraction. Isolate core histone proteins from cells or tissues using a protocol
designed specifically to extract and retain histone PTMs.

Histone charge is key to isolation
The basic structural unit of chromatinis the nucleosome, which consistsof DNA wrapped around an octamerof core histone proteins (H2A, H2B,
H3 and H4). The interaction between
histones and DNA occurs because
histones are positively charged proteinsthat are attracted to the negatively
charged DNA backbone.
The highly basic nature of histone
proteins often precludes them fromisolation using traditional methods forprotein extraction. Instead, histones
must be isolated under acidic conditions
to retain solubility. The inclusion of
inhibitors helps to preserve histone
modifications such as phosphorylation,acetylation and methylation at specificamino acid residues on the N-terminal
histone tails.
Histone Extraction Kit Highlights• Acid extraction protocol isolates
highly basic histone proteinsfrom cell culture, tissue, andprimary cells (e.g. T-cells)• Preserves histone acetylation,
methylation and phosphorylation• Compatible with downstream
analysis by Western blot, ELISA
and Active Motif’s Histone H3
PTM Multiplex Kit

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Active Motif’s new Histone Extraction
Kit provides a simple method to acidprecipitate histones from cell culture,
tissue (including brain), and primarycells (e.g. T-cells) while preserving
histone modifications (Figure 1). For
cultured cells, both a one-step andtwo-step extraction protocol are
included for sample optimization.
Lysates prepared using the Histone
Extraction Kit are suitable for use
in downstream applications such asWestern blot, Histone Modification
ELISAs or Active Motif’s Histone H3
PTM Multiplex Kit.
For researchers looking for a more
purified histone lysate, we recommend
using one of Active Motif’s Histone
Purification Kits which include additional
column purification and precipitation
steps to remove non-histone proteins.
For more information on histone
isolation or PTM analysis, visit us atwww.activemotif.com/histone.

Figure 1: A Western blot confirms histone PTMs arepreserved following use of the Histone Extraction Kit toisolate lysates from untreated (-) and treated (+) HeLa
cells using the one-step extraction protocol.

What’s in the box?
The Histone Extraction Kit contains
enough reagents for 100 extractions
from cultured cell lines and tissues,or two 96-well cell culture plates.
Supplemental protocols are provided
for working with primary cells.

Product

FormatCatalog No.

Histone Extraction Kit

192 rxns

40028

Histone Purification Kit

10 rxns

40025

Histone Purification Mini Kit

20 rxns

40026

Histone H3 trimethyl Lys 4 ELISA

1 x 96 rxns

53113

Total Histone H3 ELISA

1 x 96 rxns

53110

96 rxns

33115

Histone H3 PTM Multiplex Kit

9DNA METHYLATION ANALYSISNEW

RRBS Service for Genome-scale DNA Methylation
Profiling
Active Motif has now expanded it’s suite of DNA methylation services with the introductionof our new RRBS (Reduced Representation Bisulfite Sequencing) Service. The additionof RRBS along with our existing MeDIP-Seq and Targeted Bisulfite Sequencing services
provides a broad choice of applications for DNA methylation analysis. Our RRBS Service
queries the methylation status of more than 4 million individual CpGs using only 1 μgof starting material. As with all of our services, customers only need to provide DNA,tissues or cells, and we will perform the entire experiment from sample preparation
to bioinformatic analysis.
RRBS Service
Customers submit DNA, cell pellets
or frozen tissues, then we perform:
1 DNA purification
2 DNA digestion
3 Sequencing adapter ligation
4 Bisulfite conversion
5 PCR amplification
6 Sequencing on Illumina® platform
7 Bioinformatic analysis

Why use RRBS for DNA methylation
analysis?
DNA methylation is a stable andheritable epigenetic modification thatis involved in the regulation of gene
expression. DNA methylation patterns
are cell-type specific and are indicativeof cellular origin or fate. Alterations
in DNA methylation patterns can beprognostic of disease and even provide
information about the underlying
mechanisms and cause of disease.
Therefore, comparative analysis of
DNA methylation patterns from normal
and diseased or treated vs. untreatedsamples can be a productive strategy forthe identification of biomarkers.

