Genome-wide localization of exosome components
to active promoters and chromatin insulators
Su Jun Lim, Patrick J. Boyle, Madoka Chinen, Ryan K. Dale and Elissa P. Lei*
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA
Received October 31, 2012; Revised January 6, 2013; Accepted January 7, 2013
Chromatin insulators are functionally conserved
DNA–protein complexes situated throughout the
genome that organize independent transcriptional
domains. Previous work implicated RNA as an
important cofactor in chromatin insulator activity,
although the precise mechanisms are not yet under-
stood. Here we identify the exosome, the highly
conserved major cellular 30to 50RNA degradation
machinery, as a physical interactor of CP190-
Drosophila. Genome-wide profiling of exosome by
ChIP-seq in two different embryonic cell lines
reveals extensive and specific overlap with the
CP190, BEAF-32 and CTCF insulator proteins.
Colocalization occurs mainly at promoters but also
boundary elements such as Mcp, Fab-8, scs and
scs0, which overlaps with a promoter. Surprisingly,
exosome associates primarily with promoters but
not gene bodies of active genes, arguing against
simple cotranscriptional recruitment to RNA sub-
strates. Similar to insulator proteins, exosome is
also significantly enriched at divergently transcribed
promoters. Directed ChIP of exosome in cell lines
depleted of insulator proteins shows that CTCF is
required specifically for exosome association at
Mcp and Fab-8 but not other sites, suggesting that
alternate mechanisms must also contribute to
exosome chromatin recruitment. Taken together,
our results reveal a novel positive relationship
throughout the genome.
The exosome is a multisubunit complex conserved from
archaea to humans that is the major cellular 30to 50RNA
degradation machinery. Involved in the turnover of
normal as well as aberrant RNAs, the exosome addition-
ally plays a major role in RNA processing and maturation
[reviewed in (1)]. The exosome consists of a core complex
including a hexameric ring of RNase PH homology
domain-containing subunits (Ski6/Rrp41, Rrp42, Rrp43,
Rrp45, Rrp46 and Mtr3) capped by a trimer of S1/KH
domain-containing subunits (Csl4, Rrp4 and Rrp40)
[reviewed in (2)]. It has been shown that yeast exosomes
channel RNA through the center of the core complex (3),
but it is the association of either of the hydrolytic RNases
Dis3/Rrp44 and Rrp6 with the yeast and human core
complex. A contrasting view in Drosophila suggests that
exosome subunits can function independently or form a
continuum of various functional complexes (4).
Although the core exosome and Dis3 localize to both
the nucleus and the cytoplasm, the Rrp6 component is
predominantly nuclear, suggesting specialized activities
for the exosome in the nucleus. Rrp6 alone or the entire
RNA quality control and surveillance pathways [reviewed
in (5)]. Depletion of exosome levels or mutation of
exosome components leads to stabilization of cryptic
unstable transcripts (CUTs) in yeast, antisense promoter
transcripts in mammals (6,7), as well as other aberrant
RNAs. In yeast, Nrd1-dependent transcription termin-
ation of certain non-coding genes and CUTs from
intergenic regions also involves recruitment of the
exosome to promote transcript degradation (8–10),
raising the possibility of chromatin proximal exosome
*To whom correspondence should be addressed. Tel: +1 301 435 8989; Fax: +1 301 496 5239; Email: firstname.lastname@example.org
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Published online 28 January 2013 Nucleic Acids Research, 2013, Vol. 41, No. 52963–2980
Published by Oxford University Press 2013.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
activity. In these cases, it is not known whether the
exosome associates with chromatin in order to carry out
its surveillance activities. Toward this end, an over-
expressed tagged version of Rrp6 was shown to associate
with chromatin of yeast protein coding genes using whole
Drosophila, it was demonstrated that certain exosome
subunits associate with at least some actively transcribed
genes (12,13) and may be recruited to chromatin through
interaction with RNA polymerase II (Pol II) elongation
factors Spt5 and Spt6 (14). Nevertheless, a high-resolution
genome-wide study of exosome chromatin association has
yet to be performed in any organism.
insulators are DNA–protein complexes that organize
chromatin into independent transcriptional domains. In
Drosophila, there are at least five chromatin insulator
families that can be categorized by the association of par-
ticular DNA-binding proteins. These classes include the C
CCTC-binding factor (CTCF), Suppressor of Hairy-wing
(Su(Hw)), GAGA factor (GAF), Zeste-white 5 (Zw5) and
boundary element-associated factor (BEAF-32) [reviewed
in (15,16)]. For example, the hsp70 genes at 87A7 are
located between the scs and scs0boundary elements,
bound by Zw5 and BEAF-32, respectively, (17,18). Zw5
and BEAF-32 interactwith
interaction may promote chromatin looping observed
between scs and scs0(19). In addition, the Fab-8 insulator
is a well-characterized cis-regulatory region of the
Abdominal-B (Abd-B) locus in the bithorax complex that
harbors binding sites for the zinc-finger DNA-binding
protein CTCF (20,21). CTCF is required for Fab-8 insu-
lator function and looping interactions among insulators,
enhancers and promoters at Abd-B (22–25). Multiple in-
Centrosomal protein 190 (CP190), a zinc-finger and
BTB/POZ domain-containing protein that may play a
global role in chromatin organization (22,26). Since en-
hancers must often activate their target promoters from
long distances, it is likely that insulators act as tethering
sites for chromosomal loops that can constrain enhancer–
promoter interactions and possibly protect a region from
its surrounding chromatin environment.
Depending on their context, chromatin insulators could
either repress or promote transcription based on the
nature of the higher order chromatin interactions to
which they contribute. Recent studies show that several
insulator proteins, particularly CP190 and BEAF-32,
associate with certain transcriptionally active promoters
(27–29). In fact, BEAF-32 appears to be required for tran-
scription at a number of the promoters to which it binds
(28). How insulator proteins are targeted to specific pro-
moters is unknown; however, insulator protein recruit-
ment correlates with specific transcription initiation
patterns (30). The precise role of insulator proteins at
the promoter is still unclear, but these findings suggest
that insulator proteins may regulate transcription in a
more direct manner than previously suspected.
Earlier work suggests that the catalog of insulator-
associated factors and potential regulators is not yet com-
plete. The CP190/CTCF class of insulators interacts with
the genome, chromatin
Argonaute2, which promotes Fab-8 activity as well as in-
sulator-dependent looping at this site (25). Moreover, the
gypsy class of insulator proteins has been shown to phys-
ically interact with the ubiquitin and SUMO ligase Topors
and Lamin, a major component of the nuclear matrix (31).
Recent work also showed the association of Top2 with
gypsy insulator complexes, and this factor appears to be
important for the stability of the Mod(mdg4)2.2 protein
(32). Of particular interest to this study is the finding that
the Rm62 helicase interacts with CP190 in an RNA-
dependent manner, suggesting that RNA may be a com-
ponent of the gypsy insulator complex (33). Finally, the
RNA-binding protein Shep was recently identified as the
first known tissue-specific regulator of gypsy insulator
activity (34). Here, we sought to identify additional
RNA-related chromatin insulator factors and examine
their potential functional significance.
