Topics in Eukaryotic Regulation
Topics in Eukaryotic Regulation BIOCHEM 703
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E CD i Sausu An Epigenetic Role for Maternally Inherited piRNAs in Transposon Silencing Julius Brennecke et al Science 322 1387 2008 DOI 101126science1165171 Science The following resources related to this article are available online at wwwsciencemag org this information is current as of January 25 2009 Updated information and services including highresolution gures can be found in the online version of this article at httpwwwsciencemagorgcgicontentfu32259061387 Supporting Online Material can be found at httpwwwsciencemagorgcgicontentfu32259061387DC1 A list of selected additional articles on the Science Web sites related to this article can be found at httpwwwsciencemagorgcgicontentfu32259061387relatedcontent This article cites 28 articles 14 of which can be accessed for free httpwwwsciencemagorgcgicontentfull32259061387otherarticles This article appears in the following subject collections Molecular Biology httpwwwsciencemagorgcgicollectionmolecbiol Information about obtaining reprints of this article or about obtaining permission to reproduce this article in whole or in part can be found at httpwwwsciencemagorgaboutpermissionsdtl Science print ISSN 00368075 online ISSN 10959203 is published weekly except the last week in December by the American Association for the Advancement of Science 1200 New York Avenue NW Washington DC 20005 Copyright 2008 by the American Association for the Advancement of Science all rights reserved The title Science is a registered trademark of AAAS Downloaded from wwwsciencemagorg on January 25 2009 with class B 14 17 18 Consistent with a role for Cap H2 in antagonizing homolog pairing the Uhch I Ub p enotype was dominantly enhanced by Cap H2 mutations Fig 3A com pare class D with class B Fig 3C and table S1 Conversely Cap H2 overexpression suppressed the Uhch I UbXIi i wing phenotype closer to wild type Fig 3A compare class D with class B Fig 3C and tables S3 to 85 Cup H2 mutant enhancement of the Uhch I beI phenotype was suppressed irl a chromosomal rearrangement backgron Rbecbquot I beIl t that is thought to disrupt allelic associations be tween bea x I beI and wild type be Fig A compare class B with class C Fig 3C table s2 7 The bea I UbXICap H2 and RbeCbquot I UbXICap H2 ies only vary by the reciprocal translocation that moves 3R bear ing becbquot I be to 2R and vice versa This suggests that Cap H2 enhancement of the beam UbXI phenotype is through increasing the association of homologous loci Alternatively Cap H2 function may follow trans chromosomal interactions for example acting locally to enable enhancer interactions irl trans or as a general transcriptional rep es or Although either is for mally possible Cap H2 s ability to globally dis rupt aligned polytene structure suggests it carries out a related function irl diploid cells to antago nize trans chromosomal interactions Cap H2 was tested irl a second transvection system involving mutant alleles of the gene yellow y In yMZWW and y W 8 ies there is mini mal cuticle pigmentation yet when placed irl trans to one another y82 9218 complementation occurs with partial restoration of pigment nearer to wild type levels The W8 allele is a deletion of the yellow promoter and the y g allele a deletion of upstream enhancer elements It is thought that partial complernentation occurs in y 9y1 8 through the ability of ng s enhancers to act in trans to associate with the intact promoter of y 72 9 and then to activate yellow 19 20 As are bech d UbAJ transvection of y g ym8 is en hanced irl a Cop H2 mutant background which leads to darker pigmentation of the abdominal stripes relative to controls Fig 3 B and D 7 Tran 39 enhanced by slowing the rate of cell division 2 The percent of Cap H2 homozygous mutant cells speci cally in mitosis was cytologically