Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila
© Kavi and Birchler; licensee BioMed Central Ltd. 2009
Received: 22 April 2009
Accepted: 16 November 2009
Published: 16 November 2009
Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure.
The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.
Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in Drosophila. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in Drosophila.
The metazoan chromosome consists of two distinct functional compartments based mainly on their transcriptional competence and higher order chromatin packaging. Heterochromatin is tightly packed and has a paucity of actively transcribed genes. It plays a vital role in biological functions such as determining the distribution of meiotic recombination, telomere maintenance and sister chromatid cohesion [1–3]. The metazoan chromosome is also interspersed with facultative heterochromatin, which has the potential to become transcriptionally competent. This fine tuning ensures gene regulation in a cell specific and spatio-temporal manner during development.
The long held notion that heterochromatin is refractory to transcription was reversed in recent experiments performed in Schizosaccharomyces pombe and mouse cells [4, 5]. It was demonstrated that centromeric heterochromatic repeats are transcribed in the late S phase of the cell cycle and this transcription of heterochromatic repeats is essential for the structural maintenance of centromeric heterochromatin. It was also shown that heterochromatin is a versatile platform with proteins such as SWI6, which prevents access of RNA Polymerase II to centromeric repeats, in dynamic equilibrium with Epe1, which promotes transcription . During transcription through the heterochromatic arrays, H3S10phos increases and SWI6 deposition is decreased, thus decondensing the heterochromatin structure. During the late S phase, transcription of heterochromatic repeats by RNA Pol II occurs and an increase in the deposition of Ago1, Clr4 and Rik1 is also observed . The accumulation of Clr4 histone methyl transferase, together with the RNAi induced transcriptional silencing (RITS) complex components, results in the processing of cen siRNAs, which would then direct the methylation of H3K9 at heterochromatic repeats. It was also shown in S. pombe that transcription of the centromeric repeats produces nascent transcripts, which are used as a template by the RNA-dependent RNA polymerase complex (RDRC) to produce dsRNA. The latter is then cleaved by Dicer to synthesize centromeric siRNA which are then loaded onto RITS, bringing about the deposition of H3K9me2 (Clr4 mediated) and SWI6 at the centromeric heterochromatin [7–10]. It was also shown that in fission yeast mutations in the second largest and fourth largest subunit of RNA polymerase II affects the synthesis of centromeric and pre-centromeric siRNAs, respectively [11, 12] and was accompanied by reduction of H3K9me2 and Swi6 at the centromeres. The mutations in RNA Pol II subunits did not cause any significant changes in global transcription but its effect was confined to the centromeric heterochromatin structure. These studies highlighted the fact that RNA Pol II performs an integral function for centromeric heterochromatin structural maintenance in conjunction with the small RNA processing machinery.
We explored the role of RNA Polymerase II on heterochromatin structure in Drosophila because of the availability of polymerase mutations and a well-developed model system for the study of heterochromatin. We employed genetic, biochemical and cytological analysis to address this issue. Our analysis indicates that the largest and second largest subunits of RNA polymerase II interact genetically and biophysically with RNA silencing machinery components. Our data also indicate that RNA Pol II mutants (largest and second largest subunits) link transcription and RNA silencing components to heterochromatin structure in metazoans.
Results and discussion
To further understand the nature of the interactions between RNA Pol II and the RNA silencing machinery, we tested the effect of an array of different RNA silencing machinery mutations on PEV including those implicated in piRNA formation, which acts independently of Dicer . In each case, different mutant alleles of each gene were tested in order to rule out any linked gene effect on PEV suppression. The PEV analysis indicated genetic interactions between dcr-2(L811fsX), dcr-2(G173E), ago-2(414), piwi, piwi, hls , hls , aub [QC42 ], aub [P3a] and RNA Pol II alleles (Figure 1A and 1B and Additional file 1). In each case the trans heterozygotes of RNA Pol II and RNA silencing pathway mutations exhibited stronger suppression of PEV than single heterozygotes. However, the suppression of PEV was strongest in the trans-heterozygotes of RNA Pol II and dcr-2 alleles among all the other combinations tested as indicated by the eye pigment measurements.
