LETTER

doi:10.1038/nature10492

Chromatin-associated RNA interference components
contribute to transcriptional regulation in Drosophila
Filippo M. Cernilogar1,2{*, Maria Cristina Onorati3*, Greg O. Kothe4, A. Maxwell Burroughs5, Krishna Mohan Parsi1,2,
Achim Breiling6, Federica Lo Sardo1, Alka Saxena5, Keita Miyoshi7, Haruhiko Siomi7, Mikiko C. Siomi7,8, Piero Carninci5,David S. Gilmour4, Davide F. V. Corona3 & Valerio Orlando1

RNA interference (RNAi) pathways have evolved as important
modulators of gene expression that operate in the cytoplasm bydegrading RNA target molecules through the activity of short (21–30 nucleotide) RNAs1–6. RNAi components have been reported tohave a role in the nucleus, as they are involved in epigenetic regulationand heterochromatin formation7–10. However, although RNAimediated post-transcriptional gene silencing is well documented,
the mechanisms of RNAi-mediated transcriptional gene silencing
and, in particular, the role of RNAi components in chromatin
dynamics, especially in animal multicellular organisms, are elusive.
Here we show that the key RNAi components Dicer 2 (DCR2) and
Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact
with the core transcription machinery. Notably, loss of function of
DCR2 or AGO2 showed that transcriptional defects are accompaniedby the perturbation of RNA polymerase II positioning on promoters.
Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, aswell as missense mutations that compromise the RNAi activity,
impaired the global dynamics of RNA polymerase II. Finally, the deep
sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq)
revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and othergenetic loci on both the sense and antisense DNA strands, but with astrong bias for the antisense strand, particularly after heat shock.
Taken together, our results show that DCR2 and AGO2 are globallyassociated with transcriptionally active loci and may have a pivotal
role in shaping the transcriptome by controlling the processivity of
RNA polymerase II.
Accumulating evidence indicates that RNAi components and small
RNAs function in the nucleus to control heterochromatin formation,
as well as repeat-induced gene silencing and transposable element
mobilization7–10. However, the global association of RNAi components
with chromatin and their role in transcriptional regulation remains tobe elucidated. To investigate a role for RNAi in the chromatin
dynamics of a multicellular organism and, possibly, in transcriptional
gene silencing, we first determined whether any of the key RNAicomponents are present in the nucleus of Drosophila cells. Cellular
fractionation of embryonic tissue culture cells (S2 cells) showed thatthe microRNA-processing factors DCR1 and AGO1, as well the RNAicomponent AGO2, are equally distributed between the cytoplasm andthe nucleus (Supplementary Fig. 1a). By contrast, the RNAi protein
DCR2 is greatly enriched in the nuclear fraction (Supplementary
Fig. 1a). To evaluate the association of RNAi components with thedifferent nuclear compartments, we carried out chromatin fractionation experiments11 (Supplementary Fig. 1b, c). A substantial amount of

DCR2 and AGO2 was detected in chromatin fractions, together with
RNA polymerase II (RNA Pol II), Negative elongation factor E (NELFE), Polycomb (Pc) and histone H3 (Supplementary Fig. 1c). By contrast, most of the cellular DCR1 and AGO1 was found in the Triton
X-100 soluble fraction (S1 fraction), together with tubulin, a marker
for proteins that are not associated with chromatin (Supplementary
Fig. 1c). Taken together, our data indicate that the DCR2–AGO2
complex is mainly associated with chromatin, whereas the
DCR1–AGO1 complex is mostly cytoplasmic, in accordance with its
cytoplasmic function (that is, post-transcriptional gene silencing).
To determine whether RNAi components associate with chromatin
in vivo, Drosophila polytene chromosomes were stained with AGO2and DCR2-specific monoclonal antibodies3,12,13. Both AGO2 andDCR2 were detected at several hundred sites on polytene chromosomesfrom wild-type larvae (Supplementary Figs 1d, g and 2a, b). By contrast,
little or no staining was detected in Ago2- or Dcr2-null chromosomes1,2(Supplementary Fig. 1f, i and Supplementary Table 1a). Strikingly, themajority of AGO2- and DCR2-associated loci correspond to interbands, suggesting that AGO2 and DCR2 preferentially associate witheuchromatic, transcriptionally active, loci14 (Supplementary Fig. 2a, b).
In particular, the chromatin binding of DCR2 requires AGO2, but theconverse is not true (Supplementary Fig. 3), suggesting that AGO2
also acts as the RNAi effector complex on chromatin.
Interestingly, AGO2 and DCR2 are present at the 87A and 87C
cytogenetic loci (Supplementary Fig. 1e, h). These cytogenetic locicontain copies of the heat-shock gene Hsp70, thus providing wellcharacterized inducible candidate genes with which to investigate
the role of DCR2 and AGO2 in transcription and, in particular, in
RNA Pol II pausing regulation15,16. To determine whether DCR2 andAGO2 affect Hsp70 transcription, Hsp70 transcript levels were measured in control cells and in cells depleted of DCR2 or AGO2
(Supplementary Fig. 4). The knockdown of either RNAi component
resulted in a significant increase in Hsp70 transcripts in non-heatshocked cells (Fig. 1a, b). Similar results were obtained for a second
heat-shock gene, Hsp68 (Fig. 1a, b). The increased expression of Hsp70
and Hsp68 in cells depleted of DCR2 could be reversed by the expression of a Flag-tagged wild-type copy of DCR2 (DCR2–Flag) (Fig. 1c),indicating that the change in expression was not the result of an offtarget effect of the Dcr2 RNAi. Interestingly, the levels of the endogenous AGO2 protein increased together with the expression of wild-type
DCR2–Flag (Supplementary Fig. 4a), suggesting that the DCR2–Flagprotein is integrated into the RNAi pathway and that re-establishmentof functional levels of DCR2 and AGO2 rescues the observed transcriptional defects in heat-shock genes. However, the depletion of
DCR2 and AGO2 did not affect the expression of Hsp70 and Hsp68

1

Dulbecco Telethon Institute, Epigenetics and Genome Reprogramming, IRCCS Fondazione Santa Lucia, Via del Fosso di Fiorano 64, 00143 Rome, Italy. 2Dulbecco Telethon Institute, IGB ABT-CNR, Via
Pietro Castellino 111, 80131 Naples, Italy. 3Dulbecco Telethon Institute at Università degli Studi di Palermo, Dipartimento STEMBIO, Sezione Biologia Cellulare, Viale delle Scienze, Edificio 16, 90128
Palermo, Italy. 4Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. 5RIKEN Omics
Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. 6Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im
Neuenheimer Feld 580, 69120 Heidelberg, Germany. 7Keio University School of Medicine, Tokyo 160-8582, Japan. 8JST, CREST, Saitama 332-0012, Japan. {Present address: Helmholtz Center Munich,Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, 85764 Munich-Neuherberg, Germany.
*These authors contributed equally to this work.
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RESEARCH LETTER