found to be greater relative to h ero gous controls but this was statistically insigni cant 137 n 1318 versus 095 n 3375 P gt 005 952 Furthermore with ow cytometry homozygotes and heterozygotes did not vary signi cantly irl these data do not mle out a cell cycle delay lead irlg to enhanced transvection they also do not support a major regulatory role for Cap H2 in cell cycle progression Cap H2 s ability to disassem ble the aligned structure of polytene chromosomes instead suggests that it antagonizes transvection by inhibiting homology dependent chromosomal interactions in diploid somatic cells Just as condensin mediated supercoiling has been proposed to initiate chromosome condensa tion 12 we speculate that supercoiling activity also exists in interphase nuclei and can disrupt chromosome alignment This may be through providing a force that physically disrupts irlterchro mosomal associations andor favors intrachromo sornal higher order structures that make inaccessible regions prone to trans associate g S13 This condensin activity may be a crucial aspect of gene regulation by disrupting tran communication of allelic regulatory elements References and Notes l W Duncan Anno Rev Genet 36 521 2002 F Savarese R Grosschedl Cell 126 248 2006 F Bantignies C Grimaud S Lavrov M Gabut G avalli Genes Dev 17 2406 2003 J Vazquez M Muller V Pirrotta J W Sedat Mol Biol Cell17 2158 2006 Edgar T L Orr Weaver Cell 105 297 2001 K Dej A C Spradling Development 126 293 1999 Materials and methods are available as supporting material on Science Online A Schleiffer et ol Mol Cell 11 571 2003 T Hirano Curr Biol 15 R265 2005 laxUI Jgt WNb k 7 o 3 o 3 amp aE i i u n o o 58 2003 T Hiram Not Rev Mol Cell Biol 7 311 2006 K l mura T Hirano Cell 90 625 1997 E B Lewis Am Not 89 73 955 C V Cabrera J Bolas A Garcia Bellido Nature 318 569 1985 16 R A H White M Akam Nature 318 567 1985 t t t t t t i mprg kpwm REPORTS 17 J E astelli Gair J L Micol A Garcia Bellido Genetics 126 177 1990 M J Gemkow P J Verveer D J Arndt Jovin Development 125 4541 1998 19 J R Morris en P K Geyer C T Wu Proc Natl Acod Sci USA 95 10740 1998 20 P K Geyer M M Green V G orces EMBO 9 2247 1990 M M Golic K G Golic Genetics 143 385 1996 We acknowled e T Orr Weaver for supporting the initial screen We are grateful to the following individuals C Boswell microscopy V Lien UAS Cap H2 mCherry transgenics M Hart mitotic index P Jansma fly imaging and J Blumenstiel FlSH We thank S Hawley T Orr Weaver M Lilly E Kelleher and the JMS T student forum for critical reading of the manuscript and especially C T Wu for exceptional guidance with transvection studies We are gr 1 Vazquez C Zuker P O Farrell and C T Wu for fly stocks Support was from NH GM069462 grant to 6B and TH was funded by the University of Arizona NSF lGERT integrative Graduate Education and Research Traineeships Program in Genomics and NH Grant for Graduate Training in Biochemistry and Molecular Biology n a n Supporting Online Material vwwvsciencemagorgcgicontentfull32259061384DC1 Materials and Methods Figs 51 to 13 Tables 51 to 55 References Movies 51 and 52 Au ust 2008 accepted 2 October 2008 101126science1164216 An Epigenetic Role for Maternally Inherited piRNAs in Transposon Silencing ulius Brennecke Colin D Malone Alexei A Aravin1 Ravi Sachidanandamf r Alexander Stark2 3 Gregory Hannonli In plants and mammals small RNAs indirectly mediate epigenetic inheritance by specifying cytosine methylation We found that small RNAs themselves serve as vectors for epigenetic information Crosses between Drosophila strains that differ in the presence of a particular transposon can produce sterile progeny a phenomenon called hybrid dysgenesis This phenotype manifests itself only if the transposon is paternally inherited suggesting maternal transmission of a factor that maintains fertility In both P and Ielement mediated hybrid dysgenesis models daughters show a markedly different