The suppression of PEV is a reflection of the changes in the chromatin structure of heterochromatin. The effect of RNA Pol II and RNA silencing machinery trans-heterozygote mutants was not confined to the heterochromatin environment of the chromocentre. This was shown in an experiment employing transgenic flies that have seven tandem copies of mini-white, referred to as DX1 (Figure 1C), that are located in the euchromatin of chromosome 2. Flies that are homozygous for this transgene arrangement have a heterochromatin environment around the mini-white arrays, thus silencing the expression of mini-white transgenes in mosaic fashion . We tested two combinations, namely RNA Pol II 140(A5) and piwi, as well as RNA Pol II 140 (A5) and hls  on DX1 homozygous flies. In each case the trans-heterozygotes reversed the silencing of mini-white to a much greater extent when compared to either single heterozygotes or control flies with no mutations. The exact molecular mechanism of DX1 silencing has not been elucidated but is believed that pairing sensitive silencing might be one of the contributing factors. The experiments performed with RNA Pol II mutants suggest that there is also involvement of a transcriptional silencing component.
In addition to the second largest subunit of RNA Pol II, we also studied the effect of the RNA Pol II's largest subunit mutation on H3K9me2 levels in the adult carcass (Figure 3). As the largest subunit gene (RNA Pol II215 W81) is located on the X-chromosome, a PEV analysis of male flies was not possible and the fact that a translocation balancer chromosome between the X chromosome and the second chromosome was not available precluded any immunofluoresence analysis on larval polytene nuclei. The mutant allele used was W81, which has a truncated carboxyl terminal domain (CTD) due to the presence of a premature stop codon.
Trans-heterozygotes of RNA Pol II215(W81)/+; dcr-2(L811fsX) showed a significant reduction of H3K9me2 in Western blot analysis compared to the wild type as well as RNA Pol II215(W81)/+ alone. The reduction of H3K9me2 with two different subunits of RNA Pol II, in combination with dicer-2 mutations, provides further evidence of a role of RNA pol II in heterochromatin formation in conjunction with RNA silencing genes.
With regard to piRNA genes, previous experiments involved examining HP1 mislocalization in piwi/piwi heteroallelic mutants. This combination did not show a major mislocalization of HP1 . To test the impact of RNA pol II, we introduced the RNA Pol II140(A5)/+ mutation in this background. This combination caused an obvious mislocalization of HP1 (Figure 5).
To establish whether the cytological observations represented a mislocalization or a quantitative difference, we used a Western blot analysis which indicated that HP1 protein levels were the same in the wild type and RNA Pol II 140(A5)/+; dcr-2 (G173E)/+, thus confirming that HP1 is mislocalized and not upregulated in the trans-heterozygote mutants (Additional file 3). The mislocalization can be attributed to a reduced H2K9me2 deposition at the chromocentres of mutants, which allow HP1 to associate with various low affinity binding sites. The above experiments also highlight the role of small RNAs generated by the transcription of heterochromatic repeats in guiding heterochromatin modifications (H3K9me2 and HP1) at the chromocentre.
