The genomic binding sites of a noncoding RNA
Matthew D. Simona, Charlotte I. Wangb, Peter V. Kharchenkoc, Jason A. Westa, Brad A. Chapmana,
Artyom A. Alekseyenkob, Mark L. Borowskya, Mitzi I. Kurodab, and Robert E. Kingstona,1
aDepartment of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114;
Genetics, Department of Medicine, Brigham and Women’s Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02115; and
cCenter for Biomedical Informatics, Harvard Medical School and Informatics Program, Children’s Hospital, Boston, MA 02115
Edited by* Keith R. Yamamoto, University of California, San Francisco, CA, and approved October 19, 2011 (received for review August 17, 2011)
Long noncoding RNAs (lncRNAs) have important regulatory roles
and can function at the level of chromatin. To determine where
lncRNAs bind to chromatin, we developed capture hybridization
analysis of RNA targets (CHART), a hybridization-based technique
that specifically enriches endogenous RNAs along with their tar-
gets from reversibly cross-linked chromatin extracts. CHART was
used to enrichthe DNA and protein targetsof endogenouslncRNAs
from flies and humans. This analysis was extended to genome-
wide mapping of roX2, a well-studied ncRNA involved in dosage
compensation in Drosophila. CHART revealed that roX2 binds at
specific genomic sites that coincide with the binding sites of pro-
teins from the male-specific lethal complex that affects dosage
compensation. These results reveal the genomic targets of roX2
and demonstrate how CHARTcan be used to study RNAs in a man-
ner analogous to chromatin immunoprecipitation for proteins.
chromatin-associated RNAs ∣ chromatin-modifying complexes ∣
RNase H mapping
encoded at specific loci in chromatin and trans-acting factors that
bind them (1). Although the importance of trans-acting proteins
(e.g., transcription factors) has long been appreciated, there is
growing interest in the role of long noncoding RNAs (lncRNAs)
is enhanced by the recent discovery that the majority of eukaryotic
genomes are transcribed (3) and that many of the resulting tran-
scripts are developmentally regulated (4) but do not encode pro-
teins. Although the functional scope of these RNAs remains
unknown (5–7), several lncRNAs play important regulatory roles
at the level of chromatin (8). Determining where these ncRNAs
bind on the genome is central to determining their function.
Examples of lncRNAs that influence chromatin include the
roX ncRNAs in flies and Xist in mammals, both having well-
established roles in dosage compensation (8, 9); Kcnq1ot1 and
Air ncRNAs, which are expressed from genomically imprinted
loci and affect chromatin silencing (10–13); Evf2, HSR1, and
other ncRNAs that positively regulate transcription (14–16);
lncRNAs that target the dihydrofolate reductase promoter and
the rDNA promoters through triplex formation (17, 18); and the
human HOTAIR and HOTTIP lncRNAs, which regulate poly-
comb-repressed and trithorax-activated chromatin, respectively
(19, 20). Dysregulation of several of these lncRNAs has been
associated with disease (21, 22). Our understanding of the bio-
chemical roles of these RNAs comes largely from their interac-
tions with specific proteins—insights gained from classical
biochemical techniques developed for studying translation and
RNA-processing complexes and also more recent technological
advances using RNA immunoprecipitation (23) and cross-linking
and immunoprecipitation (24–26). These experiments suggest
that several lncRNAs specifically interact with chromatin-m
odifying machinery and may act as scaffolds for multiple com-
plexes (27) or as targeting modules to direct these complexes
to specific chromatin loci (reviewed in refs. 28 and 29). There
are various modes by which an RNA can interact with a chroma-
tin locus, including direct interactions with the DNA (through
enerating cellular diversity from genetic information re-
quires the regulatory interplay between cis-acting elements
canonical Watson-Crick base pairing or nonconical structures
such as triple helices) or indirect interactions mediated through
a nascent RNA or protein (28).