10

Advantage of RRBS for biomarker
identification
RRBS is an ideal method for biomarker
identification in that it increases theefficiency of genome-scale DNA methylation profiling without compromising
the sensitivity achieved by conventional
Whole Genome Bisulfite Sequencing(WGBS). RRBS significantly lessens
the number of nucleotides analyzed
by deep sequencing by enriching forregions of high CpG content within the
genome.
This method dramatically reduces thescale and expense of WGBS yet still
provides millions of data points focusedat biologically relevant positions suchas promoters and CpG islands. Byutilizing Active Motif’s new end-to-end
RRBS Service, you can experience allof the benefits this method provides
for whole-genome DNA methylation
analysis including:> Single base resolution> Quantitation at each base
> Data at millions of locations 		
across the genome> Data enriched at promoters and
CpG islands> Significantly less expensive than
Whole Genome Bisulfite Sequencing
For more information, please visit us
at www.activemotif.com/services-rrbs.

NormalKidneyTumor
KidneyLAT1
Figure 1: RRBS data using biopsied human kidney
tumor and adjacent normal kidney. The displayed region
is a representative region from the genome-wide data
set and shows differential DNA methylation at thepromoter of the LAT1 gene. Each block is a separate
data point with copper representing a methylated cytosine and purple representing the unmethylated base.

RRBS Service Features• Low input material requirements> 1 mg DNA, 500K cells or
25 mg tissue• Data provided on > 4 million CpGs
• Data from biologically relevant
regions> Promoters> CpG islands

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DNA METHYLATION 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 are 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

GATCGACGATCGGAGCGTAGGTACGACGTTMe

MeBisulfite Conversion

Me

GATCGAUGATCGGAGUGTAGGTAUGACGTTMeMePCR AmplificationMeGATCGATGATCGGAGTGTAGGTATGACGTT
Figure 1: Schematic of bisulfite conversion illustrates
the 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 the DNA methylation profile. The
conversion reaction occurs as a 3-step
deamination of cytosine residues intouracil. As only unmethylated cytosine

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FFPE Tissue Blocks

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.

residues are susceptible to bisulfite
conversion, the original methylation
state of the DNA can be determined.
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 degradationand provide greater than 99% 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 bisulfiteconverted human or mouse DNA isalso included in the kit. Because theprimer pair only produces a PCR
product if conversion has occurred,
you can confirm the procedure worked
before starting downstream sequencing(Figure 3). For more information, 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 rxns

55021

11

DNA METHYLATION ANALYSIS

NEW

Simple High-throughput Screen to Evaluate Changes
in Global DNA Methylation
Active Motif offers two simple-to-use solutions to screen for global changes in
DNA methylation levels. The first utilizes the repetitive element, LINE-1, to quantify
5-methylcytosine (5-mC) levels in either human or murine samples. The second
specifically detects global changes in the DNA methylation variant, 5-hydroxymethylcytosine (5-hmC). Both are provided as quantitative plate-based ELISA assays toenable easy, rapid high-throughput processing of samples in a single experiment.

OD 450 nm

2.4
2
1.6
1.2
0.8
0.4
0

0%

1%

2%

4%

6%

8%

Standards (% 5-mC)

10%

0 µM 1 µM 10 µM

3T3 + 5-Aza-CdR

Figure 1: The Global DNA Methylation Mouse – LINE-1 Kit
was used to compare the methylcytosine levels between
two genomic DNA samples. Assay results show adecrease in 5-mC levels resulting from treatment of 3T3
cells with 5-Aza-2-deoxycytidine, a DNA methyltransferase
inhibitor. The % 5-mC is calculated using the included DNA
standards. Methylcytosine levels of the DNA standards are
displayed as a percentage of total cytosine content.