In plants, experiments have implied a role of WG/GW motifs as docking sites for Argonaute binding such as for AGO4 to the CTD of the largest subunit of Pol IVb subunit NRPD1B , which is a specialized RNA pol II involved with transcriptional silencing. The CTD of the Drosophila lacks any reiterative GW/WG motifs and it might be speculated that the absence of these domains could contribute to the lack of any physical interaction between dAGO2 and RNA Pol II. We could not detect any interaction between dAGO2 and RNA Pol II CTD, implying that the interaction is very weak or indirect. The analysis of the amino acid sequence of RNA Pol II 140 revealed the presence of a PxVxV site (residues 350-354) (Additional file 5). Similarly, AGO2 also contains the pentapeptide PxVxV (residues 486-490)(Additional file 5). The peptide sequence PxVxM/L/V represents the conserved sequence found in all HP1 interacting proteins . Recent experiments performed in flies demonstrate that PIWI interacts physically with the HP1 protein by virtue of the presence of the PxVxV domain . The amino acid replacement of the central valine residue of the pentapeptide abolished interaction between HP1 and PIWI, thus highlighting the importance of the pentapeptide domain for this interaction. As RNA Pol II 140 possesses a PxVxV domain, it is interesting to speculate that HP1 might bridge PIWI and AGO2 with the RNA Pol II 140 subunit. This might constitute a novel RNA Pol II complex in metazoans exclusively dedicated for silencing. We could not address this issue because of the unavailability of suitable RNA Pol II 140 antibodies for immunoprecipitation.
However, we found dAGO1, which typically binds miRNAs, co-immunoprecipitated with RNA Pol II (8WG16) CTD antibody (Figure 6), but not with antibodies against the activated CTD. The presence of dAGO1 in the pulldown fraction using 8WG16 antibodies prompted us to investigate the role of the miRNA machinery in heterochromatin modifications. dcr-1 and ago-1 are two genes that play a predominant role in miRNA metabolism in flies [27, 28]. There was no effect on H3K9me2 modification at the chromocentre of polytene chromosomes of RNA Pol II(A5)/+; dcr-1(Q1147X) compared to wild type (Additional file 6). Similarly this trans-heterozygote combination did not relieve the silencing of the mini-white array DX1 or the In(1)w [m4h] heterochromatin environment. When trans-heterozygotes of RNA Pol II140 (A5)/+; ago1(k04845)/+ were introduced into the In(1)w [m4h] background, there was moderate suppression of PEV (Additional file 7). Similarly, the chromocentre of polytene chromosomes in this background caused moderate reduction of H3K9me2 compared to wild type and there was a significant reduction in H3K9me2 levels in the Western blot analysis. The presence of AGO1 in the RNA Pol II (8WG16) pulldown fraction indicates that AGO1 might have an affinity for binding to small RNAs arising from heterochromatin. This may well be the case as there is evidence that AGO1 and AGO2 have somewhat overlapping functions and there is sharing of biochemical components among miRNA, endo-siRNA and siRNA pathways in Drosophila[29, 30].
The TAF-1/TFIID (TATA box binding protein associated factor 1) is the major component of the transcription initiation complex in eukaryotes. Trans-heterozygotes of RNA Pol II140(A5)/+; TAF-1/+ and the single heterozygote TAF-1/+ shows no effect on suppression of PEV (Additional file 10). This control shows that mutations affecting transcription factors (TAF-1 interacts with RNA Pol II as part of general transcription machinery) had no effect on suppression of PEV and implicates the specific interaction of pol II with the small RNA silencing components.
In order to test whether the double heterozygous combinations affect post-transcriptional functions of the RNA silencing machinery, selected genotypes were examined for an effect on white RNAi. The trans-heterozygotes of (1) RNA Pol II(A5)/+; piwi/+; (2) RNA Pol II (A5)/+; hls /+; and (3) RNA Pol II (A5)/+; dcr-2(L811fsX)/+ had no effect on w-IR RNAi (Additional file 11). This experiment indicates that in RNA Pol II 140(A5)/+ heterozygotes in which the dose of RNA Pol II is halved, the effect is more pronounced on the heterochromatin structure (transcriptional gene silencing (TGS), but the w-IR RNAi pathway is unaffected under these circumstances. To obtain further insight into the role of RNA Pol II in silencing, we tested for an effect on TGS silencing involving the interaction between Alcohol dehydrogenase-white hybrid transgenes . In these flies the transgene copies of w-Adh bring about silencing of Adh-w at the transcriptional level; however, the silencing is eliminated in a piwi mutant background [31, 32]. When RNA Pol II 140(A5)/+ was introduced into this genotype, there was no apparent effect on silencing (Additional file 12). The trans-heterozygotes of RNA Pol II 140(A5)/+; hls /+ also had no effect on relieving the silencing of Adh-w by w-Adh transgenes.
