SUPPLEMENTARY INFORMATION
a

C

b

N

c

Pol II

Cernilogar et al. Fig. S1
C S1 P1 S2 P2Pol II

175

175

Control (C)

Cells

Dcr1

Dcr1

175

175

TritonX-100

Dcr2

175

Dcr2

175

doi:10.1038/nature10492

Supernatant (S1)Pellet (P1)

not chromatin-boundchromatin-bound

AGO1

83
175

AGO2

AGO1
83
DNAse + Salt

AGO2
83

Pc

63

tub

Supernatant (S2)Pellet (P2)chromatin-boundmatrix-bound

45

32

*
NELF-E

16

H3

Pc

63

tub
45

d

f

e
87A

DNA
AGO2
87C

DNA
87A

87C

DNA

AGO2
87A
AGO2
87C
AGO2

merge

DNA

merge

Ago2- /-

WT

g

h
Dcr2

DNA

i
87A

DNA
Dcr2

87C
DNA
87A

DNA
87A

87C
Dcr2
87A

87A
Dcr2
87A

87C
merge

WT

87C

merge
87A

Dcr2-/-87C

87C
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Figure S1: RNAi components are associated with chromatin. a) Equivalent amounts ofcytoplasmic (C) and nuclear (N) extracts from Drosophila S2 cells were analyzed by western blot forthe presence of the indicated proteins: RNA polymerase II (Pol II), Dicer-1 (Dcr1), Dicer-2 (Dcr2),
Argonaute1 (AGO1), Argonaute2 (AGO2), Polycomb (Pc) and Tubulin (tub). b) Scheme of theprocedure used to fractionate S2 cell extracts (adapted from ref.11). Insoluble fractions weredissolved in RIPA buffer (see methods). Chromatin associated proteins should be found in fractions
P1 and S2. c) Equivalent amounts of the resulting samples were analyzed by western blot for thepresence of the indicated proteins. Tubulin (tub) serves as a chromatin unbound marker; Pol II,
Polycomb (Pc), NELF-E (* uncharacterized band) are chromatin bound markers; histone H3 (H3) is atightly chromatin bound marker that is fully released only after high salt and DNAse treatment. Three
independent biological samples have been analyzed. Shown are representative pictures. d-f)
Immunolocalization of the AGO2 protein (red) on WT (d,e) and homozygous Ago2414 (f) mutant
chromosomes. DNA is stained in blue. e) Higher magnification of the boxed area from d. f) Below thewhole chromosome is shown a higher magnification of the boxed area. g-i) Immunolocalization of the
Dcr2 protein (green) on WT (g,h) and homozygous Dcr2L811fsX (i) mutant chromosomes. h) Higher
magnification of the boxed area from g. i) Below the whole chromosome is shown a higher
magnification of the boxed area. Single signals are shown in black and white.

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Cernilogar et al. Fig. S2
a

b

DNA
AGO2

DNA
Dcr2

merge

merge

DNA

DNA

AGO2

Dcr2

split

split

Figure S2: AGO2 and Dcr2 associate with many euchromatic sites on polytene chromosomes.
Immunolocalization of AGO2 (a) and Dcr2 (b) in WT chromosomes . Blue is DNA staining. Below isshown a higher magnification of the boxed area with single signals in black and white. In order tohighlight AGO2 and Dcr2 chromatin binding, the cytoplasmic and nucleoplasmic background signals
were removed from polytene stainings (see Materials and Methods).

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Cernilogar et al. Fig. S3

a

WT

b

Dcr2-/-

DNA
AGO2

c

DNA
AGO2

WT

d

Ago2-/-

DNA
Dcr2

DNA
Dcr2

Figure S3: Chromatin binding of Dcr2 requires AGO2 but not vice versa.
a,b) Immunolocalization of AGO2

in WT (a) and homozygous Dcr2L811fsX (b) mutant

chromosomes. c,d) Immunolocalization of Dcr2 in WT (c) and homozygous Ago2414 (b) mutant
chromosomes. Blue is DNA staining. In order to highlight AGO2 and Dcr2 chromatin binding,the cytoplasmic and nucleoplasmic background signals were removed from polytene stainings(see Materials and Methods).