Non-HS exp. level

3.5
3
2.5
2
1.5
1
0.5
0

g 0.25

Hsp70

EGFP RNAi

87C

Ago2 RNAi

87A
87C

87A

87A

e

Hsp70

Hsp68
Hsp68

Hsp70
–154 +58 +471

f 10

S2

Pol II ChIP642

Dcr2 RNAi

–154

5

58

S2

4

471

S2+HS

–154

58

471

2,171

Dcr2 RNAi

0.3

NELF-E ChIP

0.2
0.1
–154

k
Dcr2 RNAi
EGFP RNAi
G/A ladder

AGO2 ChIP

3

EGFP RNAi

0.4

0

2,171

2

58

471

2,171

1

0.3
0.25
0.2

58

EGFP RNAi

471

2,171

Dcr2 RNAi
Pol II ChIP

0.15
0.1
0.05

0

72 h + DCR2–Flag

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–154

i

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+2,171S2+HS

DCR2 ChIP

0.1

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87C

S2+HS

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Hsp68

S2

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+30

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Input (%)

Ago2–/–

Dcr2–/–WT
Hsp70

Input (%)
Hsp70

Hsp70

c

d

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–154

58

471

2,171

l

Relative KMnO4 intensity

3
2.5
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Input (%)

Non-HS exp. levelb

EGFP RNAi

Input (%)

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12
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30
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Dcr2 RNAi

Figure 1 | RNA Pol II promoter-proximal pausing on Hsp70 is decreased incells treated with Dcr2 RNAi. a, b, Quantitative RT–PCR analysis of heatshock (HS) gene transcripts. RNA from S2 cells treated with enhanced green
fluorescent protein (EGFP) dsRNA (control), Dcr2 dsRNA (a) or Ago2 dsRNA(b) was analysed with primers specific for the indicated heat-shock genes.
c, Induction of the Dcr2–Flag transgene is able to revert the phenotype induced
by DCR2 depletion. S2 cells that had been stably transformed with a Dcr2–Flagtransgene were treated with EGFP dsRNA (control) or Dcr2 dsRNA. Dcr2–Flag
expression was induced only in the Dcr2 RNAi sample, by the addition ofcopper. The samples were analysed before and after a 72-h induction of thetransgene. The transcript levels are shown with respect to the EGFP control(experiment:control ratio); n 5 3. The bars represent the mean 6 s.d. d, Hsp70
DNA-FISH on polytene chromosomes from wild type (WT), homozygous
Dcr2L811fsX (Dcr22/2) or homozygous Ago2414 (Ago22/2) mutant larvae,
showing the merge of DNA (blue) and FISH (green) signals (top) and DNA
staining (bottom). e, Schematic representation of the Hsp70 transcription unit
with the position of the PCR amplicons used in this study. The numbers
indicate the middle of each amplicon with respect to the transcription start site(arrow). f–h, ChIP analysis of the heat-shock gene Hsp70. Chromatin from S2cells (S2) or S2 cells after exposure to heat shock (S21HS) was
immunoprecipitated with anti-RNA Pol II 4H8 (which recognizes thephosphorylated Ser-5 carboxyl-terminal domain), anti-DCR2 or anti-AGO2
antibodies. The bars represent the mean 6 s.d.; n 5 3. i, j, ChIP analysis of
Hsp70. Chromatin from S2 cells that had been treated with EGFP dsRNA(control) or Dcr2 dsRNA was immunoprecipitated with anti-RNA Pol II 4H8
or anti-NELF-E antibodies. The resultant DNA was analysed by quantitative
PCR. Protein binding is expressed as a percentage of input with the backgroundsignal subtracted. The bars represent the mean 6 s.d.; n 5 3. The differences in
RNA Pol II ChIP values in Fig. 2f, i are due to different batches of antibody usedin these assays. k, Permanganate (MnO4) mapping of open transcription
bubbles on Hsp70. Permanganate reacts with single-stranded thymine bases,
such as those in an open transcription bubble, uncovering a transcriptionallyengaged RNA Pol II. The autoradiograph includes a G/A ladder, which wasused to determine the position of the bands, and shows the permanganate
reactivity of thymines observed in S2 cells treated with EGFP dsRNA (control)or Dcr2 dsRNA. The hyper-reactive thymines 122 and 130 are labelled. Two
independent biological samples were analysed. l, The quantification of theautoradiograph is shown. exp., expression.