In this study, we identified the exosome as a novel
interactor of CP190 insulator complexes. Genome-wide
profiling of exosome components compared with insulator
proteins by staining of polytene chromosomes as well as
high-resolution ChIP-seq in two different cell lines reveals
highly overlapping genome-wide association profiles of
the exosome with CP190, BEAF-32 and CTCF insulator
proteins. Unexpectedly, we find that the majority of
exosome binding sites are situated at the promoters of
transcribed genes but not throughout gene bodies or 30
ends. Exosome chromatin association correlates with
exosome binding sites are not significantly changed in
response to exosome depletion. Finally, we found that de-
pletion of BEAF-32 or CP190 has no effect on exosome
chromatin association, but reduction of CTCF levels
reduces exosome association with the CTCF-dependent
Mcp and Fab-8 insulators. These results reveal a previ-
ously unknown association between exosome and chroma-
tin insulators throughout the genome.
MATERIALS AND METHODS
Chromatin immunoprecipitation and ChIP-seq
Cells were fixed by addition of 1% formaldehyde to cell
mediafor 10minat R.T.
Formaldehyde was quenched by addition of glycine to
0.125M with gentle agitation for 5min at R.T. Cells
were pelleted at 400 rcf, washed twice in PBS, and resus-
pended in 1ml ice–cold cell lysis buffer (5mM PIPES, pH
8, 85mM KCl, 0.5% NP-40) supplemented with Complete
protease inhibitors (Roche). Nuclei were released by
Dounce homogenization with pestle B and pelleted by
centrifugation at 9190 rcf for 5min at 4?C. Nuclei and
chromatin were further processed as described previously
(35). Immunoprecipitations were performed with 3ul
rabbit a-Rrp6 (14), rabbit a-Rrp40 (36), rabbit a-BEAF-
32 (37), rabbit a-CP190 (25), rabbit a-CTCF (22),
rabbit a-Su(Hw) (37) or rabbit IgG (Santa Cruz Bio-
technology) coupled to rProtein A agarose beads (GE
Healthcare). Primers used are listed in Supplementary
2964 Nucleic Acids Research, 2013,Vol.41, No. 5
Samples for ChIP-seq from input DNA, Rrp6 ChIP and
Rrp40 ChIP were prepared according to the manufac-
turer’s protocol with standard or TruSeq adapters
(Illumina). DNA was sequenced on an Illumina Genome
Analyzer II or HiSeq 2000 at the NIDDK Genomics Core
Facility. Exosome ChIP-seq data are available at Gene
Expression Omnibus (GSE41950).
Amplicons used for dsRNA knockdowns were designed
based on recommendations from the Drosophila RNAi
Screening Center. Templates were PCR amplified from
genomicDNA using primers
promoter sequence (Supplementary Table S9). DsRNAs
were produced by in vitro transcription of PCR templates
using the MEGAscript T7 kit (Ambion) and purified using
NucAway Spin Columns (Ambion). S2 and S3 cells were
grown at 25?C in Shields and Sang M3 Insect medium
(Sigma) supplemented with 0.1% yeast extract, 0.25%
bactopeptone and 10% fetal bovine serum (HyClone).
Transfections of 1?107cells using 2mg of dsRNA were
performed using Cell Line Nucleofector Kit V (Amaxa
Biosystems) transfection reagent using the G-30 protocol.
On day 2, cells were diluted with normal media ?1:5. Five
knockdown efficiency was confirmed by western blotting.
RNA purification and RNA-seq
Total RNA was isolated from S2 and S3 cells using Trizol
Reagent (Invitrogen) using the recommended protocol.
Polyadenylated RNA and ribosomal (rRNA) depleted
(Ambion)and the RiboMinus
RNA-Seq (Invitrogen), respectively. Depletion of rRNA
was ?99% by qPCR measurement of 5S rRNA.
Sequencing libraries were prepared from Poly(A)+and
rRNA depleted RNA samples according to the manufac-
turer’s protocol (Illumina), and sequencing was performed
on an Illumina Genome Analyzer II or HiSeq 2000 at the
NIDDK Genomics Core Facility. Exosome knockdown
RNA-seqdata are available
at Gene Expression
CP190 immunoaffinity purification
CP190 complex immunoaffinity purification was per-
formed from nuclear extracts from 0–24h OR embryos
as described previously (33). Columns were used only
once for western blotting analyses but reused multiple
times for RNase activity assays to conserve material,
which led to slightly altered elution profiles.
RNase activity assay
RNA substrate was synthesized by in vitro transcription
using the pAWG-su(Hw) vector containing the su(Hw)
cDNA. Primersused are
Table S9. The resulting 99nt RNA was end labeled with
[50-32P]pCp by incubation with T4 RNA ligase at 4?C O/N
and purified by running on a 12% polyacrylamide gel (8M
urea, 1x TAE) followed by extraction of RNA bands by
crushing with a motorized pestle and soaking in 0.3M
NaOAc pH 5.2. RNA was ethanol precipitated with
20mg glycogen. RNA substrate was incubated with
buffer alone, Csl4–Flag exosome complexes, or imm-
unoaffinity column fractions in 10mM Tris-HCl pH 8.0,
50mM KCl, 5mM MgCl2and 10mM DTT for 2h at
37?C. Csl4–Flag complexes were purified from transiently
transfected S2 cells as described previously (36). Samples
were then heated to 65?C for 15min, immediately cooled
on ice, and separated on a 15% polyacrylamide gel (8M
urea, 1x TAE) at 15W for 45min. Gels were then exposed
to phosphorimager plates overnight and scanned on a Fuji
Immunofluorescence of polytene chromosomes
Polytene chromosome spreads were prepared essentially as
described previously (38). Rabbit a-Rrp6, rabbit a-Rrp40,
mouse a-BEAF (19) (Developmental Studies Hybridoma
Bank), guinea pig a-CP190 (25), guinea pig a-CTCF
[generated similarly as in (22)], guinea pig a-Su(Hw) (35)
and guinea pig a-Mod(mdg4)2.2 (35) were used for
Western blotting was performed with guinea pig a-CP190,
rabbit a-CTCF, guinea pig a-Mod(mdg4)2.2, mouse
Hybridoma Bank), mouse a-BEAF, rabbit a-Rrp6,
rabbit a-Rrp40, guinea pig a-Rrp4 (36), guinea pig
a-Rrp46 (36), guinea pig a-Csl4 (36) and rat a-Ski6 (14).
Reads were mapped with Bowtie 0.12.7 (40) to the dm3
assembly, excluding chrUextra. Only the best uniquely
mapping reads allowing two mismatches were kept (par-
ameters –best –strata -m1 -n2 –tryhard -k1). Libraries
from technical replicates were merged. Duplicate reads
were collapsed with Picard’s MarkDuplicates (http://
and reads falling in repetitive regions were removed. Peaks
were called with SPP using z.thr=3, window.size=1000,
an FDR of 1% and otherwise default parameters. Similar
results were obtained using the MACS algorithm (41).
Binary heat maps
Supervised hierarchical clustering of overlap by at least
1bp was performed as in (42), using the multi-intersect
program from BEDTools (43) (with the cluster option)
and the binary_heatmap() function from pybedtools (44).
Overlap enrichment heat maps
Data files containing called peaks were downloaded and
converted to BED files (25,45–55). Endogenous siRNA
cluster coordinates were used directly as reported in sup-
plementary tables, or raw reads from Czech et al. and
Fagegaltier et al. were clustered de novo as previously
Nucleic Acids Research,2013, Vol.41, No. 52965
Enrichment scores were calculated as described previ-
ously (25). The full enrichment matrix was hierarchically
clustered using correlation as a distance metric and
complete linkage clustering as implemented in SciPy,
with rows clustered identically as columns. Selected rows
from the full clustered matrix in Supplementary Figure S1
are shown in Figure 4B. For the active regions heatmap,
active regions were first defined as any region bound by
both Pol II and H3K4me3 using data for these factors in
S2 cells from modENCODE
modencode.org/). Genome-wide features used in the full
heat map were then subsetted so that only those features
overlapping an active region by at least 1bp were con-
sidered. Enrichment scores were generated similarly to
the full heat map, except that when features were
shuffled, the new random locations were required to fall
within an active region. The same row/column ordering in
Figure 4B was applied in order to facilitate comparison.