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72h Dcr2 flag

EGFP RNAi

Dcr2 RNAia

EGFP RNAi

doi:10.1038/nature10492

Cernilogar et al. Fig. S4

b
Dcr2

Dcr2

c

Pol II
flag
AGO2
AGO2

NELF

tub

actin

d

e

Dcr2
AGO2
actin

Figure S4: Protein and transcript levels in Dcr2, AGO2 depleted cells and after the expression of
the Dcr2-flag transgene. a) Western blot showing the Dcr2, Dcr2-flag, AGO-2 and tubulin (loading
control) protein levels in the same samples analyzed in fig. 1c. b,c) The samples analyzed were S2cells treated with EGFP dsRNA (control) or Dcr2 dsRNA. b) Western blot with the indicatedantibodies. Actin is the loading control. c) Quantitative RT-PCR of Aubergine (Aub; negative control),Dcr2 and AGO2 transcripts. n=3, bars represent the mean± standard deviation. d,e) The samples
analyzed were S2 cells treated with EGFP dsRNA (control) or AGO2 dsRNA. d) Western blot with theindicated antibodies. e) Quantitative RT-PCR. n=3, bars represent the mean± standard deviation.

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Cernilogar et al. Fig. S5ab

Figure S5: hsp70 and hsp68 transcript levels in S2 cells following heat shock activation.
Heat shock activates transcription of heat shock genes. In response to heat shock, the heat shock
factor (HSF) rapidly associates with the promoter region. Quantitative RT-PCR analysis in S2
cells treated with EGFP dsRNA (control) or Dcr2 dsRNA (a) or AGO2 dsRNA (b) and exposedto heat shock (HS). n=3 independent RT-PCR experiments. Bars represent the mean±standard
deviation.

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Cernilogar et al. Fig. S6
a

DNA
AGO2

DNA
AGO2

c

d

0,25

0,25

no Ab ChIP
% input

0,20% input

Ago2-/-

b

WT

0,15
0,10
0,05
0,00

Flag ChIP

0,20
0,15
0,10
0,05
0,00

-154

58

471

2171

-154

S2

58

471

2171

S2+HS

Figure S6: Controls for the ChIP and for the specificity of the anti-AGO2 (9D6) antibody.
The anti-AGO2 (9D6) ChIP grade antibody recognizes specifically the AGO2 protein on polytene
chromosomes. Immunolocalization of AGO2 (green) in WT (a) and homozygous Ago2414 (b)mutant chromosomes. Blue is DNA staining. (c,d) As negative control for the ChIP chromatinfrom S2 cells or S2 cells after exposure to heat shock (HS) was immunoprecipitated with noantibody (c) or anti-Flag antibody (d), that has no target in S2 cells. n=3, bars represent the mean
±standard deviation.

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EGFP RNAI
Dcr2 RNAI

Naked DNA

Dcr2 RNAIEGFP RNAI+31+36 +33+23, +24+43,+44
+49
+54

+37
+40

+60
+66
+70,+71+72,+73

+84

+44

fz

+124,+125
mfas

+72

EGFP RNAi
Dcr2 RNAi

+66

+55
+57

+26

mfas

+40
+45

rho
fz

+31

+38
+47

CG9008

+37

+127

+88

rho
+127

+116

+82

+116
+119

+125
+127
+129

+116
+118,+119+36+43,+44
+54
+60
+66
+70

+110, +111

+103

+69
+73

+55
+58
+64

+120

+91
+96,+97+64, +65+66+95+105, +106

+72
+78

+55
+57

+90

+114

+55
+58
+64

+45

+84

+66,~+70+71,~+72+80,~+84+85+89+93 +95, ~+97
+99, ~+100+103, +104

+5,+6+17, ~+19+26+30, +31+35+5, +6+9, +10+11, +12+17-3, ~-1+3, ~+5+9+13 +17+20,+21
+31
+38
+44
+47
+53,~+55+56

