1978 Research Article

IntroductionThe separation of germ line and the soma allows for bothextensive specialization within the current generation of anorganism and the propagation of a totipotent genome to itsprogeny. Although this separation is most commonly describedin the context of distinct cell types in multicellular organisms,many single cell organisms maintain their germ line apart fromthe soma. In the ciliated protozoa, including Tetrahymenathermophila, each cell maintains two distinct genomes withinseparate types of nuclei (Prescott, 1994). A silent germ linegenome is housed in the micronucleus and a transcriptionallyactive somatic genome in the macronucleus. During each roundof sexual development, the old somatic macronucleus isdiscarded and a new one is generated from the micronucleus.

Tetrahymena development involves an intricate series ofnuclear events that initiate upon pairing of two cells ofcompatible mating types (Martindale et al., 1982; Ray, 1956).Within two hours of pairing, the micronucleus of each cellelongates to a crescent shape that extends throughout much ofthe cytoplasm and demarks prophase of meiosis I (see Fig. 1).After this stage, the meiotic nuclei contract and the elongatedchromosomes condense and congress near the nuclear center.Completion of two meiotic divisions in each mating partnergives rise to four haploid nuclei, three of which (the relics) are

resorbed. The remaining one serves as the gameticmicronucleus and undergoes mitotic division. One divisionproduct is then transferred to the other conjugant, where itfuses with its stationary gametic nucleus to generate a zygoticdiploid micronucleus within each cell. These zygotic nucleiperform two mitotic divisions prior to the start of nucleardifferentiation. The second of these post-zygotic nucleardivisions distributes two undifferentiated nuclei to the anteriorof the each cell and two to the posterior. Shortly thereafter, thetwo anterior products enlarge and initiate their developmentinto the new macronuclei of the final two progeny cells. Thesenew somatic macronuclei finally segregate to daughter cellsduring the first post-conjugative cell division.

Differentiation of the developing somatic macronuclei(historically called macronuclear anlagen) involves extensiveprogramming of the previously silent germ line DNA derivedfrom micronuclei to create the transcriptionally activemacronucleus for the next vegetative generation. In addition tothe establishment of chromatin modifications associated withgene regulation (Strahl et al., 1999), the development of themacronucleus involves genome-wide DNA rearrangement oftwo major types, chromosome breakage and DNA elimination(Yao et al., 2002). As a result of the first process, the fivedistinct germ-line-derived chromosomes are fragmented into

Extensive DNA rearrangements occur during thedifferentiation of the developing somatic macronucleargenome from the germ line micronuclear genome ofTetrahymena thermophila. To identify genes encodingproteins likely to be involved in this process, we devised acytological screen to find proteins that specifically localizein macronuclear anlagen (Lia proteins) at the stage whenrearrangements occur. We compared the localization ofthese with that of the chromodomain protein, Pdd1p, whichis the most abundant known participant in this genomereorganization. We show that in live cells, Pdd1p exhibitsdynamic localization, apparently shuttling from theparental to the developing nuclei through cytoplasmicbodies called conjusomes. Visualization of GFP-taggedPdd1p also highlights the substantial three-dimensionalnuclear reorganization in the formation of nuclear foci that

occur coincident with DNA rearrangements. We found thatlate in macronuclear differentiation, four of the newlyidentified proteins are organized into nuclear foci that alsocontain Pdd1p. These Lia proteins are encoded byprimarily novel genes expressed at the beginning ofmacronuclear differentiation and have properties orrecognizable domains that implicate them in chromatin ornucleic acid binding. Three of the Lia proteins also localizeto conjusomes, a result that further implicates thisstructure in the regulation of DNA rearrangement.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/12/1978/DC1

Key words: DNA rearrangement, RNAi, Ciliate, Heterochromatin,Chromodomain

Summary

Identification of novel chromatin-associated proteinsinvolved in programmed genome rearrangements inTetrahymenaMeng-Chao Yao1,*, Ching-Ho Yao1, Lia M. Halasz1, Patrick Fuller1, Charles H. Rexer2, Sidney H. Wang2,Rajat Jain2, Robert S. Coyne1,‡ and Douglas L. Chalker2,§

1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA2Department of Biology, Washington University, St. Louis, MO 63130, USA*Present address: Institute of Molecular Biology, Academia Sinica, Taipei, 11529, Taiwan‡Present address: Department of Microbial Genomics, The Institute for Genomic Research, Rockville, MD 20850, USA§Author for correspondence (e-mail: dchalker@wustl.edu)