Feature classes [TSSs (1bp transcript start position),
CDSs, introns, 50UTRs and 30UTRs] were extracted
from all annotated isoforms of all annotated genes in
FlyBase release 5.33. Intergenic regions were defined as
the remainder of dm3. Since a ChIP-seq peak can fall in
more than one class, we classified a peak by its highest
priority annotation class, where the priorities from highest
to lowest are TSS, CDS, intron, 5’UTR, 3’UTR and
Each row in the matrix in Figure 5B corresponds to the
genomic region+/? 1kb around each TSS split into 20bp
bins. Reads were extended 3’ to a total length of 200bp to
represent the fragment size. For each column i in the
matrix representing a 20bp-wide genomic location, the
input-normalized value in reads per million mapped
reads (RPMMR) was calculated as (IPi/ IPtotal)?(inputi
/ inputtotal) where IPiand inputiare the numbers of reads
overlapping that region in the IP and input, respectively,
and IPtotaland inputtotalare the total number of mapped
reads, in millions, in the libraries. Active genes were
defined as those with both Pol II and H3K4me3 within
a windowextending 250bp
downstream of the TSS. We defined ambiguous genes as
having either Pol II or H3K4me3 but not both in this
window and inactive genes as having neither Pol II nor
H3K4me3 in this window. Within each of the three
categories, rows are sorted by row means. Line plots
show the column sums of the TSS matrix (Figure 5B) or
the column sums of a similarly constructed poly (A) site
matrix in which each row is centered on the 3’-most co-
ordinate of each isoform.
Note that modENCODE supplies several data sets for
Pol II (accessions 3295 and 329), CTCF (283, 3281 and
913), CP190 (925, 280) and BEAF-32 (922, 274). In the
enrichment analysis heatmap, these data sets were treated
separately. In all other analyses, data sets have been
combined by first concatenating all data sets for a factor
and then merging any features that overlap by at least 1bp
into a single feature.
Hypergeometric overlap tests
To assess the enrichment of insulators and exosome spe-
cifically at active TSSs, we used the hypergeometric
overlap test (56) where n is the total number of
non-redundant annotated isoforms of all active genes; n1
is the number of isoforms with an exosome peak at an
active TSS, n2 is the number of isoforms with an insulator
peak at an active TSS and m is the number of isoforms
with both exosome and insulator peak at an active TSS.
Active TSSs were identified as those with both Pol II and
H3K4me3 overlapping the TSS. For each factor, the
bound active TSSs consisted of the set of active TSSs
that had at least 1bp overlap with at least one binding
site for that factor. Similar results were obtained when
considering the full set of TSSs, regardless of transcrip-
Divergent promoters were identified from coding and non-
coding genes and were defined as a minus-strand TSS
followed by a plus-strand TSS separated by no more
than threshold N bp with no intervening genic sequence
between them. Several different values of N (100, 250,
500bp) were used for analyses.
Gene ontology was assessed using DAVID (57,58) with
the genome as the background gene set and ‘high’ strin-
gency setting for clustering. We considered all genes with a
Rrp6 or Rrp40 peak anywhere in the gene. DAVID scaled
enrichment values are reported.
RNA-seq and differential expression analysis
Libraries were sequenced on both GAII and HiSeq 2000
machines in either single-end and paired-end mode. Due
to small insert sizes, only the first end of the PE libraries
was used. Adapters were clipped using cutadapt 1.0 (59).
Clipped reads were mapped with TopHat 1.4.1 (60) to the
dm3 assembly, excluding chrUextra. Mapped reads were
counted in all annotated coding and non-coding genes in
FlyBase r5.33 using HTSeq in ‘union’ mode, which only
includes reads that map unambiguously to a single gene.
Counts from technical replicates were summed. Counts
from replicates were used in DESeq using ‘local’ fit type
and ‘fit-only’ sharing mode (61). Comparisons used
the ‘per-condition’ method for estimating dispersions.
Differentially expressed genes were considered those with
Analysis of intergenic transcription, snoRNA and
knockdown, reads were counted with HTSeq in 0–500bp
upstream of gene TSSs, all intergenic exosome peaks, and
selected cis-regulatory regions of Abd-B. Subregions that
also overlapped annotated genes were subtracted in order
to focus on changes in intergenic RNA. Regions <100bp
were also removed. Bound upstream regions were defined
as gene-level TSS intersecting an exosome peak. Upstream
and intergenic peak regions were merged such that
overlapping features were considered a single feature.
2966Nucleic Acids Research, 2013,Vol.41, No. 5
considered and classified as being chromatin-associated
based on (62). Putative precursor regions for snoRNA
were defined as the largest intron overlapping each
snoRNAs, which do not have an obvious precursor,
were not considered. Precursors were then merged such
that each would be represented only once in the analysis
even though multiple snoRNAs could be in one precursor,
or both ca-snoRNAs and snoRNAs could come from the
same precursor. For each precursor, the number of pro-
cessed snoRNA-specific reads in the region was subtracted
from the total number of reads in the region in order to
isolate precursor-specific reads. These final precursor read
counts, as well as counts for all mature snoRNAs
including the ‘orphan’ snoRNAs, were examined.
Regions containing zero reads in all RNAseq libraries
were removed. For each rRNA depletion knockdown
experiment, DESeq was run as described above for
RNA-seq, and all regions were reported.
Exosome and RNase activity copurify with chromatin
Given previous evidence that RNA and RNA-binding
proteins associate with chromatin insulators, we sought
to determine whether the exosome physically associates
with CP190 chromatin insulator complexes. Using a
well-established protocol to isolate CP190 insulator com-
ponents and associated factors such as CTCF (22,25,33),
embryonic nuclear extracts were immunoaffinity purified
over control preimmune or a-CP190 antibody columns by
washing and subsequent elution with increasing MgCl2
concentrations (Figure 1A). Eluates were examined for
the presence of insulator proteins as well as exosome sub-
units. Western blotting verifies the presence of CP190 and
Mod(mdg4)2.2 insulator proteins in the a-CP190 eluates
as well as the exosome components Rrp6, Rrp4, Rrp46,
Csl4 and Ski6 but not in preimmune eluates. In contrast, a
negative control protein, HP1a, is not identified as a
specific interactor of CP190 (data not shown). Therefore,
multiple exosome components copurify with CP190 insu-
lator complexes, suggesting that the intact exosome may
be stably associated with CP190 chromatin insulator
complexes in the nucleus.
In order to assess whether physical association between
the exosome and CP190 chromatin insulator complexes is
unopurified CP190 complexes harbor ribonuclease activ-
ity. To this end, a radiolabeled 99nt in vitro transcribed
RNA was used as a substrate in in vitro degradation
assays. As a control, RNA was incubated with exosome
complexes immunopurified from S2 cells stably trans-
Examination of the RNA on a high percentage urea poly-
acrylamide gel indicates extensive degradation of the
RNA as result of incubation with exosome complexes
compared with buffer alone. Incubation of the RNA sub-
strate with preimmune or a-CP190 immunoaffinity
column fractions results in extensive degradation specific-
ally in the 1M a-CP190 elution fraction, while the
majority of RNA substrate remains intact in other frac-
tions tested. Lack of activity in the 250mM CP190
fraction was unexpected but may be due to absence of a
critical factor, presence of a repressor or minor differences
in purification procedures used for western blotting and
RNase activity assays (see Materials and Methods). These
results suggest that CP190 chromatin insulators may as-
sociate with active exosome complexes in vivo.