content of Piwiinteracting RNAS piRNAs targeting each element depending on their parents of origin Such differences persist from fertilization through adulthood This indicates that maternally deposited piRNAs are important for mounting an effective silencing response and that a lack of maternal piRNA inheritance underlies hybrid dysgenesis intercrosses between wild caught males and laboratory strain females are sterile because of defects irl gametogenesis whereas the genet ically identical progeny of wild caught females and laboratory strain males remain fertile 1 3 This phenomenon known as hybrid dysgenesis was attributed to the mobilization irl dysgenic progeny of P element or I element transposons which were present irl wild caught ies but ab In Dmsophila melanogaster the progeny of sent from laboratory strains 4 9 The disparity irl outcomes dependin on the parent of trans poson origin indicated the existence of cytoplas mically inherited determinants of the phenotype which must be transmitted through the maternal germ line 8 9 The control of mobile elements irl germ cells depends heavily on a small RNA based immune system composed of Piwi family proteins Piwi Aubergine and A603 and piRNAs 10 11 www5ciencemagiorg SCIENCE VOL322 28 NOVEMBER2008 Downloaded from wwwsciencemagorg on Janu 1388 REPORTS Both Fiwi and Aubergine Aub are deposited into o es and accum 39 developing o cyt ate in the pole plasm 12 13 implying possible transfer of einal piRN s in the lines of their progeny We therefore asked whether maternally deposited small RNAS might affect transposon suppression in a 39 le fashion and whether piRNAS might be the maternal suppressor of hybrid dysgenesis We first focused onI element induced hybrid dysgenesis 14 A cross of inducer males carry 39 ements desigtated T in Fig 1 to reac devoid ofaotiveI e ignated quot11 yielded dysgenic daughters termed SF Fig 1 and fig S1 5 6 8 15 1hese were smile I hi1 normal 39 Ste 39 39ty correlated with element expression in SF progeny termed RSF Fig 1 and g S1 1Watron sihoot ot aiologiial siienier Howard Hughex Megiial quotmime Cold Spring Harbor Laboralmy CSHL 1 Bungl wn Road Cold Spring Harbor NV11724 USA ZBroad Innitiite oi lvlarraihiirenr Innitiite oi ieihonology and Harvard Uniie 39 Cambrid e ililAu21A1 USA Zom uler siienie and Artifiiial lntelligenie laboratory Marraihhrettr h Innilule of ieihnnlngy Cam ridge MA 02139 u We seq from the ovaries of inducer wk strains 0 to 2 hour and nondysgenic crosses and ovaries from SF and RSF daughters Fig 1A and g S1A Both d e ly embryo libraries con tained similarly complex small RNA populations fig S1A 1his indicates that small RNAS were uenced 13 to 29 nucleotide RNAS WW and reactive maten y deposited because the zygotic ge nome remains inactive during most ofthe period atwe analyzed In Dmsophx39ltz piRNAs loaded into the Fiwi Aub and 3 proteins exhibit distinctive features 17 1s piRNAs occupying Fiwi and L Fig 2A However there were notable differ ences Fig 2 B and C 1he1element exhibited i w v v v the greatest disparity 21 fold between strains Fig 213 and c and fig S5A Less pronounced differences were noted for rm and gypsyu an e dif en eminoredin correspo g onic libraries withreactive mothers depositing 12 fold fewer element ducer mothers Fig 213 1his supports the hypothesis that piRNAs corre Au contain a 39 39 39 39 SZA In contrast AG03 harbors mainly Sense 39 S W1 a 5mm bias by adenoslne ax lations A comparison RNAS in mothers and embryos indicated robust maternal inheritance for the AubFiwi pool and bound piRNAs fig S2 A and B 1his obser vation is consistent with the degree of maternal 39 39 in many studies of hybrid dysgenesis 1he outcome of an inducer reactive 1 R dysgenic cross manifests itself not in embryos but in adults We therefore asked whether differ ences in maternally deposited piRNAs continued 39 2 weeks atter transposons Fig 2 B an A pro les