during heat shock, suggesting that RNAi activity is not involved
in heat-shock-gene-mediated activation (Supplementary Fig. 5a, b).
Typically, the activation of heat-shock genes results in the chromosome decondensation of heat-shock loci, forming large puffs onpolytene chromosomes17. Therefore, we used DNA fluorescence in situ
hybridization (DNA-FISH) to look at the chromatin structure of heatshock loci in polytene chromosomes with specific null mutations of
Dcr2 and Ago2: Dcr2L811fsX and Ago2414. Interestingly, we observed thatthe 87C locus is partially decondensed in Dcr2L811fsX and Ago2414
mutant chromosomes relative to the wild-type controls (Fig. 1d). The
87C locus contains four copies of Hsp70 distributed over 30 kilobases.
Decondensation was not evident at 87A, probably because this locus has
only two copies of Hsp70 within a region of less than 10 kilobases.
Because the data from PCR following reverse transcription (RT–PCR)
and from cytogenetic analyses suggested that the maintenance of RNA
Pol II pausing was defective, we used chromatin immunoprecipitation(ChIP) to measure the level of RNA Pol II with a phosphorylated Ser-5
residue that was present on the Hsp70 gene before and after heat shock(Fig. 1e, f). The primers used for quantitative PCR analysis of theimmunoprecipitated DNA spanned several regions of the Hsp70 gene.
An upstream primer set centred at 2154 bases from the transcription
start site was used to detect the heat-shock element; the 158 primer set
encompassed the region of the paused polymerase18; and two otherprimer sets were downstream within the transcription unit, extending
towards the end of the gene (Fig. 1e). Consistent with previous observations18, the RNA Pol II profile observed in S2 cells showed a peak in
the region of paused RNA Pol II (158 primer set) (Fig. 1f), which
increased after heat shock. Furthermore, ChIP analysis showed that
DCR2 and AGO2 were present at the Hsp70 promoter region (2154/
158) (Fig. 1g, h and Supplementary Fig. 6). Interestingly, after heatshock, the levels of both DCR2 and AGO2 proteins increased in the
Hsp70 promoter region (Fig. 1g, h, Supplementary Figs 1i, 6 andMethods). Consistent with RT–PCR and cytogenetic data, DCR2
depletion consistently decreased the level of RNA Pol II on Hsp70 in
the region of the paused polymerase (primer set 158) (Fig. 1i).
We extended our ChIP analysis to the NELF-E protein, which is partof the transcriptional regulatory complex that causes RNA Pol II pausing16,19. Depletion of DCR2 caused the level of NELF-E on Hsp70 todecrease in the 158 region, where RNA Pol II pauses (Fig. 1j). The
total cellular levels of RNA Pol II and NELF-E protein were not alteredby DCR2 depletion (Supplementary Fig. 4b), indicating that thediminished level of RNA Pol II and NELF-E detected on Hsp70 is
not a consequence of a decrease in the amount of available protein.
Conversely, in DCR2-depleted cells, we observed a reduction in thelevel of the AGO2 protein (Supplementary Fig. 4b). This was not
simply a result of cross-targeting of the double-stranded RNA(dsRNA) because the level of the Ago2 transcript was unaffected(Supplementary Fig. 4c). Likewise, Ago2 dsRNA resulted in reduced
DCR2 protein levels (Supplementary Fig. 4d) without affecting Dcr2
messenger RNA levels (Supplementary Fig. 4e). These results suggestthat DCR2 and AGO2 stabilize each other in a protein complex that is
important for repressing heat-shock genes in uninduced cells.
ToconfirmthatDCR2depletionaffectedRNAPolII pausingonHsp70,
we carried out permanganate footprinting analysis. Permanganate
reacts with thymine bases in regions of single-stranded DNA, revealing
the presence and location of transcriptionally engaged RNA Pol II. We
observed a significant reduction in permanganate reactivity of thymine
residues at 122 and 130 of Hsp70 in DCR2-depleted cells (Fig. 1k, l),which is in agreement with the reduced occupancy of the DNA by RNA
Pol II in this region (primer set 158) that was shown by ChIP (Fig. 1i).

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LETTER RESEARCHa

WT with HS

DNA

AGO2

Pol II

Merge

DNA
Pol II
AGO2

93D

95D

93D

87A
87C

93D

93D

87A
87C

87C

87C

95D

87A

87A
95D

95D

Ago2–/– with HSbDNA

Pol II

Merge

AGO2

DNA Pol II AGO2

95D

95D
87A/C

93D

c

DNA
Pol II

WT

95D
87A/C

87A/C

93D

Ago2V966M

d

DNA
Pol II

93De

DNA
Pol II

Dcr2L188F

f

Dcr2P1496L

DNA
Pol II+HS

Hence, DCR2 is involved in maintaining paused RNA Pol II at theobserved Hsp70 loci.
Heat-shock gene expression and loss of paused RNA Pol II on
depletion of DCR2 could be a consequence of stress induced by depletion of the RNAi machinery in the cell. Therefore, we evaluated theimpact of perturbing the RNAi pathway on the transcription of nonheat-shock genes. Four non-heat-shock genes (CG9008, fz, rho andmfas) that have paused RNA Pol II were analysed by permanganate
footprinting19,20. Depletion of DCR2 was found also to alter the distribution of RNA Pol II in the promoter-proximal region of these genes(Supplementary Fig. 7). These changes were also accompanied bydifferences in transcript levels (Supplementary Fig. 8). Notably, theobserved changes in RNA Pol II resulted both in upregulation (for
CG9008, fz and rho) and downregulation (mfas) of the correspondingtranscripts (Supplementary Fig. 8). Thus, the RNAi machinery can
influence RNA Pol II pausing at non-heat-shock genes, arguing against
a nonspecific DCR2-induced heat-shock response.
During heat shock, most of the Drosophila genome is transcriptionallyquiescent. The elongating RNA Pol II dissociates from euchromaticinterbands and accumulates at heat-shock loci21. We used this dynamic
RNA Pol II relocalization behaviour to follow the chromatin binding anddistribution of elongating RNA Pol II (which is Ser-2-phosphorylated),
as well as AGO2 and DCR2, in vivo. In agreement with the ChIP results(Fig. 1h), we found that, after heat shock, AGO2 accumulated at heatshock loci (Fig. 2a). However, in contrast to RNA Pol II, the associationof AGO2 with other loci appears unchanged (Fig. 2a). When we analysed
the dynamic chromatin repositioning of RNA Pol II after heat shock in
Ago2-null mutant chromosomes, we found that RNA Pol II was still
accumulated at the heat-shock loci (Fig. 2b). Strikingly, a substantial
fraction of elongating RNA Pol II in Ago2414 mutants was retained atmany euchromatic sites after heat shock (Fig. 2b). The same behaviour
was observed for the Dcr2L811fsX mutant (Supplementary Table 2). Taken
together, these results show that AGO2 and DCR2 associate with many
euchromatic sites and that their activity is probably required for thecorrect execution of global transcriptional repression after heat shock.
To dissect the role of RNAi in RNA Pol II regulation, we used threemutants that each carried a single amino acid substitution affecting
the following RNAi activities: the helicase (Dcr2L188F), the dicing(Dcr2P1496L) and the AGO2 slicing (Ago2V966M) activities22,23 (Supplementary Table 1a). To check for DCR2- and AGO2-specificrequirements, we first used DNA-FISH to analyse chromosome
decondensation at Hsp70 loci. Puffing frequencies were increased in
all three mutants, particularly in the AGO2 slicing mutant(Ago2V966M) (Supplementary Table 1b). In addition, in all of thesemutants, the Hsp70A, Hsp70B and Hsp68 transcripts were present atincreased levels before heat shock, compared with wild type
(Supplementary Fig. 9). Interestingly, the transcript increase was also
evident at 87A (Hsp70A), where the chromatin decondensation was
not appreciable at a cytogenetic level (Supplementary Fig. 9 andSupplementary Table 1b). Thus, all tested mutants induced transcript
upregulation of the Hsp70 genomic region, although this was notalways accompanied by chromatin decondensation. The uncouplingof chromatin decondensation and transcriptional activation has beenreported previously24, although in this case we cannot exclude an
RNAi-dependent post-transcriptional mechanism influencing the
Hsp70A transcript levels.
Next, we analysed RNA Pol II distribution and dynamics onpolytene chromosomes in the same Dcr2 and Ago2 mutants.
Remarkably, although with a different degree of penetrance, all threemutants failed to relocalize elongating RNA Pol II after heat shock(Fig. 2c–f and Supplementary Table 2), suggesting that RNAi enzymatic
activity is involved in the global RNA Pol II dynamics following theheat-shock stress response.
The association of RNAi components with RNA Pol II complexes
has been reported in other systems25–27. To investigate the possibility
that these proteins are part of a complex that regulates RNA Pol II