Determining the direct functions of lncRNAs requires knowl-
edge of where they act. This requirement motivates the devel-
opment of technology to generate genomic binding profiles of
lncRNAsinchromatin that isanalogous to chromatin immunopre-
cipitation (ChIP) for proteins. Ideally, this technology would (i)
provide enrichments and resolution similar to ChIP, (ii) use cross-
linking conditions that are reversible and allow for analysis of
RNA, DNA, and protein from the same enriched sample, and (iii)
provide adequate controls to distinguish RNA targets from the
background signal. Although there are several techniques that
localize RNAs on chromatin, none fulfill all these criteria. For
example, both fluorescence in situ hybridization (FISH) (30) and
a related biochemical approach (31), which relies on indirect bio-
tinylation of biomolecules near the target RNA, are important
techniques that localize RNAs to genomic loci, but neither has
demonstrated high resolution across the genome. The ability of
nucleic-acid probes to retrieve lncRNAs from cross-linked extracts
or rather due to direct interactions of the long capture oligos with
complementary regions found in nearby DNA. Either way, the effi-
ciency and specificity of these technologies have not allowed the
precision required for high-resolution genome-wide profiling.
We report the development of CHART (capture hybridization
analysis of RNA targets), a hybridization-based purification
strategy that can be used to map the genomic binding sites for
endogenous RNAs. We used CHART to purify lncRNAs and
their associated protein and DNA targets and to determine the
genome-wide localization of roX2 RNA in chromatin. We began
by identifying regions of the target RNA available for hybridiza-
tion to short, complementary oligonucleotides. We then designed
affinity-tagged versions of these oligonucleotides to retrieve the
target RNA along with its associated factors from reversibly
cross-linked chromatin extracts under optimized CHARTcondi-
tions. By isolating and purifying the CHART-enriched DNA frag-
ments, analogous to ChIP, CHART allows the identification of
the genomic binding sites of endogenous RNAs (Fig. 1). These
data definitively demonstrate that a lncRNA, roX2, localizes to
the samesites across thegenome as the chromatin-modifying pro-
tein complex with which it is proposed to act. Together, these data
demonstrate the utility of CHARTas a tool in the study of RNAs.
Author contributions: M.D.S., C.I.W., J.A.W., A.A.A., M.I.K., and R.E.K. designed research;
M.D.S., C.I.W., and J.A.W. performed research; M.D.S. and A.A.A. contributed new
reagents/analytic tools; M.D.S., P.V.K., B.A.C., and M.L.B. analyzed data; and M.D.S.,
J.A.W., M.I.K., and R.E.K. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The data from genome-wide CHART analyses have been deposited at
the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession
whom correspondenceshouldbe addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1113536108PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20497–20502
Design and Development of CHART. We sought to affinity purify
an RNA together with its targets by using oligonucleotides that
are complementary to the RNA sequence and developed this
technology for roX2, an approximately 600-nt ncRNA that regu-
lates dosage compensation in Drosophila (9). Guided by the
success of a chromatin-purification strategy that uses short, affi-
nity-tagged oligonucleotides (C-oligos) to enrich genomic loci
through hybridization to DNA in cross-linked extracts (33), we
pursued a similar strategy using C-oligos to capture endogenous
roX2 RNA along with its associated targets in reversibly cross-
linked extracts (Fig. 1).
We first sought to ensure that these C-oligos would target
stretches of roX2 RNA available for hybridization and not oc-
cluded by protein binding or secondary structure. We adapted an
RNase-H mapping assay (34–36) to probe sites on roX2 available
to hybridization in the context of cross-linked chromatin extracts.
RNase-H specifically hydrolyzes the RNA strand of a DNA-RNA
hybrid (37). As RNase-H is not active when exposed to the deter-
gents present in many chromatin extraction procedures, we
determined assay conditions ideal for both solubilization of the
chromatin and RNase-H mapping (Fig. S1A).Exposing chromatin
extracts to 20-mer DNA oligonucleotides one at a time and mea-
suring hybridization to roX2 by sensitivity to RNase-H revealed
regions of roX2 that were significantly and reproducibly more
available for C-oligo hybridization than others (Fig. S1 B and C).