0.01 0.251 0.01 0.251
a l F
- 4 l s
i e r
i e r
0.01 0.251 M MgCl2
i e r
k r a
Figure 1. CP190 insulator complexes
exosome and copurify with RNase activity. (A) Exosome subunits phys-
ically associate with CP190 insulator complexes. Western blotting of
embryonic nuclear extracts (lane 1, 0.4% of input) bound to a
control preimmune column (lanes 2–4) or a-CP190 column (lanes
5–7) and step-eluted with increasing MgCl2concentrations as indicated.
Note the presence of the IgG light chain (Lc) in both 1M MgCl2
elutions in the a-Rrp4 blot. For CP190 blots, 4% of the eluates were
probed. For all other blots, 96% of the eluates were probed. (B) RNase
activity copurifies with CP190 insulator complexes. Shown are DNA
marker (lane 1), intact in vitro transcribed RNA substrate (lane 2),
RNA substrate after incubation with Csl4–Flag purified control
complex (lane 3), or RNA substrate after incubation with complexes
step-eluted from either preimmune antibody column (lanes 4, 6, 8) or
a-CP190 column (lanes 5, 7, 9) at MgCl2concentrations as indicated.
Samples were run on a 15% PAGE urea denaturing gel and exposed to
a phosphorimager plate.
associate with the nuclear
Nucleic Acids Research,2013, Vol.41, No. 52967
Exosome subunits colocalize with certain chromatin
insulator proteins on polytene chromosomes
Given physical interaction between CP190 and the
exosome, we wishedto
binding profiles of the exosome compared with insulator
proteins. To this end, we performed immunostaining of
highly replicated interphase polytene chromosomes from
third instar larval salivary glands. Wildtype chromosomes
were stained with either a-Rrp6 or a-Rrp40 and costained
for a-CP190, a-BEAF-32, a-CTCF, a-Mod(mdg4)2.2 or
a-Su(Hw) insulator proteins (Figures 2–3). Both Rrp6 and
Rrp40 localize to DAPI interbands, indicative of localiza-
tion to transcribed regions of the genome, similar to a
previous study of the exosome component Ski6 (14).
Since both exosome antibodies were generated in the
same organism, double staining of Rrp6 and Rrp40 was
not possible, but the localization patterns of both proteins
are highly similar. For example, both antibodies intensely
label the actively transcribed ‘gooseneck’ region at cytolo-
gical position 31. Extensive colocalization is observed for
Rrp6 compared with CP190 (Figure 2A) and to a higher
extent with BEAF-32 (Figure 2B). In contrast, at this level
of resolution, Rrp6 does not appear to colocalize consid-
erably withthe insulator
Mod(mdg4)2.2 (Figure 2C and D). Furthermore, no ap-
preciable overlap was observed between Rrp40 and
Su(Hw) (Figure 3). The same results were obtained with
both a-Rrp6 and a-Rrp40 antibodies (data not shown).
These results suggest that the exosome colocalizes with
actively transcribed regions and specific classes of insula-
tor proteins throughout the genome.
High-resolution genome-wide mapping of exosome
subunits by ChIP-seq
In order to obtain a high-resolution map of exosome
ChIP-seq of exosome components in two embryonic cell
lines, S2 and S3 cells. We determined Rrp6 and Rrp40
ChIP-seq profiles using Illumina sequencing and peak
calling using the SPP algorithm at 1% FDR (63).
Greater than 2700 exosome peaks were observed for
Rrp6 or Rrp40 in either S2 or S3 cells (Figure 4A). High
correspondence was observed between Rrp6 and Rrp40 in
the same cell type; 82% of Rrp6 in S2 overlaps with
Rrp40, and 84% of Rrp6 in S3 overlaps with Rrp40.
Similar profiles obtained with antibodies to two distinct
exosome components suggests that the intact exosome
may associate with chromatin at these sites and further-
more verifies the specificity of the antibodies. Antibodies
directed against other exosome components did not suc-
cessfully detect signal on either polytene chromosomes or
by ChIP or were not tested due to limited availability
(data not shown). We subsequently refer to either
specific subunit profiles or a common set of ‘exosome’
binding sites for a particular cell type at which both
Rrp6 and Rrp40 are bound.
We next examined the overlap of exosome components
with that of insulator proteins and other chromatin-
associated factors or other features previously profiled in
S2 cells. We calculated enrichment scores for two-way
overlaps between all factors based on comparison with
random shuffling of sites within the same chromosome.
As expected, Rrp6 and Rrp40 profiles display among the
highest levels ofenrichment
compared with all other tested factors (Figure 4B).
Consistent with polytene chromosome staining, statistic-
ally significant levels of enrichment are observed for both
exosome factors with CP190 and BEAF-32 (78% and
84% of exosome sites, respectively, Supplementary Table
S1). Substantial enrichment is also observed with CTCF
(41%), whereas little to no enrichment is observed
between either exosome component compared with
Su(Hw) and Mod(mdg4)2.2 (20% and 3%, respectively).
Unsupervised hierarchical clustering was performed in
order to group factors based on overall similarity of en-
analysis further indicates correspondence of exosome
components with the CP190 and BEAF-32 insulator pro-
teins. Notably, we identified high levels of overlap enrich-
ment as well as hierarchical clustering with Chromator/
Chriz, a factor recently implicated in boundary formation
in a genome-wide chromatin conformation study (64). In
summary, exosome colocalizes significantly with specific
insulator proteins on a genome-wide level.
Exosome recruitment correlates with active transcription
Our comparative analyses also revealed extensive overlap
of exosome components
factors and marks indicative of active transcription.
Strong enrichment between Rrp6 and Rrp40 is observed
with factors such as RNA Pol II and H3K4me3 (Figure
4B). These results were not unexpected given previous
work indicating physical interaction between exosome
and transcription elongation factors (14). Based on this
work as well as another study suggesting that the Rrp4
exosome component associates with gene bodies (12), we
anticipated that exosome peaks would be observed at the
middle and/or 30-end of genes. In striking contrast, we
found that the distribution of exosome peaks with
respect to genes is heavily biased to the TSS and not
gene bodies (Figures 4C and D). Plots of average Rrp6
or Rrp40 signal in S2 cells normalized to input within 1kb
of an annotated TSS reveal a strong enrichment of
exosome components slightly upstream of TSSs, whereas
little to no enrichment is observed in the vicinity of
polyadenylation sites (Figure 4E). Similar TSS enrichment
is also observed for exosome binding in S3 cells (data not
Based on preferential binding to the TSS and the fact
that the exosome is the major RNA degradation machin-
ery, we hypothesized that exosome recruitment to chro-
matin may occur in a transcription-dependent manner.
When exosome signal in S2 cells is examined at TSSs
separated into transcriptionally active or inactive based
on the presence of Pol II and H3K4me3, enrichment is
observed only for active TSSs (Figure 5A). In order to
explore the relationship of binding with gene expression
level in more detail, matrices were generated for input
normalized ChIP signal in 100 bins+/? 1kb of annotated
TSSs (Figure 5B). As expected, a clear bias toward
2968 Nucleic Acids Research, 2013,Vol.41, No. 5
Figure 2. Exosome colocalizes extensively with CP190 and BEAF-32 insulator proteins on polytene chromosomes. Rabbit a-Rrp6 was detected with
a-rabbit conjugated Alexa-594 (red). Guinea pig a-CP190 (A), mouse a-BEAF-32 (B), guinea pig a-CTCF (C) or guinea pig a-Mod(mdg4)2.2
(D) were detected with a-guinea pig or a-mouse conjugated Alexa-488 (green) as appropriate. DAPI-stained DNA is shown in blue in the merged
image (right). White arrowheads point to the transcriptionally active ‘gooseneck’ region at cytological position 31. Yellow signal in the merged
images indicates a high degree of overlap of Rrp6 with CP190 and BEAF-32.