for many elements had adjusted to a stable Thexe a nlribuled eguahy to thir worh teins figs S3 and S4 12 13 opment As an example for 1731 a ninefold 10029 USA V V 1T0 whom iorrerpondenie rhohld he addrerred E mail quot3 I S5BJInRSF fmales hannonirhlegh similari between inducer and reactive strains element iRNAs dro ed twofold ascom ared 1 pp 1 Fig 1 The H hybrid A non dysgenic cross dysgenic cross sis i Crossing schenietu genr X 039 d x h law d h t h d ared Sundysaequot Wine WP hihhitoheo WWW MR in amed Small l i g 839 W4 3 33 branes were made from g g g 30 g 3 30 flies indicated by as gaff g D a D teri s Bar diagra 0 5 g 5 dicate iertility analysis 0 embryos g 40 0r2h embryos g 40 a pa ntal a g g ieniales an the basisai 3 2W g 20 egg h ing rates ii 3 A a o 0 number a counted parental Fl parental Fl 954 i quot aunt HSFdaughlels generation generation SFdaughlels generation generation heago quot215 quot401 quot228 RSF F1 daughters daughters OD ouryte NC nurse tells 28 NOVEMBER 2008 VOL 322 SCIENCE SF F1 daughters wwwsciencemagorg Downloaded from wwwsciencemagorg on January 25 2009 60 gt ovarian piRNA distribution on Repbase transposons roe 50 tilstrain reactive I WW8 strain Inducer per million small RNAs 8 10 Ireomen sequenced piRNAs in thousands B piRNAs from 2 arental ovaries 9 0 p WiiiS 9 2 99 Qquot mv transin rndga ide 1x nomad ZAM 1 v sle MA stalkeut doc doc 3 gym 100A RTlA stalker Grelemenl Bee higher in inducer strain RT B gypsv2 copia uasvgodo in 5 blood vasvl 11am irelemenl 1 higher in reactive strain hobo Srelemenl DNARepl PiotoP 110151151101151201 transposon piRNA ratios Wiiislvw 1 WlllglMpiHNA ratios Fig 2 lR hybrid dysgenesis correlates with maternal piRNA inheritance A Normalized piRNA counts for Rep ase transposons are plotte for ill1118 inducer and reactive ovaries B Left Fold differences in piRNA counts comparing ill1118 and 111 mothers are shown red line indicates a 11 ratio wwwtsciencemagtorg SCIENCE REPORTS 1 731 s 12 quotmm mlcropia gyp y I miiiillH 75 most highly targeted elements used In analysis below i E parental ovaries 0 element 102 103 104 1o5 108 reads from M reactive strain F1 ovaries element reads from RSF ovaries 8 3 4 5 6 10 10 10 10 10 reads from SF ovaries Right Transposon piRNA ratios for mothers embryos and F1 progeny SF RSF ratio are s own as a heat map C Scatter plots indicating transposon piRNA correlations between 118 and 111 mothers top and their respective intercross progeny bottom VOL 322 28 NOVEMBER 2008 Downloaded from wwwsciencemagorg on January 25 2009 1389 REP 1 390 O RTS with their inducer mothers g SSB This par alleled the overall reduction in active I element load as the inducer genome was diluted by that of the reactive strain However limits on the adapt ability of the system are seen in SF daughters for the I element and to a lesser degree for timrit and gypsy12 Fig 2 B and C and g S5 Though SF daughters contained 16 fold more I element piRNAs than their reactive mothers gt Fig 3 A piRNA amplification loop between active Ielements and ancestral fragments A Den sity profile of piRNAs matching the Ielement with up to five mismatches left and profiles for those species with zero top right or at least one mismatch below e active element sense 50 00 sequenced piRNAs indicate species that have the potential upper or must lower derive from the 42AB piRNA cluster Ielement fragments con tained within the 42AB cluster are indicated in red B Shown are fractions of Ielement piRNAs from 118 mothers and SF daughters right that match the active sequence with the indi cated number of mismatches split 9 into sense and antisense C Right All Ielement frag ments in the Release 5 genome c sequence split into modern inser tions blue and ancestral frag ments black and sorted by chromosomal position are shown gray coloring indicates hetero D we a o m o c 0 element piRNAs V w o o o W induwr Strain uniquely mapping IreemenrpiRNAs