These differences could be due to differences in accessibility of
roX2 or to factors independent of roX2, such as other competing
sequences in the extract. Because both roX2-dependent and roX2-
independent mechanisms that lead to low RNase-H sensitivity
could also interfere with efficient hybridization to C-oligos in
the context of CHARTenrichment, we focused on accessible sites
with high RNase-H sensitivity for C-oligo design.
We next sought conditions to specifically enrich roX2 RNA
together with its associated targets and tested a range of hy-
bridization conditions and C-oligo chemistries (including O2′-
methylated ribonucleotides and locked nucleic acids) on the basis
of related applications (33, 35, 38, 39). In these experiments we
used desthiobiotin-conjugated C-oligos (Fig. S1D), which allow
for gentle biotin elution (33, 40). Determining CHART hybridi-
zation conditions required balancing the solubility of the chroma-
tin extract, the stability of duplex formed upon C-oligo binding to
RNA, and the stringency required to directly capture only the
desired RNA. Using the design illustrated in Fig. S1D, we found
that a cocktail of three approximately 25-mer DNA-based C-oli-
gos provided a low background signal and high specific yields of
roX2 in a buffer with high ionic strength and high concentrations
of denaturants (Fig. 2A). Approximately half of roX2 RNA input
could be retrieved from the cross-linked chromatin extract. This
enrichment was specific; CHARTusing a scrambled control C-oli-
go did not enrich roX2, and control RNAs were not enriched by
roX2 CHART. We conclude that DNA-based C-oligos hybridizing
to RNase-H–sensitive locations on a target RNA can specifically
enrich the RNAs from a cross-linked chromatin extract.
CHART Enrichment of roX2 Targets. Having established CHART
enrichment of roX2 RNA itself, we tested whether proteins and
DNA loci associated with roX2 were also enriched. We first exam-
inedcandidate genomic sitesofroX2 binding. We foundthatDNA
was enriched for both the endogenous roX2 locus and a known
regulatory site of dosage compensation, chromatin entry site 5C2
(CES-5C2) (41) but not control sites (Fig. 2B). To test whether the
CHART-enrichment of DNA was RNA-dependent, and not an ar-
DNA, we treated the extract with RNase prior to C-oligo hybridi-
zation. The majority of the enrichment at the endogenous locus
(approximately 93%), and essentially all of the enrichment at
the trans-acting locus (>99%), was RNA-mediated (Fig. 2B); only
a minority of the DNA enriched at the endogenous roX2 locus
(approximately 7%) could be accounted for by direct binding of
the C-oligos to DNA. The RNA-mediated enrichment of the reg-
ulatory site (CES-5C2) was substantial (>100-fold over a control
locus and >1000-fold over the sense-oligo control), and the yields
(approximately 1–2%) were similar to those retrieved by ChIP.
As further support of the specificity of CHART, a control ex-
periment using sense oligos (therefore not complementary to
roX2 RNA) did not enrich the DNA locus where roX2 binds
in trans and displayed low levels of enrichment of the endogenous
roX2 locus (consistent with the levels of direct DNA binding ob-
served in the RNase control). Also, individual C-oligos were each
successful at specifically enriching the appropriate loci (Fig. S2).
Using individual C-oligos led to substantially lower yields, how-
ever, demonstrating that the cocktail of three C-oligos acts syner-
gistically (Fig. S2).
In addition to DNA, we analyzed the proteins enriched by
roX2 CHARTand found that a subunit of the male-specific lethal
complex (MSL3) was enriched relative to a scrambled control by
roX2 CHART (Fig. 2C). The yield of MSL3 protein (approxi-
mately 1%) is similar to the enrichment observed for the DNA
oligonucleotides to purify the RNA together with its targets from reversibly
cross-linked extracts. The cartoon here shows the scenario where the RNA is
bound in direct contact with the DNA together with proteins, but other con-
figurations are also possible (see the text). CHART-enriched material can be
analyzed in various ways; the two examples depicted here are (Left) sequen-
cing the DNA to determine genomic loci where the RNA is bound and (Right)
analyzing the protein content by Western blot analysis.