Figure 2 | Chromatin localization of RNA Pol II and AGO2 after heatshock. a, b, Co-immunolocalization of AGO2 (red) and elongating RNA Pol II(green) in WT (a) and homozygous Ago2414 (Ago22/2) mutant chromosomes(b) after heat shock (HS). DNA is stained in blue. Single signals are shown inblack and white. The bottom row shows a higher magnification of the boxed area
(top right). c–f, Immunolocalization of elongating RNA Pol II (green) in WT(c) and homozygous Ago2V966M (d), homozygous Dcr2L188F (e) and homozygous
Dcr2P1496L (f) chromosomes shows that missense mutations in DCR2 or AGO2
influence the behaviour of elongating RNA Pol II. DNA is stained in blue.
Chromosomes in the bottom panel have been exposed to heat shock.

elongation, thus establishing a link with the observed ‘pausing’ defects,
we tested for interactions between DCR2, NELF-E and RNA Pol II in
nuclear extracts derived from Drosophila S2 cells. Immunoprecipitatedfractions were evaluated by western blotting for the presence of RNA
Pol II, DCR2, AGO2 and NELF-E (Fig. 3a). As expected, DCR2 interacted with AGO2 (ref. 28) (Fig. 3a). In addition, we found that DCR2
and AGO2 interacted with RNA Pol II and NELF-E (Fig. 3a). All ofthese interactions were resistant to RNase treatment, indicating thatthey are not an indirect consequence of protein trapping associatedwith emerging RNA molecules (Supplementary Fig. 10a). Comparedwith general transcription factors, the amount of RNA Pol II interacting with DCR2 seems to be in the same range as for the TATA-binding
protein (TBP), confirming the association of DCR2 with many active
loci on polytene chromosomes (Fig. 3b). When we tested the sameinteraction in DCR2-depleted cells, we observed a decrease in the levelsof NELF-E and AGO2 associated with RNA Pol II (Supplementary Fig. 10b). These observations indicate that the DCR2–AGO2
complex influences the association of NELF-E with RNA Pol II,
thereby providing a possible explanation for the observation that the
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RESEARCH LETTERPol II

DCR2

lgG

5% Input

Pol II

175

DCR2

175

Longerexposure

DCR2

175
175

AGO2

NELF-E32

Next, we tried to gauge shifts in small RNA tags derived fromdiscrete transcribed regions at all gene loci in response to heat shock(Supplementary Fig. 14a). We observed little or no relative enrichment
along different length intervals, suggesting that these tags are notderived exclusively from promoter regions but are equally distributed
along the transcription unit (Supplementary Fig. 13). Although there
was a sharp increase in AGO2-associated small RNAs in the heatshock condition relative to the heat-shock condition negative control
immunoprecipitation (confirming the specific association of thesetags with AGO2), only a small increase was observed in the heatshock condition AGO2 immunoprecipitation relative to the normal
condition AGO2 immunoprecipitation, suggesting that these transcriptassociating tags are present even under normal conditions (Supplementary Fig. 14a). These data suggest a role for AGO2 in regulating
RNA Pol II processivity, in addition to promoter regulation.
We then analysed the strand bias of AGO2-associated small RNAs.
Sense and antisense tags had a specular distribution within individuallibraries, with the ratio of the sums of the sense and antisense small
RNA tag counts mapping to heat-shock loci and the set of all
Drosophila genes being equal to approximately 1 (Supplementary Fig.
14b). However, a comparison of normalized sense and antisense tag
counts across AGO2-immunoprecipitation and negative-controlimmunoprecipitation libraries showed a pronounced enrichment in
antisense tags and little or no enrichment in sense tags, suggesting that
antisense tags are highly specific in their association with AGO2 but thatadepletion of DCR2 alters the behaviour of RNA Pol II in the promoterproximal region of several genes.
Our data indicate an involvement of the RNAi machinery in theheat-shock stress response. To evaluate possible heat-shock-inducedchanges in the expression signature of AGO2-dependent small RNAs,
we sequenced small RNAs bound to AGO2 before and after heat shock.
Small RNA libraries from S2 cells were generated from RNA fractions
obtained by immunoprecipitation with antibodies against AGO2 orwith control IgG, with or without heat shock (Supplementary Fig. 11).
Basic statistical parameters for the four libraries are listed in Supplementary Table 3. Many sequenced tags mapped to repeat regions
in more than one location (Supplementary Fig. 12), which is consistent
with previous Drosophila deep-sequenced AGO2-immunoprecipitation libraries3,5. First, we focused our attention on the heat-shock promoter regions (500 base pairs (bp) upstream and 50 bp downstream of
the transcription start site). We found that heat-shock loci promoter
regions were enriched in small RNAs and showed a marked increase insmall RNA enrichment on heat shock. This was most noticeable for
Hsp23 and for all Hsp70 loci (Supplementary Tables 4 and 5). Thisobservation prompted us to consider whether the level of small RNAs
increased across all promoters29. Strikingly, the most enriched promoters were the Hsp70B loci, and Hsp23 was also one of the 20 mostenriched loci, suggesting a direct role for AGO2 at these promoters(Fig. 4a). The possible function or role in the heat-shock response ofother loci present in the top 20 list is not known.

Sense

Vm26Ab-RA

Hsp23-RA

CG16940-RA

Ubp64E-RB

CG11617-RA

CG15701-RA

CG16718-RB

CG13998-RA

xmas-1-RA

CG8008-RA

WASp-RB

CG7829-RA

CG3071-RA

PSR-RAb 30

CG7071-RB

Hsp70Bc-RA

Hsp70Bb-RA

1

CG5608-RA

10

Hsp70Bbb-RA

Figure 3 | DCR2 and the RNAi effector protein AGO2 associate with RNA
Pol II and NELF-E. a, Nuclear extracts from Drosophila S2 cells were
immunoprecipitated with antibodies specific for the indicated proteins (withIgG as a negative control, top), and the immunoprecipitates (IPs) were analysed
by western blotting for the presence of RNA Pol II, DCR2, AGO2 and NELF-E
proteins. b, Nuclear extracts from S2 cells were immunoprecipitated with
antibodies specific for the indicated proteins (with IgG as a negative control,top) and analysed by western blotting for the presence of RNA Pol II.