per nt per million small RNAs if reactive strain 0 05 0 0A 0 02 0 5 these were still sevenfold less abundant than in RSF daughters This de cit ultimately results in de repression of the paternally inherited active I elements and in sterility Fig 1 A an Though active I elements are con ned to in ducer strains all D melanogaster strains contain ancestral Irelated fragments 6 I9 2 These are i picall quot 1 1 i J 1 it 80 to 95 identity to the modem I element 0 WWW M 7m 50 all ireementpiRNAs 1118 W W inducerstiaii i 2000 i Such fragments have been proposed to mediate adaptation to and suppression of I elements in inducer strains 15 22 24 To understand the lack of adaptation to I elements in SF daughters we probed the nature of interaction between active and ancestral I element sequences The ping pong model of piRNA biogenesis J quot 39 J quot Slicer dependent arripli cation cycle between active transposons and o mismatch to active ieiemenx 40 60 gt l mismatch to active Ireemery OO 90 iia merits Within 42 B piRNA cluster match to 42AB iragmems 4pm umque to 42AB iragmems 3000 4000 5000 e000 rit i i i i 1000 2000 3000 4000 5000 nt WK reactive SF SUaIi daughters tr of mismatches to active ieiemerri as 0 i 2 3 4 5 6 6 9 piRNA orientation 0 i000 i i ORFl WW9 inducer strain 0 A O 2 chromatic 39 p HC pericentromeric HC Red fragments map to the 42AB piRNA cluster Left Bar diagrams indicate the density of piRNAs 42AB fragments in red As frag ments with high piRNA density were not more divergent overall clustering in 42AB is not an amemwm l artifact of analysis fig S7 12 EpiRNAciusiei 28 NOVEMBER 2008 VOL 322 Ireemenr ORF2 h romat ic insertions of modern elements ancient leeemenrfragments divergence 642 SCIENCE www5ciencemagrorg Downloaded from wwwsciencemagorg on January 25 2009 trans oson fragments resident in piRNA ciusters 17 18 Sequence features tting this modei wee obvious in piRNAs from the induce strain 39 deviated from the modern sequence and here Fig 3A or those piRNAs the overwhelming majority 90 wee antisense As a whoie sense oriented species showed a strong tendency Because the reactive strain Lacks active I ement copies no ping pong ampiirication oc curred and piRNAs mapping to the sense or 39 39 7 De SF daughters we still observed a clear trend for i i i i Y O copies which were paternaiiy transmitted Fig 3BThis39 39 39h h 4739 copies Fig 313 he 0 P I cies must have originated from haemchmma c agmems Fig 4 Suppression at A with ddysgenesismrr relates iih39th niatemauy 0 de usited iRN A a alizedpn mber at 5 Ha ovarian blade and early g Ur enihryuniciuiange piRNAs a NA item the indiiated strains i W niappi g u the e 17 W and Prelemems iihth up 0 mm m ihree F and I Dr wiih me P misma es Tu E Har the tight sense upper cu Lk a sen lawerd ities 5 Di piRNAs are displayed 3 NA aver the Prelem tin par WW rentalavari uithe Har M and m strains The 0 atthe tiun af Har meielumericPrmsemun in 5 Lk the m strain isi iiated 5 The immnrexun structu a NA atthe relem is shuwn CL me heiuw Fluvarian murr phuluay PW PWE m dys progeny are shown NA x Har LKX Har Wiiigx Ha wwwsc39iencemagorg SCIENCE VOL 322 pIRNAs per million small RNAs i0000 i000 39 daugh ters although they uitimateiy failed to siieice the eiement REPORTS I In both reactive and induce strains the AZAB ciuste represents a major source of piRNAs tar geting avariety ofmobile eiements g SS 17 Ofaii hMevm hv mati 7 39 sequenced dam seven Lie within mm M ma amen ii with our reactive and induce strains fig 59 None of the o h ti39 rrra jn the remaining 19 most active ciusters In the in duce strain the majority of heteochromatin derived Ieiement piRNAs arose from ancestral agments within AZAB inciuding many of the most abundant species Fig 3 A and C Despite pIRNA densnles parental across Peelemenl 20000 2 100 z c a 50 u a o g 0 2 g Hat 2000 3000 4000 m 50 Lk tinncaticn 2 00 mNAsUam I paientai OVaHeS 072h embiyos iFi