CHART is a hybridization-based strategy that uses complementary
www.pnas.org/cgi/doi/10.1073/pnas.1113536108Simon et al.
protein, DSP1 (yield <0.1%), which was not enriched in the roX2
CHART compared with a scrambled control CHART. We con-
clude that enrichment of roX2 by CHARTsimultaneously enriches
protein and DNA representing roX2 targets, and this enrichment
Extending CHART to a Mammalian RNA. Because roX2 CHARTsuc-
cessfully enriched roX2-associated targets, we tested whether
these same conditions are general for enrichment of other RNAs,
including longer mammalian lncRNAs. We applied CHART to
endogenous NEAT1 (3.8 kb), a lncRNA found in human cells,
and compared the enrichment to another human lncRNA, MA-
LAT1 (>6.5 kb) in two different cell lines (42–48). Although
these lncRNAs are both retained in the nucleus, undergo similar
processing, and are encoded next to each other in the genome,
they have distinct localizations in the nucleus, NEAT1 localizing
to paraspeckles and MALAT1 to nuclear speckles (49, 50). By
RNase-H mapping these RNAs from HeLa cells to reveal regions
available for hybridization (Fig. S3A) and applying the CHART
protocol developed for roX2, we found that both RNAs could be
enriched from cross-linked chromatin extracts derived from two
human cell lines (Fig. 3A and Fig. S3B). These RNA yields were
lower than observed for roX2, which may be due to differences in
vetting of C-oligos (only subregions of these RNAs were mapped
by RNase-H), shearing of longer RNAs, or in the complexity or
age of the chromatin extract. Regardless of the reason for the
modest (approximately threefold) differences in RNA yield, both
extracts led to similar CHART enrichment of MALAT1- and
NEAT1-associated DNA (see below).
NEAT1 assembles cotranscriptionally with paraspeckle pro-
teins, and fluorescence-imaging experiments suggest that NEAT1
is retained at its endogenous locus (51). Both NEAT1 and
MALAT1 CHART demonstrated specific enrichment of their
own endogenous genomic loci but not the other’s (Fig. 3B and
Fig. S3C). Pretreatment of the extract with RNase abrogates the
CHART signal (Fig. 3B and Fig. S3C), demonstrating that
CHART enrichment is RNA-mediated. In addition to retrieving
the endogenous NEAT1 locus, we expected NEAT1 CHART to
enrich paraspeckle proteins. Indeed, we found robust and specific
RNA-dependent enrichment of both PSPC1 and p54/nrb
(Fig. 3C), two proteins found in paraspeckles that interact with
NEAT1 (43, 46, 47). Thus, the analysis of the DNA and proteins
enriched by NEAT1 CHART demonstratesthat the conditions de-
veloped for roX2 CHART also work for a longer endogenous
lncRNA from human cells, supporting the generality of CHART.
The observed enrichment of RNAs together with their targets
indicate that CHART might be combined with high-throughput
sequencing to determine the genome-wide binding profile of an
RNA. We tested this conjecture by sequencing the DNA enriched
by roX2 CHART to study its genome-wide localization.
Extension of roX2 CHART to Genome-Wide Analysis. We sequenced
the roX2 CHART-enriched DNA to generate a genome-wide
binding profile for roX2. roX2 is known to localize to the X chro-
mosome (chrX) (52–54), where it acts together with the MSL
complex (including protein subunits MSL1, MSL2, MSL3, MLE,
and MOF) (9). The MSL complex affects dosage compensation,
at least in part, by regulating acetylation of histone H4 lysine 16
(H4K16) in the bodies of active genes (55–60) and influencing
transcriptional elongation (61). Therefore we expected strong
enrichment of the roX2 CHART-seq signal on chrX and sought
to learn more about roX2 by examining its distribution in com-
parison with ChIP results for proteins and modifications asso-
ciated with dosage compensation.
Upon aligning the sequenced reads to the fly genome, the
predominant signals from roX2 CHART-seq were a series of in-
tense peaks on chrX (Fig. 4A), consistent with FISH data (52, 53).