100

Hsp70Ba-RA

Pol II

1,000
Fold change

Pol II

TBP

lgG

1% Input

5% Input

DCR2

IPs

b

Antisense

25

Fold changekDa

NELF-E

IPs

a

20
15
10
5
0

Heat-shock Normal Heat-shock Normal Heat-shock Normal
AGO2-IP AGO2-IP AGO2-IP AGO2-IP
AGO2-IP AGO2-IP
vs neg-IP vs neg-IP vs neg-IP vs neg-IP vs neg-IP vs neg-IP
Heat-shock genes

Heat-shock promoters

All Drosophila genes

Figure 4 | Features of AGO2-associated small RNAs. a, The 20 promotersthat are the most enriched in small RNAs under heat-shock versus non-heatshock (normal) conditions. A comparison of the small RNA fold enrichment in
the AGO2-immunoprecipitation (AGO2-IP) libraries between normal andheat-shock conditions is shown for all promoters. b, Relative enrichment
calculated as fold change (y-axis) for the sum of all sense or antisense tags acrossdifferent conditions. The analysed gene/promoter definitions and conditions
are labelled below the graph. Little to no sense tag enrichment is observed acrossgene/promoter definitions, whereas antisense tags show enrichment across thegroups, particularly under heat-shock conditions. Neg-IP, negative control
immunoprecipitation.

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LETTER RESEARCH
sense tags are not. Additionally, the observed enrichment implies that
antisense tag association is strongest in the heat-shock condition (Fig. 4b).
Next, we examined cap analysis of gene expression (CAGE) datathat had been generated in Drosophila embryos for the modENCODE
project29, to survey the extent of antisense transcriptional activity atheat-shock loci (Supplementary Table 6). Antisense transcriptional
activity was observed at almost all heat-shock loci and, surprisingly,
many Hsp70 loci had roughly equivalent levels of sense and antisensetranscription (Supplementary Table 6), suggesting a possible source forthe observed small RNA tags. Taken together, these data indicate thatthe observed small RNAs are probably derived from dsRNA precursors
and that sequences that are antisense to the loci preferentially associatewith AGO2, implying that sense targeting occurs.
In this study, we show that, in Drosophila, RNAi components operate
in the nucleus, associate with transcriptionally active (rather theninactive) gene loci, interact with RNA Pol II and contribute to transcriptional control in a multicellular organism, particularly during theheat-shock stress response. Our results reveal that RNAi function has
also evolved as a key nuclear pathway that operates in the context of
pervasive sense and antisense transcription to regulate RNA-level
homeostasis in the cell (see also Supplementary Discussion).

METHODS SUMMARY
Wild-type Drosophila melanogaster (Canton-S or w1118) and the mutant stocks
Dcr2L811fsX, Ago2V966M, Ago2414, Dcr2L188F and Dcr2P1496L (refs 1, 2, 22, 23) weremaintained under standard conditions. S2 cells were grown in serum-free insect
culture medium. Protein–protein interactions were analysed by immunoprecipitations from nuclear extracts with specific antibodies. The production of dsRNAs was
performed in vitro by T7 transcription, and the dsRNAs were transfected into S2cells using FuGENE reagent (Roche). ChIP experiments were performed using
formaldehyde-crosslinked chromatin from S2 cells. ChIP-purified DNA was analysed by using quantitative PCR with primer pairs specific for the Hsp70 locus. Total
RNA from S2 cells or larvae was reverse transcribed and analysed by quantitative
PCR. Polytene chromosomes were prepared from third-instar larvae grown at 18 uC
and were used for FISH. FISH signals were quantified by densitometric analysis
using QFluoro software (Leica). To analyse RNA Pol II pausing on the promoters of
Hsp70 and non-heat-shock genes in S2 cells, permanganate footprinting was used.
AGO2-associated RNAs were isolated from S2 cells as previously described6. Small
RNA libraries were prepared and sequenced using a Genome Analyzer IIx(Illumina) in two lanes. For additional details see the Methods.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 13 July 2009; accepted 17 August 2011.
Published online 6 November 2011.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We are deeply grateful to P. Macino for discussions. We also thankR. Carthew, E. Lai, Q. Liu, R. Paro, Y. Sik Lee, and J. T. Kadonaga for reagents. This work was
supported by grants from the following: the National Institutes of Health (GM47477) to
D.S.G.; the Deutsche Forschungsgemeinschaft (SPP 1356) to A.B.; the Japan Society for
the Promotion of Science (JSPS) through the ‘Funding Program for Next Generation
World-Leading Researchers’ (NEXT Program) (a Grant-in-Aid for Scientific Research (A)No. 20241047) and the Council for Science and Technology Policy to P.C.; the NEXT
Program (a Research Grant-in-Aid to the RIKEN OSC) to K.M., M.C.S. and H.S.; Core
Research for Evolutional Science and Technology (CREST) from the Japan Science andTechnology Agency to M.C.S.; Fondazione Telethon, the Giovanni Armenise Harvard
Foundation, FIRB-MIUR, Associazione Italiana Ricerca Cancro (AIRC), the Human
Frontier Science Program CDA and the EMBO Young investigator program to D.F.V.C.;
Fondazione Telethon, AIRC and the EU FP6 Epigenome Network of Excellence to V.O.
This work was also made possible with the contribution of the Italian Ministry of Foreign
Affairs, ‘Direzione Generale per la Promozione e la Cooperazione Culturale’ to V.O. A.S. is
supported by a JSPS fellowship (ID P09745). Sequencing was provided by the Genas
service (RIKEN Omics Science Center).
Author Contributions F.M.C. and V.O. conceived the study. F.M.C. and A.B. performed
the ChIP experiments. F.M.C. and K.M.P. carried out the chromatin fractionation assays
and the western blotting. F.M.C. carried out the quantitative RT–PCR on S2 cells. K.M.P.
and F.L.S. performed the quantitative RT–PCR on mutant larvae prepared by M.C.O.
F.M.C. performed the co-immunoprecipitations and contributed reagents for thechromosome and permanganate footprinting experiments. M.C.O. performed thepolytene chromosome experiments. G.O.K. and D.S.G. performed the permanganate
footprinting experiments. A.M.B. performed the bioinformatic analysis. A.S. and P.C.
performed the deep sequencing. K.M., H.S. and M.C.S. performed the purification of the
AGO2-associated small RNAs. F.M.C., M.C.O., D.S.G., D.F.V.C. and V.O. designed theexperiments and interpreted the results. F.M.C., D.S.G., D.F.V.C. and V.O. wrote themanuscript with the contribution of M.C.O., A.B. and A.M.B., as well as input from theother co-authors.
Author Information Sequence data have been deposited in the DNA Data Bank ofJapan under accession code DRA000418. Reprints and permissions information isavailable at www.nature.com/reprints. The authors declare no competing financial
interests. Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should be
addressed to V.O (vorlando@dti.telethon.it).