Accepted 17 April 2007Journal of Cell Science 120, 1978-1989 Published by The Company of Biologists 2007doi:10.1242/jcs.006502

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nearly 250 unique somatic chromosomes by chromosomebreakage at sites identified by a highly conserved 15 bpsequence (Fan and Yao, 2000; Hamilton et al., 2006; Yao et al.,1987). In the second process, DNA segments are excised froman estimated 6000 loci dispersed throughout the chromosomes(Yao et al., 1984). These internal eliminated sequences (IESs)consist of both unique and repetitive sequences that comprise15-20 Mbp of the germ line DNA (Yao and Gorovsky, 1974).Both these DNA rearrangements occur relatively late inconjugation (12-14 hours, see Fig. 1) (Austerberry et al., 1984;Duharcourt and Yao, 2002). After these events, themacronuclear chromosomes are amplified to 45-50 copies eachand the progeny begin vegetative growth.

Although whole genome rearrangement is ratherunconventional, it is mechanistically related to the formationof heterochromatin in other eukaryotes. Prior to the removal ofIESs, their associated chromatin is marked by dimethylation(me2) of Lys9 (K9) of histone H3 (Taverna et al., 2002). Thisheterochromatin-associated chromatin modification is targetedto these loci by an RNA interference (RNAi)-relatedmechanism (Malone et al., 2005; Meyer and Chalker, 2007;Mochizuki et al., 2002; Mochizuki and Gorovsky, 2004b; Yaoand Chao, 2005; Yao et al., 2003) and is required for efficientIES removal from the macronuclear anlagen (Liu et al., 2004).Bi-directional transcription of IESs in meiotic micronuclei(Chalker and Yao, 2001), provides the precursors of germ-line-enriched small RNAs that are generated by the Dicer-like 1(Dcl1) ribonuclease (Malone et al., 2005; Mochizuki andGorovsky, 2005). The small RNAs then associate with thePIWI-family protein, Twi1p, and are carried into macronuclearanlagen (Mochizuki et al., 2002; Mochizuki and Gorovsky,2004a), where it is believed that they direct theheterochromatin-associated chromatin modifications leadingto elimination of the marked sequences. Thus the somaticmacronucleus does not just silence its heterochromatin, buteliminates it altogether.

Two chromodomain-containing proteins that bindH3K9me2-modified chromatin, encoded by programmed DNAdegradation genes, PDD1 and PDD3 (Taverna et al., 2002), aremembers of only a handful of proteins that are known to berequired for these massive DNA rearrangements. These wereidentified biochemically, along with a novel protein, Pdd2p, asdevelopmentally expressed proteins that specifically localize todeveloping macronuclei (Madireddi et al., 1996; Madireddi etal., 1994; Nikiforov et al., 2000; Smothers et al., 1997b). Pdd1pis a very abundant protein that appears to play diverse rolesduring development. This protein begins to accumulate withinthe first few hours of conjugation, when it localizes within bothmeiotic micronuclei and parental macronuclei (Coyne et al.,1999). Near the start of post-zygotic stages, Pdd1p has beenobserved in a transient cytoplasmic structure called theconjusome (Janetopoulos et al., 1999) and in the newlyemerging macronuclear anlagen. Late in development, theprotein is concentrated in foci in the developing macronucleiwhere DNA rearrangement is presumed to occur as they alsocontain IESs and associated H3K9me2-modified chromatin(Madireddi et al., 1996; Taverna et al., 2002). Removal of Pdd1from parental macronuclei abolishes pre-zygotic expressionand leads to the absence of H3K9me2-modified chromatin andfailure of DNA rearrangement (Coyne et al., 1999; Taverna etal., 2002).

Although the exact functions of Pdd1p in macronucleardifferentiation are yet to be determined, its diverse actionshighlight the dynamic nature of this process. Specificchromatin marks must be placed on dispersed IESs, which arethen assembled into foci with Pdd1p and the rest of the DNArearrangement machinery. The composition of this machinerybeyond the three Pdd proteins is unknown. This leaves anextensive gap in our understanding of the activities necessaryto carry out such dramatic genome reorganization. To identifyadditional components involved in these events, we developeda cytological screen that allowed us to identify genes encodingproteins that localize specifically in macronuclear anlagenwhen DNA rearrangements occur. From this screen, we haveidentified novel genes that are specifically expressed at thebeginning of macronuclear differentiation. We provideevidence that at least four of these genes are involved in DNArearrangement by their specific localization to DNArearrangement foci and have thus begun to further uncover therepertoire of proteins involved in forming the Tetrahymenasomatic nucleus.