CG9008

Naked DNA

Dcr2 RNAIEGFP RNAI

Naked DNA

EGFP RNAI

Dcr2 RNAI

Naked DNA

Cernilogar et al. Fig. S7

Figure S7: Transcription of non-heat shock genes is perturbed in Dcr2 depleted cells. Upper partshows the permanganate footprinting analysis of the indicated genes. Each of these panels includes thepermanganate reactivity of naked DNA as a control (Naked DNA) and the permanganate reactivity ofthymine residues observed in S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA. Hyperreactive thymines (T) are indicated. Lower part shows the quantification of the permanganate reactivity.
Some hyper-reactive T are indicated.
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Cernilogar et al. Fig. S8

Figure S8: Quantitative RT-PCR analysis of the indicated genes in EGFP and Dcr2 RNAi
samples. n=3 independent RT-PCR experiments. Bars represent the mean±standard deviation.

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Cernilogar et al. Fig. S9

Figure S9:. Missense mutations in Dcr2 or AGO2 change the expression levels of heat-shock
genes. Quantitative RT-PCR of hsp70A, hsp70B and hsp68 transcript levels in WT, homozygous
Ago2V966M, homozygous Dcr2L188F, homozygous Dcr2P1496L larvae. n=3 independent experiments.
Bars represent the mean±standard error of the mean.

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Cernilogar et al. Fig. S10a

Pol II +RNAse

Pol II

NELF-E +RNAse

NELF-E

Dcr2+RNAse

Dcr2

IgG

5% inputIPs

Pol II
175
175

AGO2

Pol II

NELF-E

Dcr2

IgG

1% input

5% input

Pol IIIPs

NELF-E

Dcr2

1% input

5% input

IgG

IPs

b

Pol II
Dcr2

AGO2
NELF-E

EGFP RNAi

Dcr2 RNAi

Figure S10: Dcr2 and the RNAi effector protein AGO2 associate with Pol II and NELF. a)
Nuclear extracts from S2 cells were immunoprecipitated and analyzed by western blot for thepresence of Pol II and AGO2. Where indicated the samples have been treated with a RNase
cocktail (RNase-A and T1). b) Nuclear extracts from S2 cells treated with EGFP dsRNA (control)or Dcr2 dsRNA were immunoprecipitated and analyzed by western blot for the presence of Pol II,
Dcr2, AGO2, NELF-E. We observed also the reduction of AGO2 levels upon Dcr2 depletion.

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Cernilogar et al. Fig. S11

n.i.

220

37 °C
anti-AGO2

26°C

anti-AGO2

anti-AGO2

n.i.

n.i.

37 °C

26°C

IP from S2 lysate

n.i.

b

IP from S2 lysate

anti-AGO2

a

100nt
80nt

160
AGO2

60nt

120
40nt

100
90

30nt

80

70

20nt

60
h.c.
50

10nt

40

(kDa)

Figure S11: Purification of AGO2-associated small RNAs. (a) S2 cells were lysed before andafter heat-shock and AGO2 was immunopurified from the lysates using anti-AGO2 antibody.
Immunoprecipitated AGO2 was visualized by silver staining. n.i.: non-immune IgG (negative
control), h.c.: heavy chains of antibodies. (b) Small RNAs co-immunopurified with AGO2 in (a)were visualized by [32P]ATP-labeling.

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Cernilogar et al. Fig. S12

Figure S12: Comparisons of genome locations to which tags mapped. All mapped tags wereassigned to various genome annotations derived from FlyBase and the UCSC genome browser.