Nucleic Acids Research,2013, Vol.41, No. 52969
expressed genes is observed for exosome ChIP signal at the
TSS. Strong enrichment is seen for exosome at a set of
promoters that display promoter-proximal enriched Pol II
(PPEP, Figure 4B), suggesting that exosome preferentially
associates with genes with paused Pol II. Taken together,
we conclude that exosome chromatin association correl-
ates with active transcription.
Overlap of exosome with insulator proteins at specific
transcriptionally active sites
Given that exosome and certain insulator proteins are
enriched at transcriptionally active regions and TSSs, we
wanted to verify that enrichment of overlap observed
throughout the genome is not due simply to a general
preference for active regions. We first assessed the
overlap of exosome with insulator proteins at genomic
sites corresponding to active TSS, inactive/ambiguous
TSS and non-TSS sites in S2 cells (Supplementary
Figure S2, Supplementary Table S1). As expected, the
majority of sites at which exosome and insulator
proteins overlap corresponds to active TSSs. However, it
is apparent that many active TSSs are not co-occupied by
exosome and insulator proteins. Therefore, we calculated
the statistical probabilities of two-way overlap between
exosome and CP190, BEAF-32 or CTCF sites by
hypergeometric tests considering only active TSSs of all
annotated gene isoforms and indeed observed highly sig-
nificant overlap over expectation (P<3.7?10?110for
each comparison). We also determined that enrichment
scores for two-way overlaps between exosome and insula-
tor proteins are similar when considering only actively
transcribed regions ofthe
Figure S3) compared to when transcriptionally inert
regions of the genome are also considered (Figure 4B).
We conclude that colocalization of exosome and CP190,
BEAF-32 and CTCF occurs at specific regions of the
genome and is not solely due to active transcription status.
Exosome preferentially associates with divergently
transcribed genes as well as specific functional gene
classes similar to BEAF-32
Like BEAF-32, as well as CP190 and CTCF, exosome
binding is enriched at TSS of divergently transcribed
genes. Previous work showed that BEAF-32 preferentially
associates with the promoters of ‘head-to-head’ or diver-
gent gene pairs (28). Thus, we performed a series of
Fisher’s Exact tests to examine whether exosome-bound
and insulator-bound regions associate preferentially with
divergent versus non-divergent TSSs. We confirmed the
enrichment of BEAF-32 at divergent genes using different
merge Su(Hw) Rrp40
Figure 3. Exosome chromatin association is not globally affected in CTCFy+2mutants. Localization of Rrp40 (red) and the Su(Hw) insulator protein
(green) on wildtype (top) or CTCFy+2mutant (bottom) larval salivary gland polytene chromosomes. Rabbit a-Rrp40 was detected with a-rabbit
conjugated Alexa-594. Guinea pig a-Su(Hw) was detected with a-guinea pig conjugated Alexa-488. DAPI-stained DNA is shown in blue in the
merged image (right). White arrowheads point to the transcriptionally active ‘gooseneck’ region at cytological position 31.
2970 Nucleic Acids Research, 2013,Vol.41, No. 5
Figure 4. ChIP-seq analysis of exosome components in two embryonic cell lines. (A) Binary heat map of Rrp6 and Rrp40 binding sites in S2 and S3
cells ordered by supervised hierarchical clustering. Each column represents a single genomic location, and a black mark in a row represents the
presence of a particular factor at that site. Total peak counts are indicated for each factor (right). (B) Heat map of log2enrichment scores for
pairwise comparisons of binding sites for Rrp6, Rrp40 and exosome with additional data sets in S2 cells. Color scale corresponding to enrichment
value is indicated (bottom). Positive values indicate significant enrichment whereas negative values indicate significant negative enrichment. Self–self
Nucleic Acids Research,2013, Vol.41, No. 52971
threshold distances and also observed enrichment for
CP190, CTCF and exosome (Supplementary Figure S4).
In contrast, Su(Hw) and Mod(mdg4)2.2 display minimal
or negative enrichment for divergent promoters, respect-
ively. Interestingly, we found that divergent genes display
notably higher expression than non-divergent genes
(Supplementary Figure S5). Indeed, Pol II is also highly
enriched at divergent TSSs (Supplementary Figure S4),
raising the possibility that enrichment at divergent pro-
moters may be at least partially due to elevated transcrip-
tional activity at these regions.
In an attempt to better understand the biological
significance of exosome chromatin association, we per-
formed gene ontology analysis to identify functional
classes of genes enriched for exosome binding. DAVID
analysis (57) on exosome binding sites reveals extensive
Figure 4. Continued
comparisons are indicated in gray, and pairwise comparisons that are not statistically significant (P>0.001) are indicated in white. Numbers to the
right of each row indicate the total number of features in each data set, and the number of sites overlapping with exosome are indicated in
parentheses. Data sets in bold were generated in this study. Data generated by modENCODE consortium is marked with an asterisk. Other data
are from studies as indicated (25,48–55). The ca-snoRNA data set was defined in embryos (62). Full heat map with hierarchical clustering is shown in
Supplementary Figure S1. (C) Classification of Rrp6 and Rrp40 ChIP-seq peaks in S2 and S3 cells with respect to annotated genes. Number of sites
and percentage of total in parentheses corresponding to TSS, transcription start site; CDS, coding sequence; 50UTR, 50untranslated region; 30UTR,
30untranslated region. See methods for classification hierarchy of overlapping categories. (D) Screenshots of input, Rrp6 and Rrp40 ChIP-seq signals
at the polo, CycB and bel loci in S3 cells. The bottom of each scale bar indicates 0. Black bars indicate primer sets used for directed ChIP in
Figure 6B. (E) Genome-wide profiles of Rrp6 and Rrp40 S2 input normalized ChIP-seq tag density around transcription start sites (orange) or poly
(A) sites (green) for all genes.
Figure 5. Genome-wide exosome chromatin association correlates strongly with active transcription. (A) Exosome associates preferentially with
active promoters. Profile of S2 Rrp6 and Rrp40 input normalized ChIP-seq tag density around TSSs associated (red) or not associated (blue) with
H3K4me3 and Pol II 250bp upstream or 750bp downstream of the TSS. (B) Matrices of Rrp6 and Rrp40 S2 ChIP signal across all annotated TSSs
+/? 1kb. Each row corresponds to an annotated gene, and red lines indicate grouping of rows into active, ambiguous or inactive TSSs. Rows are
ordered by mean signal from highest to lowest within each subgroup. Active genes were defined as genes with Pol II and H3K4me3 within 250bp
upstream or 750bp downstream of the TSS, ambiguous genes had one of these marks within this window, and inactive genes had neither mark in the
same window. Signal intensity is shown in input-normalized enriched RPMMR. Color scale corresponding to enrichment is indicated (right).
2972Nucleic Acids Research, 2013,Vol.41, No. 5
overrepresentation of cell cycle and ribosomal protein
genes in both S2 and S3 cells (Supplementary Table S2).
Consistent with our finding that exosome colocalizes with
BEAF-32 genome-wide, BEAF-32 has previously been
shown to associate with cell cycle genes (29). Overall,
these results demonstrate that the genome-wide exosome
chromatin binding profile shares features with that of
CP190, BEAF-32 and CTCF insulator proteins.