ResultsGFP-Pdd1p fusions shows dynamic localization duringconjugationDifferentiation of the somatic macronucleus from the germ linemicronuclear genome involves extensive DNA rearrangementsand chromosome reorganization. As Pdd1p is an abundantprotein that is essential for these events, we hypothesized thatgenes whose products exhibit similar localization duringconjugation probably also participate in this genomeremodeling. Although Pdd1p localization at specific stages ofdevelopment has been reported in various studies (Coyne et al.,1999; Janetopoulos et al., 1999; Madireddi et al., 1996;Madireddi et al., 1994), a comprehensive characterization ofits behavior in live cells throughout conjugation has not beenperformed. To better understand the dynamic nature of nucleardevelopment, we visualized the localization of GFP- or CFP-tagged Pdd1p in mating Tetrahymena cells. We examined bothan N-terminal GFP fusion expressed from a fragment of thePDD1 promoter and a C-terminal CFP fusion expressed fromthe CdCl2-inducible MTT1 promoter. Even though these fusionproteins are expressed from high-copy rDNA-based vectors,their abundance is comparable to endogenous Pdd1p levels(supplementary material Fig. S1). Both fusion proteins showedindistinguishable localization patterns; however, the MTT1promoter allowed us to examine localization during pre-zygotic development because early conjugation expressionfrom a partial PDD1 promoter fragment in our GFP vector wasnot robust. Expression of Pdd1p-CFP in cells carrying adefective Pdd1 allele (Pdd1-Ntag) rescued the conjugationdefects upon induction of tagged protein expression, whichprovides functional significance to the localization patternsdescribed (supplementary material Table S1).

Prezygotic developmental stagesAt the beginning of conjugation, Pdd1p was detected in bothparental macronuclei and in micronuclei as they enter meiosis(Fig. 1A) (Coyne et al., 1999). In meiotic nuclei, Pdd1p wasuniformly distributed in the nucleoplasm and appeared to beexcluded from the condensed chromosomes in both prophase(Fig. 1A) and during meiotic division (Fig. 1B, note the

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absence of GFP fluorescence overlapping with DAPIfluorescence). Exclusion from chromatin was somewhatunexpected as this was not apparent in studies with fixed cells(Coyne et al., 1999); however, this pattern has also beenobserved with Dcl1p, the DNA rearrangement-associatedDicer-like protein (Malone et al., 2005). Localization withinmicronuclei continued through meiosis, but was observed to alesser degree in post-meiotic, gametic micronuclei and in therelics – the three discarded products of meiosis (Fig. 1C).Pdd1p was also found in the post-zygotic micronuclei prior tothe start of differentiation of these nuclei into either micro ormacronuclei (data not shown). By the second post-zygoticnuclear division (~6 hours into conjugation) that gives rise tothe immediate precursors of the new micronuclei andmacronuclei (also called macronuclear anlagen), Pdd1p wasabsent from the micronuclei for the remainder of development.

In contrast to the homogeneous localization of Pdd1p inmeiotic micronuclei, this protein is found within the oldmacronuclei concentrated in a large number of discrete foci,the function of which is unknown. However, it is tempting to

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speculate that this localization is associated with the genomecomparison between the germ line and somatic genomesthrough which sequences in the old macronucleus canepigenetically alter DNA rearrangement patterns (Chalker etal., 2005; Chalker and Yao, 1996). Pdd1p remained stronglylocalized within these old macronuclei throughout pre-zygoticdevelopment. However, upon the second post-zygotic division,after which new macronuclei emerge, the protein waspredominantly lost from old macronuclei and rapidly appearedin the developing macronuclear anlagen as they enlarged in theanterior of the mating pairs (compare Fig. 1C to 1D). This swifttransition indicates that that the existing protein was eitherdegraded or that it relocalized to the new developing nuclei.