0 0 M O N T H 2 0 1 1 | VO L 0 0 0 | N AT U R E | 5©2011 Macmillan Publishers Limited. All rights reserved

RESEARCH LETTERMETHODS
Fly stocks. Flies (Drosophila melanogaster) were maintained under standard protocols. Canton-S or w1118 were used as wild-type strains. The Dcr2L811fsX andAgo2V966M stocks2,23 were obtained from R. W. Carthew. The Ago2414 stock1 was
obtained from E. Lai. The Dcr2L188Fand Dcr2P1496L stocks22 were obtained from Y.
Sik Lee.
Cytoplasmic and nuclear fractionation. S2 cells were washed twice in ice cold
PBS, resuspended in solution I (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM MgCl2,
0.1 mM EDTA, 0.1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulphonyl
fluoride (PMSF) and Roche protease inhibitor cocktail) and passed seven times
through a 25G syringe. After centrifugation (2,000 r.p.m. for 10 min at 4 uC), thesupernatant was collected as the cytoplasmic fraction. The pellet was washed insolution I four times, resuspended in solution II (10 mM HEPES, pH 7.9, 400 mM
NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF, 5% glycerol
and Roche protease inhibitor cocktail) and incubated on ice for 30 min withoccasional flicking. After centrifugation (12,000 r.p.m. for 20 min at 4 uC), thesupernatant was collected as the nuclear fraction; 20 mg each sample was analysed
by western blotting.
Preparation of the nuclear extract and immunoprecipitation. The nuclear
extracts were obtained from S2 cells, and the protein complexes were immunoprecipitated from the extracts, according to ref. 30 with minor modifications.
Briefly, for each immunoprecipitation, 300 ml nuclear extract (600–800 mg) was
mixed with 300 ml TEA150 buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM
EDTA, 1 mM DTT, 1 mM PMSF, 0.1% NP40, 2 mg ml21 leupeptin, 2 mg ml21
aprotinin, 2 mg ml21 pepstatin) and 30 ml Protein A/G PLUS-agarose beads(Santa Cruz Biotechnology), and then incubated for 1 h at 4 uC for pre-clearing.
The supernatant was transferred to a new tube, and the appropriate amount of oneof the following antibodies was added to the supernatant: 5 mg anti-DCR2(Abcam) and/or 2 mg mouse anti-DCR2 antibody; 2 mg anti-Pol-II antibody(4H8); 10 ml anti-NELF-E antibody; 1–2 ml anti-TBP antibody; or 2 mg rabbit
IgG. The samples were incubated for 3 h on a wheel at 4 uC. Then, 40 ml ofProtein A/G PLUS-agarose beads was added. After incubation for 2 h, the beads
were washed five times with 500 ml of TEA150 buffer then resuspended in SDS–
PAGE loading buffer and analysed by western blotting. Proteins were detectedwith the SuperSignal West Dura substrate (Pierce). For single-strand RNase treatment, 2 ml RNase cocktail (RNase A1T1, Ambion) was added during one of thewashing steps, and the samples were incubated for 20 min at 30 uC.
RNAi in S2 cells. S2 cells were grown in serum-free insect culture medium (HyQ
SFX, HyClone). The production of dsRNAs (400–500 bp) of EGFP, Dcr2 and Ago2
was performed in vitro by T7 transcription. Sense and antisense RNAs weredenatured by heating and were re-annealed in water to become dsRNAs. RNAi
was performed as described previously31 and lasted 8–10 days. The primers for the
production of T7 templates were as follows: EGFP-F, 59-ACGTAAACGGCCACAAGTTC-39; EGFP-R, 59-TGCTCAGGTAGTGGTTGTCG39; DCR2-F, 59-GTTC
CGCTTTGGTCAACAAT-39; DCR2-R, 59-TGATCGTCTTTTCCATGCAG-39;
DCR2UTR-F, 59-ATGATGATTCCAGCCCAGTC-39; DCR2UTR-R, 59-TTATTTCGACCCAAGGTAAC-39; AGO2-F, 59-GCTGCAATACTTCCAGCACA-39;
and AGO2-R, 59-CTCGGCCTTCTGCTTAATTG-39.
Antibodies. The mouse anti-RNA Pol II antibody (4H8, Abcam ab5408) recognizes the Ser-5-phosphorylated C-terminal domain32. The mouse anti-RNA Pol II
antibody (H5, Covance) recognizes the elongating RNA Pol II, which is phosphorylated at Ser 2 in the C-terminal domain18. The rabbit anti-DCR1 (ab4735),
anti-DCR2 (ab4732), anti-histone H3 (ab1791) and mouse anti-actin (ab8224)
antibodies were from Abcam. The mouse anti-b-tubulin antibody (E7) was fromthe Hybridoma Bank at the University of Iowa. The mouse anti-Flag (M2) antibody
was purchased from Sigma. The mouse anti-AGO1, anti-AGO2 and anti-DCR2
antibodies have been described previously3,12,13. The rabbit anti-Pc antibody33 wasprovided by R. Paro. The rabbit anti-TBP antibody34 was a gift from J. T. Kadonaga.
The anti-NELF-E antibody was described previously16. In western blotting experiments, a non-commercial anti-DCR2 antibody, kindly provided by Q. Liu35, wasalso used.
ChIP. Chromatin was prepared as described previously36 from non-treated S2cells and heat-shocked S2 cells (as described below) with a fixation step of 15–
25 min at room temperature. The following antibodies were used for immunoprecipitation (approximately 8 3 106 cells per immunoprecipitation): anti-RNA
Pol II (4H8, 2 ml), anti-NELF-E (5 ml), anti-AGO2 (9D6, 3 ml), mouse anti-DCR2(4 ml) and anti-Flag M2 (as a negative control, 4 ml) antibodies. The specificity of
the ChIP-grade anti-AGO2 (9D6) antibody is shown in Supplementary Fig. 6. The
anti-DCR2 antibody used for the ChIP experiment is the same one used for the
immunofluorescence experiments shown in Supplementary Fig. 1i. Quantitative
PCR was performed in a DNA Engine Opticon 2 (MJ Research, Bio-Rad) instrument using the QuantiTect SYBR Green PCR Kit (QIAGEN) or in a LightCycler
480 (Roche) using the ABsolute QPCR SYBR Green Mix (Thermo Scientific)