The role of small RNAs in maintaining genomic stability and chromosome structure is receiving increasing attention. It has now been well articulated that in Drosophila, S. pombe and mouse defects in the RNAi machinery lead to a compromised heterochromatin structure and aberrant regulation of transposable elements. Recent studies in S. pombe, Arabidopsis and mouse have further elucidated the mechanism by which small RNAs arising from heterochromatic repeat transcription modify histone methylation in the heterochromatin. Plants have an exclusive RNA Pol V dedicated for heterochromatin maintenance [33, 34]. The case of centromeric heterochromatin formation in S. pombe is best studied in regard to the relationship between the RNAi machinery and RNA Pol II transcription.
We observed genetic and biochemical interaction between the second largest subunit of RNA Pol II and RNA silencing genes. The genetic interaction had a chromatin component in terms of suppressing PEV and reduction of H3K9me2 levels. The interaction between RNA Pol II and Dicer-2 was the strongest in terms of suppressing PEV of w [m4h]. In contrast, dcr-1, which has a key role in miRNA metabolism, had no role in determination of heterochromatin structure in our studies. The interaction of RNA Pol II with other proteins in the RNA silencing pathway displayed a similar level of effect on heterochromatin structure.
An interesting aspect of this study is the involvement of piwi and aub in suppressing PEV and H3K9me2 reduction in combination with RNA Pol II. PIWI and Aub associate with a unique class of rasiRNAs called piRNAs that are found predominantly in the germline cells and are independent of the Dicer processing machinery [15, 35]. However, piwi mutations also have effects in somatic cells and the PIWI protein has been demonstrated to be present in the soma and piRNAs have been detected in somatic tissues [26, 36]. Also, the fact that Dicer-2 co-immunoprecipitates with RNA Pol II and their genetic interaction strongly suppress PEV might indicate a possible role for endo-siRNAs in regulating heterochromatin structure.
Recent experiments in plants reveal the presence of a RNA Pol V complex which shares some subunits of RNA Pol II and participates in RNA silencing . Because animals lack RNA Pol IV and Pol V, it is possible that they possess a specialized RNA Pol II complex involved in RNA mediated silencing. The interaction of Dicer-2 with the transcriptionally competent form of RNA Pol II might reflect one such form. It might also represent an additional mechanism of gene regulation by degrading aberrant transcripts during the elongation process. The involvement of dAGO1 with RNA Pol II in co-immunoprecipitation experiments suggests an additional level of complexity regarding the involvement of 'Argonaute slicer' in the cleavage of small RNAs. While this result might implicate miRNAs being involved in heterochromatin formation, the following observations suggest otherwise: (1) trans-heterozygotes of RNA Pol II and dcr-1 have no effect on w [m4h] PEV suppression and H3K9me2 levels; and (2) miRNAs have a central bulge making it difficult to explain how specificity is achieved when miRNAs base pairs imperfectly with nascent centromeric transcripts/centromeric DNA. The involvement of AGO1 probably suggests that small RNAs arising out of cleavage of nascent heterochromatic transcripts have some affinity for AGO1.
Our experiments suggest that RNA Pol II transcription through centromeric heterochromatin results in a nascent transcript that could fold into a stem loop structure by virtue of the presence of many direct and inverted repeats present in the heterochromatin. This acts as a substrate for Dicer-2 generating endo-siRNAs, which guide chromatin modifications at the heterochromatin. The interaction of Dicer with RDRC has been shown to be crucial for centromeric heterochromatin structure in S. pombe. The interaction between RNA Pol II and Dicer-2 in Drosophila reflects a similar mechanism. The exact role of the RNA Pol II second largest subunit mutations on small RNA synthesis from the centromere is not known but it is possible that it disrupts the biochemical interaction between the CTD of the largest subunit and Dicer-2. The heterochromatin specific histone modifications are dependent on both the RNA pol II complex as well as small RNA silencing machinery components.