Macronuclear differentiationUpon the emergence of developing macronuclear anlagen, weobserved consistent, strong localization of tagged Pdd1palmost exclusively to these nuclei despite the concurrentpresence of old macronuclei and developing micronuclei (Fig.1D,E). Initially, GFP-Pdd1p was rather evenly distributed

Fig. 1. Pdd1p shows dynamic localization during development. C-terminal CFP (A-D) and N-terminal GFP (E-H) Pdd1p fusion protein(constructs shown) localization was visualized by fluorescence microscopy of live cells, co-stained with 4�,6-Diamidino-2-phenylindole(DAPI). (DAPI accumulation in vacuoles can produce significant cytoplasmic fluorescence in addition to nuclear staining.) Differentialinterference contrast images (DIC) of cells are paired with GFP or CFP and DAPI fluorescence. The observed nuclear Pdd1p localization isillustrated in the chronological progression through conjugation in the top diagram as gray shading. Image panels corresponding to theillustrations are: (A) prophase Meiosis I; (B) metaphase Meiosis I; (C) nuclear exchange (data not shown for post-zygotic divisions and newmacronuclei emergence – Mac I); (D,E) early macronuclear differentiation (Mac II); (F-H) macronuclear development after pair separation(exconjugants Mac II). Selected nuclei are labeled in the illustration and on the DIC images as: om, old macronuclei; mic, micronuclei; gmi,gametic micronuclei; relic, discarded meiotic products; zmi, zygotic micronuclei; NM, new macronuclear anlagen.

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throughout the macronuclear anlagen until just prior to matingpair separation. As macronuclei differentiation continuedwithin each exconjugant, GFP-Pdd1p fluorescence becameincreasingly more punctate (Fig. 1F,G) until Pdd1p-containingfoci eventually decreased in abundance and then disappearedaltogether (Fig. 1H) (Madireddi et al., 1996; Madireddi et al.,1994). Closer examination of the late-stage, Pdd1p-containingnuclear foci by differential interference contrast (DIC)microscopy together with GFP fluorescence shows that thesefoci are spherical structures or doughnut-shaped bodies withindeveloping macronuclei (Fig. 2E-G). The three-dimensionalnature of these apparent DNA elimination structures, asvisualized in live cells, highlights the extensive nuclearreorganization that occurs during the elimination of 15-20 Mbpof DNA.

Cytoplasmic bodiesIn addition to anlagen localization, we detected transientlocalization of GFP-Pdd1p in the cytoplasm near the time (6-7 hours after pairing) that macronuclear anlagen emerged (Fig.2A-D). In some cases a single cytoplasmic focus was observednear the anterior of the mating pair (see Fig. 1D and the rightside partner of mating pair in Fig. 2B) that resembles theconjusome, a large (diameter up to 7 �m), anteriorly localized,subcellular structure that is temporally and spacially correlatedwith the appearance of anlagen and is known to contain Pdd1p

(Janetopoulos et al., 1999). Although conjusome-like foci wereobserved, more often, multiple smaller GFP-Pdd1pcytoplasmic foci were apparent (Fig. 2C,D). These multiplefoci were primarily observed just prior to (see Fig. 1B) orduring the stage of development when conjusomes have beendescribed. This observation may suggest that the conjusomecan exist as a single subcellular structure or as multiple entities.Both the timing and concentration of these cytoplasmicstructures near the anterior where anlagen initially begin theirdevelopment is consistent with a role of these structures astrafficking centers for Pdd1p and other anlagen-localizedproteins. This examination of fluorescently tagged Pdd1preinforces and expands prior descriptions of the dynamicbehavior of this protein during Tetrahymena conjugation.

Identification of development-specific nuclear localizedproteinsMutant screens have revealed that a number of loci are requiredfor Tetrahymena development (Cole et al., 1997; Cole andSoelter, 1997), yet few proteins and their corresponding geneshave been identified that have putative roles in somatic genomedevelopment. The Pdd proteins, encoded by PDD1, PDD2 andPDD3, were discovered biochemically by identifyingdevelopmentally expressed proteins that were enriched inisolated macronuclear anlagen (Madireddi et al., 1996;Madireddi et al., 1994; Nikiforov et al., 2000; Smothers et al.,1997b). These three proteins and two proteins involved inRNAi, Twi1 and Dcl1, are currently the only proteinsdemonstrated to be required for DNA rearrangement (Maloneet al., 2005; Mochizuki et al., 2002; Mochizuki and Gorovsky,2005). Given that the zygotic genome must be rapidlytransformed into the new somatic genome once anlagen areformed, we reasoned that a significant fraction of thedevelopmentally expressed proteins that specifically localize todifferentiating macronuclei are likely to be important for DNArearrangements or other aspects of nuclear development. Toidentify such proteins, we devised a screening strategy (Fig.3A) in which a library of cDNAs, generated from polyA-selected, developmentally expressed RNA, was fused to the C-terminus of GFP in vector pCGF-1, such that the resultingfusion proteins could be expressed in conjugatingTetrahymena. This GFP-tagged cDNA library was transformedinto Tetrahymena to produce a panel of transformants that werearrayed in 96-well plates and examined for specific proteinlocalization. Once the transformants matured to matingcompetence, replicates of the panels were mated to wild-typecells, and wells were surveyed using an inverted fluorescencemicroscope for mating pairs showing nuclear GFP localization.The initial transformant panels were plated at a density atwhich most of the 96 wells contained drug-resistant cells; thus,based on a Poisson distribution, each well contained, onaverage, four or more independent transformants. This allowedus to screen fewer plates; however, once GFP localization wasobserved, it was necessary to subclone transformants from thepositive wells and perform secondary screens to identify thespecific transformant responsible for the desired localization.