Some roX2 CHARTsignals, however, coincide with the peaks in
the control sense-oligo profiles (for examples, see Fig. 4A and the
autosomal signals in Fig. S4A). We interpret these peaks as sites
where the C-oligos directly enrich DNA. When normalized by the
sense-oligo control and ordered by significance, the top 173 roX2
CHART peaks were all found on chrX (Fig. S4B). This strong
enrichment of roX2 CHART signals on chrX is consistent with
the role of roX2 in dosage compensation.
The enrichment of peaks on chrX was encouraging, but the
autosomal signals revealed that CHART eluant contains con-
taminants from nonspecific hybridization. Many of the artifactual
measured by RT-qPCR. (B) Enrichment of DNA loci by roX2 CHART. CES-5C2 is a regulatory site enriched by roX2 CHART. The enrichment values are labeled for
comparison of CES-5C2 by roX2 CHART with sense-oligo CHART and also with roX2 CHART at a control site, Pka. RNase-positive lanes represent CHART en-
richment from extracts pretreated with RNase to eliminate RNA-mediated signal. Error bars represent ±SEM for three qPCR experiments. Primers are listed in
Table S3. (C) Specific enrichment of a tagged MSL subunit, MSL3-TAP, by roX2 CHART. DSP1 antisera (64) is used as a negative control because of its sensitivity.
CHARTallows specific enrichment of roX2 along with its associated targets. (A) Enrichment of RNAs by roX2 CHART (using C-oligos listed in Table S2) as
from HeLa chromatin extracts by either N, NEAT1 CHART; M, MALAT1 CHART; or O, a mock (no C-oligo) control as measured by RT-qPCR. (B) Similar to A, but
enrichment ofassociated DNA loci as determined byqPCR. Error bars represent ±SEM for three independent CHARTexperiments. (C)Specific enrichment oftwo
paraspeckle proteins, p54/nrb and PSPC1, by NEAT1 CHART from MCF7 extract. Histone H3 was chosen as a negative control because it is a highly sensitive
antiserum and NEAT1 is not expected to be predominantly chromatin bound.
NEAT1 CHART, but not MALAT1 CHART, specifically enriches NEAT1 RNA along with its protein and DNA targets. (A) Enrichment of the indicated RNAs
Simon et al. PNAS
December 20, 2011
peaks could be filtered by using extra controls and post hoc
computational approaches. In this case, setting the appropriate
thresholds was viable given our strong expectation of chrX
enrichment, but ideally CHART could be performed and inter-
preted for lncRNAs without such expectations. We sought to
minimize retrieval of these contaminants by increasing the bio-
chemical specificity of CHART, thereby increasing the interpret-
ability of the raw mapped CHART reads.
To improve the CHART protocol and minimize purification
of products from direct binding of C-oligos to DNA, we removed
the heated hybridization step to avoid denaturing the DNA and
eluted the roX2 CHART material enzymatically with RNase-H.
In this alternative to biotin elution, the DNA bound via roX2
RNA should elute from the resin, but DNA directly bound to
the C-oligo should not elute. Because we were no longer using
a biotin elution, we used biotinylated rather than desthiobiotiny-
lated C-oligos (Fig. S1E). These modifications maintained the
specific enrichment of the endogenous roX2 locus and CES-5C2
(Fig. S4C), leading us to sequence two independent RNase-
H–eluted roX2 CHART replicates.
It was immediately evident from the raw, mapped sequencing
reads that the RNase-H–eluted CHARTsamples greatly reduced
background from nonspecific hybridization (Fig. 4A). This con-
clusion is supported by statistical analyses that reveal a decrease
in raw autosomal read intensities in comparison to the previous
biotin-eluted CHART sample (Fig. S4D). The two RNase-H–
eluted CHARTsamples showed excellent agreement (Fig. S4E),
and ordering the peaks by input-normalized significance (i.e.,
without other CHART controls) demonstrated that the top 214
peaks from these data are on chrX (Fig. S4F).