Fly stocks and PEV analysis
All flies were grown at 25°C on standard food medium. The genotypes were:
y w; RNA Pol II 140(A5)/TM3, Sb
RNA PolI 215(W81)/FM7a
ru h th stcuRpII140 [wimp] sr[1[ e [s] ca/TM3, Sb
y w; ago2(414)
y w; eyFLP; FRT 42 D dcr-2(G173E)/CyO
y w; eyFLP FRT 42 D dcr-2(L811fsX)/CyO
y w; eyFLP FRT 82B dcr-1(Q1147X)/TM3, Ser
cn1 P(ry t 7.2) ago-1(04845)/CyO
ru st hls [E616] e ca/TM3, Sb e
ry cv c sbd hls [DE8]/TM2, Ubx ry e
w1; hls  e/TM3, Sb e
w1; aub Df(P3a)/CyO
y w; piwi/CyO
y w; piwi/CyO
y w; DX1/CyO
The male flies of each of the genotypes mentioned above were crossed to female In(1)w [m4h]; SM6a/Gla; TM3, Sb/Ser. The single heterozygotes of RNA Pol II 140 in the w [m4h] background were then mated with RNA silencing mutants in a similar background. The F2 males were then sorted into different single and double heterozygous groups and analysed for PEV.
In order to analyse the effect of piwi on DX1 silencing, the DX1(lac w) transgene array was recombined with Sco and balanced over CyO to produce DX1 Sco/CyO flies. The males of DX1 Sco/CyO were then crossed to female piwi/CyO. In the next generation, female non-curly DX1 Sco/piwi females were selected, which then were crossed to male w; Gla/SM6a; TM3, Ser/Sb. In the next generation non-Sco flies DX1 piwi/CyO were obtained.
Measurement of eye pigment
Fly heads from 10 animals were homogenized in methanol containing 0.1% hydrochloric acid (HCl). The absorbance of the supernatant was measured at 480 nm after the centrifugation of the fly head homogenate. Three independent experiments were performed in each case.
Immunofluoresence analysis of polytene chromosomes
To analyse trans-heterozygotes of RNA Pol II 140 and dcr-2, piwi and hls, we crossed them into a T(2;3)CyO Tb background. In the next step, males of RNA Pol II 140 and females of piwi, dcr-2(G173E), hls  (each balanced over T(2;3) CyO Tb) were crossed and non-Tb third instar larvae were selected for further analysis. Three to four pairs of salivary glands each from control and trans-heterozygotes were dissected in 0.7% NaCl. The glands were then fixed for about a minute in 3.7% formaldehyde in phosphate buffered saline (PBS; ice-cold). The glands were then kept in a solution of 45% acetic acid and 3.7% formaldehyde for about 2 min and then squashed. The slide was placed on dry-ice for 20 min, the cover slip removed and then washed twice in PBS for 10 min each and blocked for 30 min in a solution of PBS containing bovine serum albumin (BSA). The following antibodies were used at 1:100 dilutions: Sxl (Hybridoma bank, University of Iowa, USA), HP1 (from Dr S Elgin) and H3K9me2 (Upstate). The primary antibodies were incubated overnight at 4°C. On the following day, the slides were washed twice in PBS and blocked in PBS-BSA solution. The slides were then blocked with 5% goat serum for 30 min at 37°C. The secondary antibodies (1:100 goat anti rabbit-conjugated with fluorescein isothiocyanate and 1:200 goat anti-mouse conjugated with Texas red) were then applied to the slide for about 1 h at 37°C. The slides were washed twice in PBS and visualized using fluorescence microscopy after application of 4', 6-diamidino-2-phenylindole. The images were adjusted using Photoshop CS3 version software.