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Cernilogar et al. Fig. S13

Figure S13: Gene browser view of the Hsp70Bbb, Hsp70Bb, and Hsp70Bc gene loci.
Normalized tag counts mapping to the displayed FlyBase gene loci are visualized for eachcondition; condition labels are given to the right. Sense and antisense tags are depicted in green
and purple, respectively. Tags are displayed by summing over the total counts at a location along
the entire length of the tag. The same scale is used for each library; scales are provided to the left.
Regions containing the bulk of the tags found in the promoter regions are outlined in blue.
Regions corresponding to the boundaries of the genes are outlined in red. While an increase intags is observed in the 3’ region of the Hsp70Bbb and Hsp70Bb loci, this is not a consistently
observed trend (see global analysis in Fig. S14a) Genome visualization provided by the ZENBU
Omics Data Integration and Visualization System (Severin J, http://fantom.gsc.riken.jp/zenbu/).

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Cernilogar et al. Fig. S14ab

Figure S14: Features of AGO2-associated small RNAs. a) Comparison of short RNA tags
mapping to different locations of transcribed regions. To ascertain whether short RNAs derived
from transcribed regions were biased in their origins for specific spatial locations, all transcripts(defined by Flybase) were divided into four segments: a fixed promoter length (-500/+50)
centered around the TSS and three equivalently-sized sections normalized to the length of a given
transcript yielding a 5’ region, a midsection region, and a 3’ region. Total tag counts were summed
across all segments and relative fold enrichment (y-axis) was compared across the libraries listed
at the bottom of the figure. b) Sense/antisense tag ratio for the indicated short RNA IP-seq
libraries; ratios calculated by summing all tag counts across the indicated gene definitions in boththe sense and antisense directions (see Methods for more information).

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Cernilogar et al. Table S1a
Mutant’s name

Molecular lesion

Additional information

Dcr2L811fsx (null)

Premature stop codon

Full length protein:NO
Dicing activity:NO
RNAi activity:severely reduced

Dcr2L188F

Amino acid substitution
in the DExH helicase
domainFull length protein:YES
Dicing activity:YES
RNAi activity: reduced

Dcr2P1496L

Amino acid substitution
in the RNAse III
domainFull length protein:YES
Dicing activity:NO
RNAi activity: reduced

Ago2414 (null)

Deletion of exons 1 and
2

Protein:NO
Slicing activity:NO
RNAi activity:severely reduced

Ago2V966M

Amino acid substitution
in the PIWI domain

Full length protein:YES
Slicing activity:NO
RNAi activity: reducedb

Table S1: a) mutants analyzed in this study. b) results of the hsp70 DNA-FISH. The relative
puffing ratio existing between 87C and 87A was calculated by quantitative densitometric analysis.
Any deviation of 33% from the normal average relative puffing ratio existing in wild type at the
87C/87A loci was considered an increase in puffing at the 87C relative to 87A in the Ago2 andDcr2 mutants analyzed.

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Cernilogar et al. Table S2Table S2: number of bands, corresponding to active Pol II, on polytene chromosomes after heatshock. Five/six chromosomes for each genotype have been analyzed.

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Cernilogar et al. Table S3

Table S3: A summary of the basic statistics for the four libraries is given .

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Cernilogar et al. Table S4

Table S4: Short RNA raw (left) and normalized (right) counts mapping to heat shock promoter
regions -500/+50 around transcriptional start sites. Tag counts are strandles; derived from the senseor anti-sense direction of the loci. Tags are normalized by calculating parts per million (tags per
million or tpm). Loci for which no tags were recovered are not shown in the table. As an additional
normalization strategy, tags per million miRNA counts were also tabulated for each library (Table
S5) because miRNA percentages were largely unaffected by heat shock treatment (Figure S12); foldenrichments calculated across conditions with these values were consistent with tpm normalization(data not shown).