Lack of stabilized transcripts at exosome-bound regions
We wished to address whether chromatin regions bound
by the exosome correspond directly to sites of active RNA
degradation. A previous study in human cells revealed the
stabilization of an ?1.5kb polyadenlyated divergent tran-
script in the opposite orientation at promoters of many
genes (6). In order to determine whether a similar phe-
nomenon occurs in Drosophila, we performed dsRNA
knockdown of GFP as a control, as well as Rrp6 and
Rrp40 in S3 cells, harvested total RNA, and performed
rRNA depletion followed by non-directional RNA-seq.
Figure S6), although residual protein could be detected
by ChIP (data not shown). Visual inspection of our
RNA-seq data did not uncover obvious evidence of
upstream divergent transcription in either control or
exosome knockdown libraries (data not shown). RNA-
seq libraries sequenced to a depth of over 20M reads
from two biological replicates each of control versus
knockdown cell lines were analysed for differential expres-
sion specifically in the 500bp intergenic region upstream
of exosome-bound TSSs using the DESeq algorithm, and
no statistically significant upregulation of these exosome-
bound upstream regions was observed (Supplementary
Tables S3 and S4).
As an alternate approach, we also examined distribu-
tions of genome-wide RNA fold changes upon exosome
subunit knockdown at sites upstream of TSSs either
bound or unbound by exosome. For exosome-bound
upstream regions, an approximately equal number of
sites show an increase or decrease of RNA-seq reads in
Rrp6 or Rrp40 depleted cells compared with control cells
(Supplementary Figure S7). In contrast, regions upstream
of TSSs unbound by exosome actually display slightly
increased median expression in Rrp6 depleted cells.
Furthermore, no statistically significant changes in expres-
sion were detected at intergenic exosome peaks by DESeq
analysis (Supplementary Tables S3 and S4). Similar results
were obtained with oligo-dT selected RNA-seq libraries
from either S2 or S3 cells (data not shown). Therefore,
we conclude that exosome chromatin recruitment does
not necessarily correspond to regions of transcript degrad-
ation, and we find no clear evidence for extensive diver-
gent transcription even when the major cellular RNA
degradation machinery is depleted.
maturation in yeast, we examined whether depletion of
exosome subunits leads to stabilization of snoRNA or
of snoRNAs are derived from introns of protein coding
or non-coding genes (65). We used DESeq expression
analysis to examine mature snoRNAs and intron regions
knockdown versus control S3 libraries. We were unable
to detect evidence for genome-wide stabilization of mature
snoRNA or precursors (Supplementary Tables S3 and S4);
however, we did observe accumulation of two likely
snoRNA precursors with 30extensions when either Rrp6
or Rrp40 is knocked down (Supplementary Figure S8).
Both of these snoRNA genes are bound by exosome, in
addition to 61 of 249 annotated snoRNAs genome-wide
considering either S2 or S3 cells. Two-way overlap
between exosome and snoRNA genes is enriched over ran-
dom expectation (Figure 4B), and interestingly, exosome
preferentially associates with a class of chromatin-
associated snoRNAs (ca-snoRNAs, 21 of 40) suggested
to be involved in maintaining open chromatin structure
(62). These results suggest that the Drosophila exosome
machinery affects processing of at least a subset of
snoRNA transcripts and might perform this function in
association with chromatin.
Exosome binding at the TSS does not correlate with
exosome-dependent transcript regulation genome-wide
We wondered whether exosome may be recruited to genes
to enhance transcript degradation. In order to address this
possibility, we performed mRNA expression profiling
using control, Rrp6, and/or Rrp40 knockdown RNA-seq
libraries from S2 and S3 cells using the DESeq algorithm,
this time at the gene-level for annotated genes. Similar to a
previous study (66), we identified hundreds of transcripts
with altered expression profiles upon knockdown, the
majority of which are stabilized upon exosome depletion
(Supplementary Tables S5–S7). Strikingly, we found that
transcripts that increase in exosome knockdowns are
actually less likely to harbor an exosome peak at the
TSS compared with transcripts unaffected by knockdown
(Supplementary Table S8). Negative enrichment is likely
due to the fact that exosome associates with higher expres-
sion genes whereas transcript stabilization is biased
Figure S9). Hence, no obvious genome-wide correlation
is apparent between exosome-dependent effects on gene
expression and exosome chromatin association.
Curiously, depletion of exosome in S3 cells led to sta-
bilization of an aberrant transcript at the Abd-B locus.
The Abd-B gene is expressed in posterior cells later in em-
bryonic development and is regulated by a host of down-
CTCF-dependent boundary elements Mcp and Fab-8.
Upon close visual inspection, we noted stabilization of
an ?20kb polyadenylated transcript extending between
downstream of the RE isoform promoter to beyond the
RA isoform promoter particularly in Rrp6 but also in
Rrp40 depleted cells (Supplementary Figure S10). We
verified by directional RT-PCR that the transcript is
transcribed in the same orientation as the Abd-B gene
(data not shown), which is normally expressed in S3 but
not S2 cells. Transcription in this region was not observed
in exosome depleted S2 cells, suggesting that the transcript
Nucleic Acids Research,2013, Vol.41, No. 52973
requires the same factors needed for normal Abd-B expres-
sion. We also observed evidence for expression of a similar
transcript during the 4–10h window of embryonic devel-
opment based on modEncode oligo-dT selected RNA-seq
data from whole embryos (Supplementary Figure S10).
Aberrant expression of this nature was not observed
extensively throughout the genome, and the functional
or mechanistic significance of this stabilized transcript is
Exosome subunits are essential in Drosophila
To date, specific mutations in exosome subunit genes have
not been reported in Drosophila, precluding in vivo
analyses of exosome function in the context of organismal
development. We obtained a panel of UAS-inducible
transgenic double-stranded RNA (dsRNA) hairpin lines
directed against exosome subunit genes from the Vienna
Drosophila RNAi Center and tested the effects of
knockdowns using various Gal4-drivers on general devel-
opment and adult viability. As expected, expression of
RNAi hairpins against Dis3, Mtr3, Rrp6, Rrp42 or Ski6
using the strong ubiquitous Act5C::Gal4 driver results in
adult lethality (Table 1), indicating that individual
exosome subunits are essential for viability and are
non-redundant. We additionally tested RNAi hairpins in
limited subsets of tissue and observed necrosis of pre-
sumptive head tissue in pupae when driving with the
GMR::Gal4 eye-restricted driver, although this phenotype
was not fully penetrant or observed with all knockdown
lines. Furthermore, driving of all hairpins using the ves-
tigial wing margin enhancer vgM::Gal4 resulted in
necrosis and severe wing blistering as well as loss or dis-
rupted arrangement of scutellar bristles. Based on these
results, we were unable to utilize exosome hairpin lines
to assay insulator-dependent
Nevertheless, these findings confirm the essential role of
the exosome in general fly development, similar to the
insulator proteins CP190 (26) and CTCF (22,23).
CTCF is required for exosome recruitment to certain
sites including Fab-8
Given physical association between the exosome and
genome-wide overlap, we next examined whether insulator
proteins are required for exosome chromatin association.
In order to deplete insulator proteins, S3 cells were trans-
fected with dsRNAs to GFP as a control, BEAF-32,
CP190 or CTCF. Western blotting verified highly efficient
knockdown of insulator proteins but no change in levels
of the exosome components Rrp6 or Rrp40 (Figure 6A).
as wellas extensive
Directed ChIP followed by quantitative PCR was per-
formed for a variety of well-characterized chromatin insu-
lator sites, such as scs, scs0, Mcp and Fab-8 as well as
several non-divergent cell cycle gene promoters strongly
enriched for exosome binding. We also examined the
highly transcribed RpL32 coding region, as a control site.