The gently squashed polytene spreads  were prepared in the same manner except that solution II (45% acetic acid in PBS) was omitted. The glands were squashed in 0.7% NaCl and the fixed in 3.7% formaldehyde for 20 min at 4°C.
Western blot analysis
Adult flies (aged 12-15) were dissected to remove the ovaries. The carcasses were then homogenized in HEPES buffer containing protease inhibitor cocktail (Pierce). The homogenate was then acidified with HCl to a final concentration of 0.2 N HCl and kept on ice for 1 h. The homogenate was then centrifuged at 11,000 g for 15 min and the supernatant was then neutralized with NaOH. The histone enriched protein lysate was then boiled with Laemmli sodium dodecyl sulphate sample buffer and loaded onto the gel. The Western blot analysis was performed by standard procedures . The antibodies used were rabbit polyclonal H3K9me2 (1:1000) and H4 loading control (1:1000). Supersignal pico chemiluminescent substrate kit (Pierce) was used to observe the bands and Image gauge (NIH) software was used to measure the density of bands.
Wild type embryos at 6-18 h were homogenized in total lysis buffer containing 1 M Tris pH 8, 150 mM NaCl, 10 mM EDTA, 10% glycerol and protease inhibitor cocktail (Pierce). The homogenate was then kept for 15 min on ice. After centrifugation the lysate (about 500 μg) was applied to the activated amino link resin beads. The beads had RNA Pol II monoclonal antibodies 8WG16 (50 μg from Covance) covalently linked to the beads. The lysate was incubated with the beads with gentle mixing at 4°C overnight. The interacting proteins were eluted and analysed by western blots. Native mouse serum was used as a negative control. The antibodies used were Dicer-2 and Ago-1 rabbit polyclonal antibodies.
Immuno-co-localization using blocking peptide on polytene chromosomes
Salivary glands from wild type larvae were squashed and fixed in formaldehyde and acetic acid solutions as described above. AGO1 antibody (Abcam) was diluted 1:4 in PBT (PBS+Triton X-100). In order to test for the specificity of the antibody the specific peptide bound by the AGO1 antibodies was used at 1:50 dilution, mixed with AGO1 and incubated at room temp for 30 min with occasional gentle shaking. Sxl antibody (Hybridoma bank, Iowa, USA) was used as an internal control. The nuclei were observed by standard methods as described above.
The ChIP method was adapted from a previously published protocol . For each ChIP, a reference sample (Mock) corresponded to a ChIP performed at the same time without the addition of the specific antibody. Formaldehyde, at a final concentration of 1.8% in buffer A1, that is, 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES (pH 7.6), 0.5% Triton X-100, 0.5 mM DTT, 10 mM sodium butyrate, protease inhibitor cocktail (Roche, Basel, Switzerland), was used for crosslinking while crushing whole Drosophila animals (adults) for 10 min at room temperature. After blocking the reaction with glycine, and after three washes (5 min each at 4°C with buffer A1), subsequent steps were performed as described in . For immunoprecipitation (IP) reactions, 5 μl H3K9me2 (Millipore) was used per reaction.
Real time polymerase chain reaction analysis
Two microlitresl of DNA sample was used for the analysis. All samples were analysed in triplicate. The threshold values (Ct) were used to calculate the fold change differences by the delta delta Ct method. The IP and mock samples were normalized to the input. The ABI 7300 (Applied Biosystems Inc) was used for the analysis. The SYBR green master mix was purchased from Applied Biosystems Inc. The primer sequences used were:
bovine serum albumin
carboxyl terminal domain
phosphate buffered saline
RNA-dependent RNA polymerase complex
RNAi-induced transcriptional silencing
transcriptional gene silencing.
The authors express their gratitude to the following individuals for their generous supply of antibodies and fly stocks - Dr Mark Mortin, Dr Mikkiko Siomi, Dr Qinghua Liu, Dr Richard Carthew, Dr Sarah Elgin, Dr Nicole Riddle and Dr Phillip Zamore. This work was supported by NSF grants MCB-0211376 and 0641204.
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