according to manufacturer’s instructions. Relative enrichments were calculated
as a percentage of the input. The Hsp70 primer sequences were as follows: the2154 and 158 primers were described previously18; 1471forward, 59-GATCTG
GGCACCACCTACTC-39; 1471reverse, 59-TGGGAGTCGTTGAAGTAGGC-39;
12171forward, 59-CACGATCAAGAACGACAAGG-39; and 12171reverse,
59-CTTTGGCCTTAGTCGACCTC-39. The names of the primer pairs indicate
the distance of the middle of each amplicon from the Hsp70 transcription start site.
Heat-shock induction. Third-instar larvae were transferred to preheated 1.5-ml
microcentrifuge tubes and submerged in a 37 uC water bath for 40 min. S2 cells
were transferred to pre-heated medium and submerged in a 37 uC water bath for
40 min.
Quantitative RT–PCR analysis. Total RNA from S2 cells or larvae was isolatedwith TRIzol reagent (Invitrogen). RNA from each sample was subjected to cDNA
synthesis using a QuantiTect Reverse Transcription Kit (QIAGEN). All primers
were annealed at 60 uC. Quantitative PCR was performed with a DNA Engine
Opticon 2. Quantification was normalized to the housekeeping gene GAPDH1,
and relative expression levels were calculated using the following equation:
A 5 2[Ct(ref) 2 Ct(ref 2 control)] 2 [Ct(sample) 2 Ct(sample 2 control)]. The primer sequenceswere as follows: GAPDH1-F, 59-ATCGTCGAGGGTCTGATGAC-39; GAPDH1R, 59-ACCGAACTCGTTGTCGTACC-39; HSP70, see 1471 primers in the ChIP
section; HSP70A-F, 59-GTGTCTACCAACATGGCAAG-39; HSP70A-R, 59-CTG
TGTTTCTGGGGTTCATG-39; HSP70B-F, 59-GGTTGAGATTATCGCCAAT
GAC-39; HSP70B-R, 59-GTCGTATTTTCGGCCGATGAG-39; HSP68-F, 59-GA
GAAGGCACTCAAGGACGC-39; HSP68-R, 59-GAAGGTCTTGGACTGCTT
GC-39; MFAS-F, 59-AGAAACCGTGGACACCTTTG-39; MFAS-R, 59-TGTGAAGTCAGCGTTTCTGG-39; CG9008-F, 59-GTTGGCAGAACATCGAAGG-39;
CG9008-R, 59-AGCGCAGCTTGAGTTTTTGT-39; RHO-F, 59-CGCGAGATCATCAAACTGAG-39; RHO-R, 59-CTCGTAAATCCAGGAGCTTG-39; FZ-F,
59-GTCAGCGTATGCCCTACCAT-39; FZ-R, 59-ACTTTTTCGCACGGGACT
TA-39; AGO2-F, 59-CTGGATGATGGATACGAGGC-39; AGO2-R, 59-GCGA
CTGTGGAAGTAGGACG-39; DCR2-F, 59-GCAATAGCGATGCAGTATGG39; DCR2-R, 59-CGTCCTAGAAGAAGGCATCG-39; AUB-F, 59-AAACCAACTGGGCTGATGTC-39; and AUB-R, 59-GCTCTGGCAAGGTAATAACG-39.
FISH analysis. Cytological preparations and FISH were carried out as described
previously37. FISH signals were quantified by densitometric analysis using
QFluoro software (Leica). Any deviation of 33% or greater from the normal
average relative puffing ratio of the 87C locus to the 87A locus in wild-type flies
was considered an increase in puffing at the 87C locus relative to the 87A locus in
the Ago2 and Dcr2 mutants analysed.
Immunostaining of salivary gland polytene chromosomes. Polytene chromosomes were prepared from third-instar larvae grown at 18 uC. Single and double
immunostaining was carried out as described previously38. The Fab-fragment
blocking method was used to stain the chromosomes with anti-AGO2 and antiRNA Pol II antibodies, both raised in mice. Antibodies were used at the following
dilutions: anti-RNA Pol II antibody (H5), 1:200; anti-AGO2 antibody, 1:50; antiAGO2 antibody (9D6), 1:50; mouse anti-DCR2 antibody, 1:100; and Fab fragment(anti-mouse), 1:100. Given the specificity of the anti-AGO2 and anti-DCR2
antibodies in polytene staining, the cytoplasmic and nucleoplasmic backgroundsignals were removed (using the Lasso tool in Photoshop; Adobe), to highlight
AGO2 and DCR2 chromatin binding.
Permanganate footprinting. S2 cells (2 ml, approximately 4 3 106 cells ml21)
were treated with permanganate, essentially as described previously19. The permanganate cleavage pattern was analysed as described previously39. The primer
sequences were as follows: CG90081237R_LM1, 59-CGCATTTACATAATGTTTGC-39; CG90081226R_LM2, 59-AATGTTTGCAAAAATTATTCTGC-39;
CG90081220R_LM3, 59-TGCAAAAATTATTCTGCACACAAAG-39; FZ1221R_LM1,
59-AAGTACCGTTAGCTGTATATCATT-39; FZ1213R_LM2, 59-TTAGCTGTATATCATTTATGCCTCC-39; FZ1197R_LM3, 59-TATGCCTCCCCCGCTTT-39;
MFAS1178_LM1, 59-TTATTGTCAATTGTCTTGTTTTTAC-39; MFAS1159_LM2,
59-TTTTACAAAAATTGGCACACACT-39; MFAS1155_LM3, 59-ACAAA
AATTGGCACACACTCAATTA-39; RHO1210R_LM1, 59-CTTAGTTTTGCTGCTCGTAA-39; RHO1204R_LM2, 59-TTTGCTGCTCGTAAATCCA-39;
RHO1198R_LM3, 59-GCTCGTAAATCCAGGAGCTTGT-39; for HSP70 87A,
87C/A250_LM1, 59-GCAGGCATTGTGTGTGAGT-39; 87C250_LM2, 59-GGC
ATTGTGTGTGAGTTCTTCTTT-39; 87C250_LM3, 59-TGTGTGAGTTCTTCTTTCTCGGTAACTTG-39; linker A9, 59-GCGGTGATTTAAAAGATCTGAA
TTC-39; and linker B, 59-GAATTCAGATC-39. LM1 was the first primer usedin each set. LM2 was used for amplification. LM3 was used for labelling. Linker A9and linker B are the two primers that are annealed and ligated to the blunt ends
generated by primer extension with LM1. Quantification of the autoradiographs
was carried out using ImageQuant software (Bio-Rad).
DCR2–Flag rescue assay. To selectively inhibit the endogenous Dcr2 gene, aspecific dsRNA targeting the 39-untranslated region sequence missing in the©2011 Macmillan Publishers Limited. All rights reserved