These data demonstrate that roX2 CHARTcan be combined
with sequencing to map the binding sites of a lncRNA, as exem-
plified by the robust, RNA-mediated enrichment of a series of
sites highly enriched on chrX found by roX2 CHART. To further
validate the CHART-seq technology and explore the localization
of roX2, we used these data to test at molecular resolution
whether roX2 localization coincides with specific features of do-
sage-compensated chromatin, especially sites bound by the MSL
Analysis of roX2 CHART-seq–Enriched Sites. The MSL complex is
thought to find its binding sites through at least two different
mechanisms. Genetic and molecular experiments have revealed
a set of 150–300 high-occupancy sites containing a GA-rich se-
quence motif(41, 62). These sites may actas chromatin entry sites
for initial, sequence-specific recognition, followed by spreading
to sites on the chrX located in active genes (9). This second class
of sites is thought to be recognized through general marks of ac-
tive transcription, such as H3K36me3, because active autosomal
genes can acquire MSL binding when inserted on X (63). Gen-
ome-wide CHART of roX2 allowed us to test whether roX2
RNA has the same preference for chromatin entry sites as the
MSL complex. When compared to ChIP-chip or ChIP-seq for
chromatin modifications associated with dosage compensation
(H4K16ac and H3K36me3) or with the ChIP signal observed
for a tagged version of MSL3, the roX2 CHART signal was no-
table for its coincidence with MSL high-occupancy sites (Fig. 4 A
and B). The lower significance autosomal signals did not line up
with previously proposed MSL binding sites (62) and were not
enriched for MSL binding, which suggests they are unlikely to
be real roX2 binding sites. Statistical analyses demonstrated that
the top roX2 CHART peaks are all enriched for MSL binding
(Fig. S5A), and known MSL binding sites have a higher roX2
CHARTsignal than non-MSL-binding sites (Fig. S5B). Not only
does the roX2 CHARTsignal overlap with the MSL-ChIP signal,
but also the datasets correlate (Fig. 4C) and the intense peaks
reads from roX2 and sense-oligo CHART data (performed from S2 cells expressing MSL3-TAP) (55) compared to MSL3-TAP ChIP data from MSL3-TAP Clone 8
(41). Both mapped read numbers and normalized read numbers are listed. Note the RNase-H–eluted roX2 CHART has higher peaks signals at roX2 binding sites
and required a different scale than the other three sequencing tracks. Below, ChIP-chip data for the indicated histone modifications are shown (S2 cells,
ModENCODE) (65). (B) Finer-scale examples and comparisons of roX2 CHART data, with normalized read depth, except Far Right where normalized for peak
height. (C) Correlation between the roX2 CHART signal and MSL3-TAP ChIP signal (41) by plotting the conservative enrichment magnitudes (relative to cor-
responding inputs) on a log2scale of roX2 CHART peaks (from combined RNase-H-elution replicates) and MSL3-TAP ChIP peaks. Peaks from chrX are shown in
red and autosomal peaks in blue, but the Pearson r was determined including both sets of peaks. (D) A motif identified from the top roX2 CHART peaks,
depicted here as a motif logo in comparison with a nearly identical motif previously determined from MSL3-TAP ChIP-chip data (41).
roX2 CHART-seq reveals robust enrichment of roX2 on chrX and precise localization to sites of MSL binding. (A) Top four rows, mapped sequencing
www.pnas.org/cgi/doi/10.1073/pnas.1113536108Simon et al.
align precisely (Fig. S5C). Inspection of the data also reveals that
the CHART signal typically mirrors the contours of the MSL3-
ChIP signal (Fig. 4B). These data are consistent with roX2 acting
as an integral subunit of the MSL complex while the complex
is bound to chromatin.
If roX2 is binding to the samespectrum ofchromatin entry sites
as MSL3, one prediction is that the locations of roX2 CHART
peaks can be used to find a DNA motif associated with roX2
binding, and this motif should be similar to the motif previously
derived for sites of MSL3 binding. Indeed, motif analysis of the
CHART data for roX2 yields a nearly identical motif to that de-
rived from the ChIP analysis of MSL3 (Fig. 4D). This sequence
can attract local MSL activity when inserted onto an autosome
(41). In sum, these data demonstrate that CHART allows the
determination of the genome-wide binding sites of a ncRNA.