Using global DNA methylation
changes to study disease
Population studies have revealed astrong correlation between global
DNA methylation levels and factors
such as environmental exposures,
lifestyle or clinical outcomes. The
ability to screen patient samples forchanges in global 5-mC or 5-hmClevels will provide much needed
insight into our understanding of therelationship between DNA methylationand gene expression and its correlation
with diseases such as cancer.
DNA methylation is characterized by amethyl group within the cytosine of a
CpG dinucleotide. These CpG sites
are often found within repetitive regions

12

and are normally methylated torepress transcription, while global
hypomethylation of repetitive elements
is observed in cancer. Likewise, the DNA
methylation variant 5-hmC is known toplay a role in transcriptional regulationand embryonic development and may
also serve as a prognostic indicator incertain neurodegenerative disorders.

Global 5-hmC quantification
The Global 5-hmC Quantification Kit
uses a DNA binding agent to capture
enzymatically digested DNA fragments
to the plate and an antibody specific for
5-hmC for detection. This colorimetricassay works with as little as 20 ng ofgenomic DNA and includes DNA
standards for quantification (Figure 2).

Global 5-mC quantification
Active Motif’s simple, plate-based
Global DNA Methylation – LINE-1 Kit
uses a consensus sequence within
the human or mouse repetitive LongInterspersed Nucleotide Element 1(LINE-1) as a surrogate readout for
global 5-mC levels. First, genomic
DNA is enzymatically digested to create
LINE-1 fragments. The DNA is thenhybridized with a LINE-1 consensus
probe. Following capture onto a
96-well plate, a 5-mC antibody andcolorimetric detection reagents areused to generate a signal that is easily
measured with a microplate reader.
The assay includes DNA standards for
5-mC quantification and can detect as
little as 0.5% methylcytosine (Figure 1).

0.9

Global 5-hmC Quantification

0.8
0.7
0.6

% 5-hmC

Global 5-mC Mouse LINE-1

Global 5-hmC ELISA
Mass Spectrometry

0.5
0.4
0.3
0.2
0.1
0
mouse mouse mouse mouse mousebrain heart kidney liverlunghuman human humanbrain kidney liver

Figure 2: The Global 5-hmC Quantification Kit was used
to determine the % 5-hmC in genomic DNA isolated
from mouse and human tissues. Genomic DNA wastested at 20 ng/well for human brain and 50 ng/well forall other DNA samples. The % 5-hmC was determinedusing the included DNA standards. Results werecompared to mass spectrometry data obtained from 500 ng
of the same DNA samples. Results show the Global
5-hmC Kit provides equivalent quantification using only afraction of the starting material.

Product

FormatCatalog No.

NEW: Global DNA Methylation Mouse – LINE-1

1 x 96 rxns

55022

Global DNA Methylation – LINE-1 Kit

1 x 96 rxns

55017

Global 5-hmC Quantification Kit

1 x 96 rxns

55018

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DIRECT PROTEIN & ANTIBODY LABELINGNEW

Sortase 2.0 – A Versatile Platform for Rapid, Controlled,
Site-specific Protein Modification
The proteome is diverse with many nuances surrounding structure and function, andpost-translational modifications (PTMs) compound an already complex system.
Identification, characterization and mapping of protein modifications enables insight
into protein function, unlocking disease pathways and aiding in identification of drug
targets. Currently, researchers have few options for modifying proteins for functional
research, and each comes with its own set of limitations. To overcome these limitations,
Active Motif now offers a proprietary labeling technology, Sortase 2.0.

Sortase 2.0 Advantage• 10X higher activity thanwild-type Sortase A• Fast, simple site-directed labeling• N- or C-terminal reaction• Works with multiple expression
systems

Deciphering protein structure andfunction with better tools is vital tounlocking disease pathways or
identifying novel drug targets. However,conventional techniques have theirshortcomings. Genetic based
approaches can alter a protein’sbiophysical properties and do notallow incorporation of synthetic
modifications. Traditional chemical
modification methods are not sitespecific and may lead to a loss of proteinfunction. Active Motif’s Sortase 2.0
is a novel technology for protein
modification and labeling that canachieve markedly higher activity inless time than the wild-type Sortase A
while still providing site-specific control
over the target modification.
Sortase is a transpeptidase found inGram-positive bacteria that functions
to anchor surface proteins to thebacterial cell wall. Sortase A modifies