LETTER RESEARCH
Dcr2–Flag transgene was used. After 8 days of dsRNA treatment, the expression of
the fusion protein was induced by adding 1 mM CuSO4.
Chromatin-binding assay. The chromatin-binding assay protocol was essentiallyas described previously11. S2 cells (60 ml, approximately 3–6 3 106 ml21) were
washed with cold PBS. One-tenth of the cell suspension (control fraction, C)
was resuspended in RIPA buffer (150 mM Tris-HCl, pH 8.0, 150 mM NaCl,
0.5% DOC, 0.1% (w/v) SDS, 1% (v/v) NP-40, 2 mg ml21 leupeptin, 2 mg ml21
aprotinin, 2 mg ml21 pepstatin, 1 mM PMSF, 1 mM NaF and 1mM sodium
orthovanadate) and left for 30 min on ice. The remaining fraction was lysed for
15 min on ice in cold CSKI buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 1 mM
EDTA, 300 mM sucrose, 1 mM MgCl2, 1 mM DTT, 0.5% (v/v) Triton X-100,
2 mg ml21 leupeptin, 2 mg ml21 aprotinin, 2 mg ml21 pepstatin, 1 mM PMSF,
1 mM NaF and 1mM sodium orthovanadate). The cell lysate was divided into
two portions, which were centrifuged at 500g at 4 uC for 3 min. The supernatants(S1 fraction), which contain Triton-soluble proteins, were further analysed. One ofthe pellets was washed twice in CSKI buffer and then resuspended in RIPA buffer(the P1 fraction). The second pellet, after washing in CSKI buffer, was resuspended
in CSKII buffer (10 mM PIPES, pH 6.8, 50 mM NaCl, 300 mM sucrose, 6 mM
MgCl2, 1 mM DTT, 2 mg ml21 leupeptin, 2 mg ml21 aprotinin, 2 mg ml21 pepstatin,
1 mM NaF and 1mM sodium orthovanadate), then treated with DNase (Promega)for 30 min and extracted with 250 mM NH2SO4 for 10 min at 25 uC. The sample
treated with DNase and salt was then centrifuged at 1,200g for 6 min at 4 uC, andthe supernatant (S2 fraction) and pellet (P2 fraction) were collected. The P2
fraction was also resuspended in RIPA buffer. All fractions (20 mg) were analysed
by western blotting.
Purification of AGO2-associated RNAs. For heat shock, S2 cells were transferred
to pre-heated medium at 37 uC and submerged in a 37 uC water bath for 40 min.
Immunoprecipitation was performed as described previously3. Briefly, S2 cells
were washed twice in ice cold PBS, resuspended in an EMPIGEN-containing
PBS buffer (1% EMPIGEN, 1 mM EDTA, 100 mM DTT, 2 mg ml21 pepstatin,
2 mg ml21 leupeptin and 0.5% aprotinin) and incubated on ice for 10 min to lyse
cells. After centrifugation (15,000 r.p.m. for 20 min at 4 uC), the supernatant wascollected. Immunoprecipitation was performed using an anti-AGO2 antibody(9D6) or mouse non-immune IgG (negative control) immobilized on
GammaBind beads (GE Healthcare). The reaction mixture was rocked at 4 uC
for 2 h and washed five times with EMPIGEN-containing PBS buffer.
For silver staining, the washed beads were incubated with 23 sample buffer(20% glycerol, 100 mM Tris-HCl, pH 6.8, 4% SDS and 0.02% bromophenol blue)for 10 min at room temperature, and DTT (200 mM final concentration) was
added to the mixture. The sample was then incubated at 95 uC for 5 min andresolved by SDS–PAGE. After electrophoresis, the protein bands were visualized
by SilverQuest (Invitrogen).
The small RNAs associated with AGO2 were treated with phenol:chloroform
and precipitated with isopropyl alcohol. The RNAs were then dephosphorylatedwith calf intestinal alkaline phosphatase (New England BioLabs), labelled with[c-32P]ATP with T4 polynucleotide kinase (New England BioLabs) and resolved
on a 12% polyacrylamide denaturing gel.
Small RNA library sequencing and computational analysis. Small RNA libraries
were prepared as described previously40 with one of two barcodes attached to thelibraries generated from the immunoprecipitation experiments, regardless of thepresence or absence of heat shock. The libraries were sequenced using a Genome
Analyzer IIx (Illumina) in two lanes. Linkers and barcode sequences were extracted
from the raw tags; the tags were mapped to the Drosophila dm3 genome assembly
using the program Nexalign41. The genome classification of individual tags wasdetermined by overlap with existing definitions culled from publicly available
genome tracks in FlyBase and functional RNA databases42,43. Tags that mapped

to more than ten locations in the genome were removed from subsequent analyses;
tag counts for the remaining tags were distributed evenly across the total number ofmapping sites. Tags were normalized to tags per million before comparisons acrosslibraries. A summary of the basic statistical parameters for the four libraries isprovided in Supplementary Table 3. The extraction rates were between 70% and
75% per library (data not shown); mapping rates were above 90% (Supplementary
Table 3).
As an additional normalization strategy, the tags per million microRNA count
was also tabulated for each library (Supplementary Table 5) because microRNA
percentages were largely unaffected by heat-shock treatment (Supplementary
Fig. 12); fold enrichments calculated across conditions with these values wereconsistent with tags per million normalization (data not shown). Sense–antisense
distinctions were decided according to FlyBase gene definitions; overlapping genedefinitions on the same strand were merged, and the tags mapping to overlapping
sense–antisense transcripts were included in both sense and antisense totals.
Clusters of CAGE tags, which are representative of transcription start site activity,
were linked to heat-shock loci using 59 and 39 FlyBase gene definitions; each locus
was visually inspected in a genome browser, and the gene definitions were adjusted
to account for differences in transcription start sites between embryonic tissue and
FlyBase gene definitions. Large-scale genome comparisons were carried out using
the BEDTools utilities suite44.
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condensation proteins Topoisomerase II and Barren colocalise with Polycomb andmaintain Fab-7 PRE silencing. Mol. Cell 7, 127–136 (2001).
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bind promoters repressed by Polycomb group proteins. Nature 412, 651–655(2001).
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