Although recent advances have demonstrated the importance
of lncRNAs as regulatory factors and revealed that many of
these lncRNAs can act in concert with chromatin-modifying
machinery, our understanding of where these lncRNAs directly
act on chromatin has progressed more slowly. We developed
CHART and used it to examine the genomic binding sites of a
lncRNA. Because this approach is analogous to ChIP, we present
a comparison of these techniques. Depending on the antibody,
useful ChIP enrichments range from a fewfold to up to 3 orders
of magnitude for the best antisera. roX2 CHARTachieves enrich-
ments on the high end of this range, at times exceeding 3 orders
of magnitude (Fig. 2B). In theory, the resolution of CHART-seq
could have proven significantly worse than ChIP-seq because
CHARTrequires a higher degree of cross-linking. In practice, any
loss of resolution observed for CHART-seq is minor as can be
seen by comparing MSL3-TAP (where TAP is a tandem affinity
purification epitope tag) ChIP-seq signals to roX2 CHART-seq
signals (Fig. 4 A and B). Therefore CHART appears similar to
ChIP in enrichment and resolution.
The limitations of CHART also overlap with those of ChIP.
Neither provides information regarding the stoichiometry of
binding at each genomic locus—only enrichment values. Also like
ChIP, there is no guarantee that different target loci will be
enriched with equal efficiency, because at some loci the C-oligos
may have less access (e.g., if they are occluded from binding,
similar to epitope masking with ChIP). Given the utility and im-
portance of ChIP despite these caveats, it is reasonable to expect
similar utility from CHART. Importantly, both ChIPand CHART
provide information about the localization of the factor to chro-
matin loci but do not reveal the molecular basis of the interaction;
CHART-enriched targets could either be directly bound to the
RNA or bound through other factors such as bridging proteins
or RNAs. We found no evidence that roX2 binding sites are
enriched for sequences with Watson-Crick complementarily to
roX2 (Table S1), which suggests that the interactions between
roX2 and these loci are indirect, very short, or based on non-
Also similar to ChIP, CHART-enriched material can be used to
examine either candidate genomic loci or genome-wide binding
profiles. We applied both to roX2 and found roX2 localized to
dosage-compensated regions on chrX, as expected. Comparison
of the high-resolution map from roX2 CHART with published
data for the MSL complex achieved by using ChIP revealed that
roX2 binds at the same sites in chromatin as the MSL complex.
Because many lncRNAs are thought to act together with chroma-
tin-modifying machinery, this comparison allowed us to validate
the previously untested inference that a lncRNA can act at the
same sites on chromatin across the genome as its associated
CHARTwas used successfully for a longer mammalian ncRNA
from two different cell lines (Fig. 3 and Fig. S3). Few lncRNAsare
known to bind to specific genomic sites,but RNAs can be retained
near their endogenous loci, serving as a positive control for
CHART enrichment without previous knowledge of trans-acting
sites. We found that CHART analysis of endogenous loci can
be complicated by the direct DNA binding of the C-oligos, but
using RNase-pretreated extract allows this artifactual signal to
be distinguished from the desired RNA-mediated CHARTsignal.
Analysis of the RNAs examined here shows that CHART may be
successfully applied to RNAs of different lengths and origin.
Despite the successful mapping of genomic binding sites using
roX2 CHART-enriched samples, it is not yet clear how roX2 com-
pares to other chromatin-bound lncRNAs in binding mode and
stoichiometry, and therefore the generality of CHART will be
determined as it is applied to more RNAs. Although the strength
of roX2 CHART signals allows them to be easily distinguished
from nonspecific background, the use of oligonucleotides as
affinity reagents will always raise the potential of direct or indir-
ect off-target hybridization. From analysis of the autosomal bio-
tin-eluted roX2 CHART peaks, we found that particular caution
is required when interpreting sharp peaks (<600 bp) and peaks
that contain motifs with homology to the target RNA; this pattern
is indicative of likely artifacts and therefore requires further
experimentation. In the case of roX2 CHART, the CHART-iden-
tified binding sites were not found to have homology to the RNA,
which demonstrates that these potential artifacts were avoided.