We first examined the chromatin association profiles of
Rrp6 and Rrp40 compared with that of insulator proteins
in control cells. In GFP-treated cells, Rrp6 associates with
all regions except RpL32 substantially over the IgG
control (Figure 6B). For Rrp40, signal is strongest at pro-
moters, lower at Fab-8 and scs0, and essentially at back-
ground levels at scs and Mcp. The BEAF-32 insulator
protein is also very highly enriched at these promoter
regions as well as its characterized binding site scs0. We
note that the scs0insulator overlaps the aurora promoter,
which is also part of a divergently transcribed gene pair.
However, BEAF does not associate with scs, Mcp or
Fab-8 insulators. In contrast, CP190 and CTCF insulator
binding sites Mcp and Fab-8 and to a lesser extent with
certain promoters. Therefore, exosome subunit binding
partially mirrors that of insulator proteins at these insula-
tor and promoter sites.
As a control for knockdown efficiency, we next verified
that BEAF-32, CP190 and CTCF knockdowns reduce
chromatin association of the respective target protein at
all binding sites. We thus examined interdependence of
insulator proteins to their respective binding sites.
BEAF-32 is not significantly affected by knockdown of
either CP190 or CTCF at scs0or scs insulator sites or at
the polo, CycBor bel
Furthermore, CP190 chromatin association at these sites
is unaffected by BEAF-32 depletion, consistent with a
recent study showing independence of BEAF-32 and
CP190 binding despite extensive genome-wide overlap
(67). Confirming previous work, CP190 ChIP is reduced
in CTCF depleted cells at the insulator sites Mcp and
Fab-8, (25) but is mainly unaffected at the polo, CycB
and bel promoters (Figure 6B). Similar to a previous
study (67), CTCF binding is unaffected by either
BEAF-32 or CP190 depletion at all sites tested including
the Fab-8 insulator, which contrasts with dependence on
CP190 previously identified in S2 cells (25). This result
may be due to differences in cell type or expression state
of the Abd-B locus. Interestingly, Rrp6 chromatin associ-
ation is substantially reduced at Fab-8 and at Mcp and
weakly reduced at the CycB promoter uniquely in CTCF
depleted cells, whereas binding to all other loci tested
Table 1. Summary of phenotypes observed for exosome RNAi hairpin lines using indicated Gal4 drivers
VDRC stock no.Gene Act5C::Gal4 vgM::Gal4GMR::Gal4
Blistered wings, scutellar bristles missing
Blistered wings, scutellar bristles missing
Blistered wings, scutellar bristles missing
Blistered wings, scutellar bristles missing
Blistered wings, scutellar bristles missing
50% show necrosis of presumptive head tissue at pupal stage
20% show necrosis of presumptive head tissue at pupal stage
2974 Nucleic Acids Research, 2013,Vol.41, No. 5
Figure 6. CTCF is specifically required for exosome chromatin association at the Fab-8 insulator. (A) Western blotting of lysates from S3 cells
transfected with GFP (lane 1), BEAF-32 (lane 2), CP190 (lane 3) or CTCF (lane 4) dsRNA. (B) S3 cells transfected with GFP (black), BEAF-32
(dark gray), CP190 (gray) or CTCF (light gray) dsRNA were subjected to ChIP using a-BEAF-32, a-CP190, a-CTCF, a-Rrp6, a-Rrp40 or control
rabbit IgG as indicated. Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of
quadruplicate PCR measurements. Similar results were obtained for independent biological replicates.
Nucleic Acids Research,2013, Vol.41, No. 52975
(Figure 6B). Similarly, Rrp40 ChIP signal is specifically
reduced at Fab-8 in CTCF depleted cells.
In order to determine whether CTCF plays a genome-
wide role in recruitment of the exosome to chromatin, we
examined Rrp40 localization in polytene chromosomes of
wildtype compared with CTCFy+2mutants. This mutant
produces little or no CTCF protein (22) and displays
strongly reduced Abd-B looping interactions (25). We
found no significant differences at this level of resolution
(Figure 3), and Rrp6 was similarly unaffected (data not
shown). These results indicate dependence on CTCF for
exosome recruitment to the Mcp and Fab-8 insulators and
possibly the CycB promoter or other untested sites. We
were unable to assess whether insulator protein chromatin
association is dependent on exosome components using
thedepletion strategy because
knockdown could not be achieved in order to fully
remove Rrp6 or Rrp40 from chromatin (data not
shown). Our results indicate that the exosome is at least
partially dependent on CTCF for chromatin association.
Here, we present the first high-resolution map of exosome
chromatin association in any organism. In contrast to
expectations based on the prior literature, we find that
exosome binds mainly promoters but not other genic
regions of actively transcribed genes. Our results support
the conclusion that exosome associates with chromatin in
a transcription-dependent manner; however, exosome is
not recruited to all actively transcribed genes. At the
genome-wide level, exosome associates extensively with
the CP190, BEAF-32 and CTCF insulator proteins. In
support of this finding, our results uncover a previously
unknown physical association between exosome and chro-
matin insulators. At the Mcp and Fab-8 insulators,
exosome recruitment is reduced by CTCF depletion.
However, at most other sites tested, insulator proteins
are dispensable for exosome chromatin association. Our
results provide new insights into potential functions of the
nuclear exosome on chromatin.
Genome-wide profiling of exosome chromatin association
physically with CP190 insulator complexes, and both
exosome and specific insulator factors colocalize exten-
sively throughout the genome. We found that exosome
copurifies with CP190 insulator complexes isolated from
embryonic nuclear extracts. Since CP190 is a component
of multiple chromatin insulator complexes, we relied on
parallel genome-wide colocalization approaches in a
variety of cell types in order to ascertain the specificity
of the interaction. Comparison of genome-wide profiles
of exosome and insulator proteins on larval salivary
gland polytene chromosomes revealed extensive overlap
between exosome and the insulator proteins CP190 and
BEAF-32, but low correspondence with CTCF, Su(Hw)
and Mod(mdg4)2.2. Similarly, high-resolution ChIP-seq
profiling in S2 cells confirmed the specific overlap of
exosome with CP190 and BEAF-32 and was also able to
detect significant overlap with CTCF that was not
apparent in polytene chromosomes. Differential detection
of the positive correlation between exosome and CTCF
using the two techniques could either be due to differences
in cell type or developmental stage examined, or alterna-
tively, the higher resolution of the ChIP-seq method
compared with the cytological approach. We analysed
our ChIP-seq data in greater detail in order to take ad-
vantage of genome-wide profiling data of chromatin-
associated factors in the same cell type compiled by
other studies and the modENCODE consortium.
We found that exosome colocalizes with CP190,
BEAF-32 and CTCF insulator proteins at a specific
subset of active promoters. It is currently unclear how
this specificity of recruitment is achieved. Previous work
in Drosophila showed that exosome interacts physically
with transcription elongation factors and colocalizes
extensively with these factors on polytene chromosomes
(14). Another study demonstrated
epitope tagged Rrp4 and Rrp6 can crosslink to the
30-end of several genes tested, in support of a cot-
ranscriptional RNA surveillance model (12). However,
our high-resolution ChIP-seq data clearly shows that
exosome recruitment to genes occurs in sharp peaks pref-
erentially at promoters compared with coding regions.