exon0 exoni exon2 exona WK 0 i000 2000 a000rit pingepong pIRNA densities F1 progeny across Peelemenl Signature 50 40 2 30 E100 a 20 g 50 8 i0 3 0 i i i Warm a 5 i0 i5 20 25 m 50 kaHar 40 tinncaticn W in NAsUam 20 i0 j War i i i r 5 i0 i5 20 25 NAXHa 40 30 20 w m xHar i0 0 i i i i i 5 i0 i5 20 25 0 i000 2000 3000nt 5 end oveiiap m 28 NOVEMBER 2008 Downloaded from wwwsciencemagorg on January 25 2009 139 REPORTS a more than 20 fold difference in their relative levels I element piRNAs matching heterochro matic fragments were also derived from 42AB in the reactive strain Fig 3C We sought to test whether the role of mater nally inherited small RNAs in transposon silenc ing was general In P strainM strain P M hybrid dysgenesis crosses between males containing P elements P strains and females devoid of such elements M strains yield sterile progeny with severe gonadal atrophy GD sterility 3 9 We examined small RNAs from Harwich Her a P strain containing 30 to 50 P element copies and M21118 here serving as an M strain Harwic showed strong matemal deposition of P element iRN as bo 39 their 0 to 2 hour embryos lacked such species Fig 4A This contrasted with I andF element p39 As which were abundant in parents and embryos from both strains Crosses between Harwich males and M21118 emales yielded dys genic GD progeny Because of the impact of severe gonadal atrophy Fig 4B and table S1 we normalized the daughter library using piRNAs target39 th F element a transposon exhibiting consistent pro les in all strains examined Clear ly dysgenic daughters lacked prominent P element piRNAs and signatures of the ping pong ampli cation cycle Fig 4B Lerik PIA designated Lk contains two P le ts in the X TAS iRNA cluster and Nasr Allah PIA designated NA contains a single 5 truncated insertion at the same locus 25 26 Lk and NA both produce and maternally depositP element piRNAs Fig 4A the extent of its only P fragment F 25 Unlike Wm Lk and NA mothers were able to produce fertile offspring with Harwich This result correlated with robust piRNA production 39 ughter ovaries and with a strong signature 0 the ping pong ampli cation cle The 5 d of the P element largely lacked piRNAs particularly antisense species in both dysgenic ies and fertile NA Harwich progeny Fig 4B NA does not deposit maternal pi s corresponding to this region because of the trun cation of its P element in X TAS Thus maternal piRNAs are important for potent piRNA gener ation in daughters even when the P element is being effectively silenced by piRNAs matching other parts of o piRNA clusters have been envisioned as a genetic reservoir of transposon resistance with immunity being determined by the content of these loci I 7 Our data indicate that the content o iRNA clusters alone is insu icient to provide resistance to at least some elements wi i a single generation Instead maternally inherited small RNAs appear to be essential to prime the resistance system at each generation to achieve level of ancestral I fragments 29 Rearing of reactive mothers at elevated temperature or increases in maternal age raise the pr rtion of fertile progeny These observations suggest that the experience of the mother translates into a dominant effect on progeny Our data suggest that this experience may be transmitted through variations in matemally deposited small RNA populations T us transmission of instructive i A populations shaped by both genetic and environmental a ors may provide a previously unknown mechanism for epigenetic inheritance References and Notes 1 G Picard P L Heritier Drosophilo Inf Serv 46 54 1971 2 G Picard Genetics 83 107 1976 3 M G Kidwell F Kidwell A Sved Genetics 86 813 1977 4 G Rubin M G Kidwell P M Bingham Cell 29 987 1982 5 A PelissonMol Gen Genet 183 123 1981 6 A Bucheton R Faro H M Sang A Pelisson D Finnegan Cell 38 153 1984 7 N G Kidwell Proc Natl Acad Sci USA 80 1655 1983 8 S Chambeyron A Bucheton Cytogenet Genome