In addition to locating the genomic targets of an RNA,
CHARTcan also be used to examine other RNA associated fac-
tors; we have demonstrated this point by analyzing CHART-
enriched material by Western blot for protein targets (Figs. 2C
and 3C). Because CHART involves reversible cross-linking, the
enriched material can be used for the reciprocal of an RNA-IP;
instead of pulling down protein and looking for RNA, CHART
allows enrichment of the RNA and examination of which pro-
teins copurify by Western blot. Therefore, although we focused
here on the use of CHART to examine DNA targets, CHART-
enriched material can also be analyzed for other factors, and we
anticipate the extension of CHART to proteomic analyses to un-
cover RNA-associated proteins.
In summary, we have developed CHART, a technique that
allows determination of RNA targets. CHART was successfully
applied to lncRNAs of different lengths from two different organ-
isms. We were able to extend CHART to robust genome-wide
analysis and from this analysis address the previously untested
inference that a lncRNA can act across the genome at the same
sites as an associated chromatin-modifying complex. Given the
intense interest in the functionality of lncRNAs, including
their roles regulating chromatin structure and gene expression,
CHART provides a valuable tool to identify the genomic loci
directly regulated by an RNA, as exemplified here with roX2.
Materials and Methods
To accomplish CHARTenrichment, extract (250 μL, 8 × 107cell equivalent) was
adjusted to hybridization conditions (20 mM Hepes pH 7.5, 817 mM NaCl,
1.9 M urea, 0.4% SDS, 5.7 mM EDTA, 0.3 mM EGTA, 0.03% sodium deoxycho-
late, 5× Denhardt’s solution) and precleared with ultralink-streptavidin resin
(Pierce). C-oligos (800 nM each R2.1–3) were added and hybridized (55°C for
20 min; 37°C for 10 min; 45°C for 60 min; 37°C for 30 min). The bound ma-
terial was captured by using streptavidin beads [(MyOne C1; Invitrogen, over-
night, room temperature (RT)], rinsed five times with WB250 (250 mM NaCl,
10 mM Hepes pH 7.5, 2 mM EDTA, 1 mM EGTA, 0.2% SDS, 0.1% N-lauroyl-
sarcosine), and eluted with 12.5 mM biotin in WB250 for 1 h at RT. For RNase-
pretreated extract, RNase (Roche, DNase-free, 1 μL) was added to the initial
extract and allowed to incubate for 10 min at RT prior to adjusting to hybri-
dization conditions. RNase-H–eluted CHART was performed similarly, except
we omitted the prebinding to ultralink-streptavidin resin and used higher
concentrations C-oligos (1.3 μM each). For the RNase-H elution, the final rinse
was with RNase-H rinse buffer (50 mM Hepes pH 7.5, 75 mM NaCl, 3 mM
MgCl2, 0.125% N-lauroylsarcosine, 0.025% sodium deoxycholate, 20 u∕mL
SUPERasIN, 5 mM DTT). The CHART-enriched material was then resuspended
in RNase-H rinse buffer (100 μL) and RNase H (10 U) was added. The elution
Simon et al. PNAS
December 20, 2011
was allowed to proceed for 10 min with gentle shaking at RT. The beads were Download full-text
captured and the reaction stopped with EDTA before proceeding to analyze
the CHART-enriched proteins or nucleic acids. For detailed methods, see SI
Materials and Methods.
Note. During revision of this manuscript, another approach was published
describing a strategy to map the binding sites of RNAs (66).
ACKNOWLEDGMENTS. We thank Jose Antao for suggesting cell culture condi-
tions; Daniel Locker for providing the DSP1 antisera; Shangtao Liu, Jerome
Dejardin, and members of the Kingston lab for helpful discussions; Sarah
K. Bowman for critical reading of this manuscript; and the Szostak Lab for
assistance and use of their DNA synthesizer. This research was funded by
grants from the National Institutes of Health National Institute of General
Medical Sciences (5R01GM043901 to R.E.K. and GM045744 to M.I.K) and a
Helen Hay Whitney Foundation postdoctoral fellowship (M.D.S.).
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