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proteins by recognizing and cleaving
a carboxy-terminal sorting signal.
Sortase 2.0 is a proprietary engineered
variant of Sortase A that specificallylabels the C- or N-terminus of proteinsat the site of the Sortase 2.0 recognition
sequence (LP[X]TG). The activity of
the Sortase 2.0 is roughly 10X greaterthan Sortase A and is calcium independent. Furthermore, the small sizeof the recognition sequence extension
prevents its interference with proteinfunction and trafficking. Proteins
equipped with the C- or N-terminus
LP[X]TG sequence can be labeled insolution and do not require extensive
purification prior to labeling, as only
proteins carrying the this sequence
will be modified. Alternatively, proteinscarrying the LP[X]TG sequence can belabeled directly on the cell surface.
Proteins that have been geneticallyengineered to contain the LP[X]TG
motif can be labeled by Sortase 2.0
using a wide variety of tags, peptides,
DNA, carbohydrates or fluorophores.
The Sortase 2.0 N- and C-Terminal
Protein Labeling Kits provide sufficientmaterials to label up to 25 nmol ofprotein or 5x106 cells. Each kit contains
a Sortag-ready control protein with aglycine attached at the respective
terminus and a His6-tag along with a
Sortase Buffer used to dilute substrate
peptides or proteins to the desiredconcentration prior to Sortagging. The
Sortase 2.0 enzyme provided in the kit
is also modified with a His6-tag andcan easily be removed by adsorption
onto Ni-NTA resin.
For more information, please visit us
at www.activemotif.com/sortase2.0.

COMING SOON:
ABFLEX RECOMBINANT ANTIBODIES
proprietary direct labeling technology

Product

FormatCatalog No.

Sortase 2.0

250 μg

13100

Sortase 2.0 N-Terminal Protein Labeling Kit

1 Kit

13102

Sortase 2.0 C-Terminal Protein Labeling Kit

1 Kit

13103

13

miRNA TARGET IDENTIFICATIONA 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.

142

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 phenotypic statesin cancer cells. miRNAs can impact
genetic programs through post-transcriptional silencing of target genes
either by promoting degradation oftarget messenger RNAs (mRNAs) orby inhibiting their translation. miRNAs
have been implicated in the regulationof various aspects of cancer biology,
including drug resistance, cancer cell

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.

stemness, epithelial-to-mesenchymaltransition and metastasis (Sahu et al.,2016). To enable researchers to leverage
3’UTR reporter assays in their cancer
research on a high-throughput scale,
we have created a genome-widecollection of 12,000 human 3’UTR
luciferase reporters in the highly
optimized 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).
For more information, please visit
www.activemotif.com/ls-3utr.
Functional screening implicatesmiR-371-3p and PRDX6 in reversible
tolerance to cancer drugs
Acquired resistance to cancer drug
therapies almost always occurs inadvanced-stage patients, even following
a significant response to treatment. In
addition to mutational mechanisms,
various non-mutational resistancemechanisms have now been recognized.
In a recent study, Dr. Nisebita Sahu

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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.

explores the potential role for miRNAs
in transient drug tolerance.

implicating these genes as bona fidetargets of miR-371-3p (Figure 2).

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.

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).

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
DTPs (drug-tolerant persisters), did
not display any 3’ untranslated regions(3’UTRs) that could be potentiallytargeted 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 presence of miR-371-3p.
In addition, KDM-3B, which displayedpartial sequence homology in the
3’UTR with miR-371, was exploredusing the 3’UTR luciferase reporter
assay. However, miR-371 was unable
to target the 3’UTR of KDM-3B, suggesting that miR-371 does not regulate
DTPs via KDM-3B. Significantly, 21
genes displayed reduced luciferase
activity upon erlotinib treatment,

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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 andperoxiredoxin 6 in reversible tolerance to cancer drugs.
Nat. Commun. 7:12351. DOI: 10.1038/ncomms12351 (2016).

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

For more information 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

15

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Enabling Epigenetics Research