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Cernilogar et al. Table S6
CAGE
expression,
sense

CAGE
expression,
antisense

CG14207

31034.0

0.0

Hsc70-1

18.0

17.0

Hsc70-2

5.0

31.0

Hsc70-315729.0

52.0

Hsc70-4

21561.0

1.0

Hsc70-5

905.0

12.0

Hsf

3183.0

44.0

Hsp22

44.0

5.0

Hsp23

660.0

4.0

Hsp26

275.0

8.0

Hsp27

16881.0

35.5

Hsp60

1255.019.0

Hsp60B

2.0

7.0

Hsp67Ba0.0

7.0

Hsp67Bc

218.0

2.0

Hsp68

155.0

32.0

Hsp70Aa

102.3

102.0

Hsp70Ab

55.5

80.0

Hsp70Ba

44.8

3.0

Hsp70Bb

46.8

46.3

Hsp70Bbb

25.4

46.3

Hsp70Bc

250.4

50.3

Hsp83

15548.0

42.0

236.0

0.0

heat shock
locus

Hsromega

Table S6: Sense and antisense transcriptional start site activity as measured by CAGE expression at the
5’ and 3’ ends of heat shock loci (see Methods). CAGE data generated for embryonic tissue as part of
the Drosophila modENCODE project. Rows highlighted in orange correspond to loci with roughly
equivalent sense and antisense expression according to CAGE; additional loci with unusually highrelative antisense expression or lowly-expressed equivalent sense and antisense expression arehighlighted in light red.

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Supplementary discussion
Classic studies of RNAi function in heterochromatin formation have been suggesting a role ofchromatin associated RNAi in gene silencing1. However, heterochromatin formation requires
transcription, thus even heterochromatin gene silencing may involve co-transcriptional
mechanisms, perhaps via non coding RNA. Our data revealed an unexpected preferential
association of RNAi components with transcriptionally active loci and loss of function alleles
determine defects in RNA Pol II pausing and transcriptional up-regulation, particularly at
inducible heat shock genes, suggesting that RNAi function is required for correct RNA Pol IIfunction.
The precise role of RNAi and short RNAs in Pol II regulation remains to be elucidated.
However, RNA silencing mechanisms are known to act on aberrant transcripts. It is unclear
how aberrant transcripts are recognized but the mechanism seems to involve competition
between different RNA processing pathways, including exosome activity2 . The notion ofaberrant RNA remains to be fully understood. The cell may consider as aberrant either atranscript not present normally in the cell or also illegitimate levels of transcription. The lattermay require a fine tuned co-transcriptional mechanisms that may involve Pol II. Our data showthat chromatin-associated RNAi components affect Pol II, particularly after heat shock andthat loss of these components impair Pol II in heat shock stress response. It is tempting to
speculate that in Drosophila, in particular under stress conditions, aberrant RNAs are produced
and play a role in initiation of RNAi-mediated silencing. Indeed, our findings have intriguingparallelisms with a recent report regarding the role in fission yeast of RNAi in heterochromatin
formation and transcriptome. In that work, Moazed and co-workers proposed a model for
RNAi as a surveillance mechanism in which small RNAs called priRNA, derived fromdegradation of abundant transcripts, would bind AGO1 to target transcripts that result frombidirectional transcription of DNA repeats as well as other mRNAs2 . A similar model for
Dicer function was proposed, in the same system, in gene silencing control of someeuchromatic sites and transposable elements3. Our data indicate that AGO2-associated short
RNA sequences match sense and antisense transcripts at heat shock loci, suggesting that RNAi
operates on both. Of note, we found a strong bias for antisense, particularly after heat shock as
well as throughout the Drosophila genome4. Thus, we propose that in higher eukaryotes
chromatin-associated RNAi activities may operate co-transcriptionally to control thetranscriptome via sense and antisense transcripts.

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Supplementary references

1.

Grewal, S.I., Elgin, S.C. Transcription and RNA interference in the formation ofheterochromatin. Nature. 447, 399-406 (2007).

2.

Halic, M., Moazed, D. Dicer-independent primal RNAs trigger RNAi andheterochromatin formation. Cell. 140, 504-16 (2010).

3.

Woolcock, K.J., Gaidatzis, D., Punga, T., Bühler, M. Dicer associates with chromatin torepress genome activity in Schizosaccharomyces pombe. Nat Struct Mol Biol. 18, 94-9
(2011).

4.

Hoskins, R.A. et al. Genome-wide analysis of promoter architecture in Drosophilamelanogaster. Genome Res. 21,182-192 (2011).

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