Likewise, localized binding is also observed for CP190,
BEAF-32 and CTCF to promoters.
Importantly, overlap between exosome and insulator
proteins is also observed at well-characterized insulator
sequences. Exosome ChIP signal is apparent at many
known CP190, CTCF or BEAF-32-dependent insulator
sites such as scs, scs0, Mcp and Fab-8, and these were
further confirmed by directed ChIP. Despite extensive
overlap with CP190, BEAF-32 and CTCF insulator
proteins, we found that only CTCF is required for
exosome recruitment to any sites examined in this study.
Depletion of CTCF reduces exosome recruitment to both
Mcp and Fab-8 whereas other insulator proteins are dis-
pensable. Since reduction of CTCF levels reduces Abd-B
expression (23,25), it is possible that reduced exosome
chromatin association is a consequence of a change in
transcription state in the general vicinity. Finally, con-
sidering that insulator proteins do not appear to be
required for exosome association with other sites tested
including co-occupied promoters, additional alternate
mechanisms must exist to recruit exosome to chromatin.
Exosome chromatin association is dependent on active
Our results suggest that exosome is recruited to chromatin
in a transcription-dependent manner. Previous work
showed that exosome is recruited to heat shock genes
upon transcriptional induction (14). Likewise, we found
that exosome associates preferentially with active over
inactive promoters. These findings would be consistent
with an RNA-based mechanism of recruitment to chro-
matin. However, specific association of exosome with the
promoter and not gene bodies implies that exosome is not
associated with an elongating transcript since its ChIP
2976Nucleic Acids Research, 2013,Vol.41, No. 5
signal would likely extend further 30into the gene as has
been observed for a variety of RNA-binding proteins
(68,69). If exosome is indeed recruited through direct
RNA interaction, nascent RNAs may only be accessible
to the exosome during a brief window, such as prior to
capping, before assembly into protein-rich ribonucleo-
particles. However, even if recruited to a transcript
cotranscriptionally, exosome may not be able to load on
the majority of transcripts for degradation since the 30-end
would not be accessible. Another possibility is that
exosome specifically recognizes short RNAs associated
with stalled Pol II. Finally, exosome recruitment could
depend on assembly of the transcriptional apparatus but
not the RNA itself. In this case, the full or partially
formed exosome complex could be poised for degradation
at promoters but not actively engaged.
Stabilization of transcripts as a result of
We were unable to obtain evidence for substantial tran-
script stabilization corresponding to regions to which
exosome associates with chromatin. We did observe
exosome at a subset of snoRNA genes, but we only
observed a few cases of snoRNA precursor accumulation
in exosome knockdowns. Given the strong enrichment of
exosome at the TSS and previous reports that divergent
transcription is stabilized at human and yeast promoters
in exosome depleted cells (6,70,71), we looked for evidence
of stabilized transcripts using both oligo-dT selected and
knockdown cells. However, we found no evidence for di-
vergent transcription upstream of annotated promoters
regardless of exosome depletion, consistent with a
previous study of uni-directional genome-wide transcrip-
tion initiation in Drosophila (72). One possibility is that
divergent upstream transcription does not occur in
Drosophila and bidirectional transcription from a single
promoter may be specific to certain species (73). Other
purely technical possibilities are that our RNA-seq
libraries were of insufficient depth or failed to capture
aberrant transcription or altered stability since a conven-
tional size selection step was used for sequencing, which in
effect constrains the lower limit of captured RNA to 80nt.
Perhaps development and use of specialized cloning proto-
cols could successfully identify aberrant transcripts or
mild effects on transcript stabilization.
In order to test the possibility that exosome is recruited
to chromatin in order to increase the efficiency of RNA
surveillance, we also looked for evidence of stabilization
of transcripts in exosome knockdown cells at genes at
which exosome is associated at the promoter. We note
that our knockdowns could not fully remove exosome
from chromatin; therefore, sufficient depletion may not
have been achieved in order to observe an effect on
target RNAs. We did observe stabilization of many tran-
scripts in exosome depleted cells; however these genes do
not generally correspond to exosome binding sites. In fact,
we observed a negative relationship between the two data
sets likely because exosome tends to be recruited to more
highly transcribed genes whereas the transcripts stabilized
libraries of exosome
in exosome knockdown cells tend to be lowly expressed in
wildtype cells. Although we cannot rule out that cot-
ranscriptional recruitment is a mechanism to promote ef-
ficient RNA surveillance on a subset of genes, this does
not appear to be a widespread or efficient mechanism.
Another possibility is that recruitment of exosome to
chromatin poises the degradation machinery for action
under specific yet unknown conditions or to degrade tran-
scripts synthesized in trans to binding sites.
Although not a focus of our study, our expression
analysis in exosome subunit depleted cell lines revealed
mainly stabilization of low expression transcripts. Our
methodically depleted each individual exosome subunit
in S2 cells and performed gene expression profiling by
microarray (66). In that study, substantially varying ex-
pression profiles were obtained with individual subunit
knockdowns, supporting a previously suggested model
that the Drosophila exosome functions not as a singular
degradation machine but as independently functioning,
expression profiling of conditional exosome mutants in
Arabidopsis also detected limited subunit-specific effects
on transcript levels (74). We did observe markedly differ-
ent expression profiles between Rrp6 and Rrp40 knock-
downs; however, this was likely due to arrest of cell
proliferation in Rrp6 but not Rrp40 depleted cells. Our
results are consistent with cell cycle arrest specifically
reported (36), and these effects substantially confound
our expression analyses. Intriguingly, we did find that
both exosome subunits preferentially associate with pro-
moters of cell cycle genes, suggesting that exosome could
play a direct role in regulation of cell cycle gene transcript
regulation. It would be interesting to examine exosome
chromatin association at different stages of the cell cycle
given that expression of many cell cycle regulators are
tightly controlled and regulated in a cell cycle-dependent
a previous study,which
Functional relevance of exosome–insulator complex
Our findings raise the possibility that exosome contributes
to insulator activity or regulation, perhaps through an
RNA-based mechanism. RNA may contribute to higher
order insulator-dependent interactions and has been
postulated to be an important component of the gypsy
insulator complex in Drosophila (33,34) and the CTCF/
cohesin insulator complex in mammals (75). Thus associ-
ation of exosome with insulator complexes could regulate
the abundance or activity of insulator-associated RNAs,
which in turn affect RNA-mediated interactions between
insulator complexes and factors capable of regulating in-
sulator activity. A feasible scenario is that exosome regu-
lates expression of the Abd-B locus through CTCF-
dependent recruitment to the Mcp and Fab-8 insulators.
Thus far, no specific RNA has yet been implicated in in-
sulator activity at Abd-B. However, a complex array of
intergenic transcripts arising from the cis-regulatory
region of Abd-B has been shown to regulate expression
Nucleic Acids Research,2013, Vol.41, No. 5 2977
Therefore, exosome surveillance and degradation activity
may be particularly important at this complicated gene
exosome mutants should help elucidate the precise role
of exosome in chromatin insulator activity.
the downstreamhomeoticgene abd-A (76–78).
Supplementary Data are available at NAR Online:
Supplementary Tables 1–9 and Supplementary Figures
We would like to thank E. Andrulis for generously
providing exosome antibodies and the Csl4-Flag cell
line, V. Corces for a-BEAF-32 and a-CTCF antibodies,
and E. Lai for fly lines. We also thank J. Zhu and
members of the Lei laboratory for critical reading of the
Funding for open access charge: Intramural Program of
the National Institute of Diabetes and Digestive and
Kidney Diseases [DK015602-05 to E.L.].
Conflict of interest statement. None declared.
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