Res 110 215 2005 P Castro C M Carareto Genetico 121 107 2004 10 A A Aravin G Hannon Brennecke Science 318 761 2007 C Klattenhoff W Theurkauf Development 135 3 2008 H B Megosh D N Cox C Campbell H Lin Curr Biol 16 1884 2006 N Harris P M Macdonald Development 128 2823 2001 Materials and methods are available as supporting material on Science Online avarec M P Gassama T HeidmannMol Gen Genet 248 381 1995 16 M C Seleme Busseau S Malinsky A Bucheton D Teninges Genetics 151 761 1999 17 Brennecke et ol Cell 128 1089 2007 o b b F NH H w H P 18 L S Gunawardane et ol Science 315 1587 2007 published online 21 February 2007 101126 science1140494 19 P Dimitri A Bucheton Cytogenet Genome Res 110 160 2005 20 M Crozatier C Vaury l Busseau A Pelisson A Bucheton Nucleic Acids Res 16 9199 1988 21 C Vaury P Abad A Pelisson A Lenoir A Bucheton Mol Evol 31 424 1990 M P Gassama X Dramard T Heidmann 22 S Jensen Genetics 162 1197 2002 23 S Jensen M P Gassama T Heidmann Not Genet 21 209 1999 24 S Malinsky A Bucheton l Busseau Genetics 156 1147 2000 25 L Marin et ol Genetics 155 1841 2000 26 S Ronsseray M Le mann D Anxolabehere Genetics 129 50 27 P Blumenstiel D L Hartl Proc Natl Acad Sci USA 102 15965 2005 28 A Bucheton Heredity 41 357 1978 29 ama d eidmann S ensen PtoS One 2 e304 2007 30 We thank M Rooks and D McCombie CSHL for help with deep sequencing S Jensen and S Ronsseray for fly stocks and helpful discussions and D Finnegan fort e Ielement ORF 1 antibody 18 is su fellowship from The Ernst Schering Foundation CDM is a Beckman fellow of the Watson School of Biological Sciences and is supported by an NSF Graduate Research Fellowship a A S is supported by a Human Frontier Science Program fellowship This work was supported by grants from NH to GH and AAA and a kind gift from K W Davis to GH Small RNA libraries are deposited at Gene Expression Omnibus accession no GSE13081 data sets GSAB27620 to GSAB27634 Supporting Online Material vwwvsciencemagorgcgicontentfull32259061387DC1 Materials and Methods 26 Au ust 2008 accepted 27 October 2008 101126science1165171 PA824 Kills Nonreplicating Mycobacterium tuberculosis by Intracellular N0 Release Ramandeep Singh Ujjini Manjunatha1 2 Helena I M Boshoff1 Young Hwan Ha1 Pornwaratt Niyomrattanakit2 Richard Ledwidge1 Cynthia S Dowd1 Ill Young Lee1 Pilho Kim1 Liang Zhang1 Sunhee Kang1 Thomas H Keller2 an iricek2 Clifton E Barry 3rd1 r Bicyclic nitroimidazoles including PA824 are exciting candidates for the treatment of tuberculosis These prodrugs require intracellular activation for their biological function We found that Rv3547 is a deazaflavindependent nitroreductase an that converts PA824 into three primary metabolites the major one is the corresponding desnitroimidazole desnitro When derivatives of PA824 were used the amount of desnitro metabolite formed was highly correlated with anaerobic killing of Mycobocterium tuberculosis Mtb Desnitro metabolite formation generated reactive nitrogen species including nitric oxide NO which are the major effectors of the anaerobic activity of these compounds Furthermore N0 scavengers protected the bacilli from the lethal effects of the drug Thus these compounds may act as intracellular N0 donors and could augment a killing mechanism intrinsic to the innate immune system Downloaded from wwwsciencemagorg on January 25 2009 OPC 67683 which are currently in human clinical trials 2 3 These molecules are active not only against actively replicating bacteria but also against bacteria that are nonreplicating by immunity see also 2 In the system environmental factors in uence the severity of the phenotype in a dysgen ic cross 28 in a manner linked to the expression rug resistant tuberculosis TB has emerged Das a major threat to global health I Promising new candidate drugs are the bicyclic nitroimidazoles including PA 824 and 1392 28 NOVEMBER 2008 VOL
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