We screened two transformant panels arrayed in a total of8760 wells – an estimated 22,700 transformants (Table 1A).Screen 1 differed from Screen 2 in that the library used for thesecond panel was amplified by growth in E. coli prior tointroduction into Tetrahymena. Overall, an estimated one-

Fig. 2. GFP-Pdd1p localizes to the conjusome and DNArearrangement foci. GFP-Pdd1p was visualized in live cells between6 and 7 hours (A-D) or 12 and 14 hours (E-G). DIC andcorresponding views of GFP fluorescence are shown. F and G show athreefold enlargement of the region boxed in E. White arrowheadshighlight cytoplasmic structures (A-D) or nuclear (E-G) DNArearrangement foci.

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quarter to one-third of the transformant wells showed someGFP fluorescence. Fifty-five of the ~22,000 transformantsshowed nuclear localization in one or multiple types of nucleithat we could later recover in secondary screens aftersubcloning cells. We initially did not limit our screens toanlagen localization, but rather looked for any nuclearlocalization.

One important feature of this screen is that we could rapidly

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identify the gene fragments that directed GFP nuclearlocalization by PCR amplification of the cDNA from thetransformants. After amplifying and sequencing the cDNAsfrom the positive subclones, 45 of the 55 transformants werefound to contain coding sequence of previously identifiedproteins. Most of these proteins were histones or otherchromatin proteins, which are probably highly expressedduring nuclear development. Over half of these transformants(23) contained GFP fusions to one of two different cDNAs ofhistone H2B. As most of these histone H2B cDNAs wereidentified in Screen 2, biased amplification of these particularclones appeared to have occurred during growth and isolationof the library DNA. Future screens using unamplified,normalized libraries would overcome these biases. Closerexamination of these transformants revealed expectedlocalization patterns and confirmed the utility of our screen.Core histones such as H2B and H4 localized to all nuclei,whereas the micronuclear linker histone (MLH)-GFP fusionprotein was targeted specifically to micronuclei (Fig. 3B).

We also recovered cDNAs of PDD1 and PDD2 from eightand two transformants, respectively, which verifies that ourscreen can identify proteins specifically involved inmacronuclear genome differentiation (Table 1B). We did notrecover PDD3, thus our screen is clearly not saturated in termsof what can be identified. From the two screens, we identifiedten novel sequences. Nine of these ten localized specifically todeveloping macronuclei and we named these localized inmacronuclear anlagen (LIA) genes (see representative GFPlocalization for the LIA3 cDNA fusion in Fig. 3B). The tenthlocalized to micronuclei and its coding sequence had similarityto importin � proteins (Goldfarb et al., 2004). We havesubsequently found that this gene, which we have namedIMA10, is essential for micronuclear division (C. D. Maloneand D.L.C., unpublished results). Two of the anlagen-localizing cDNAs are unlikely to encode actual proteins. Onewas a segment of ribosomal RNA, and the other washomologous to the starvation-induced NgoA gene (Shen andGorovsky, 1996), but was fused to GFP in the anti-senseorientation relative to NgoA translation.

LIA genes encode primarily novel proteins with commonexpression patternsTo further assess which of these LIA genes were specificallyinvolved in macronuclear differentiation, we examined theirexpression by northern blot analysis (Fig. 4A and summarizedin Table 2). LIA1-LIA5 showed very strong development-

Fig. 3. GFP localization identifies candidate genes involved innuclear differentiation. (A) A developmental cDNA library, insertedinto GFP expression vector pCGF1, was transformed intoTetrahymena to generate panels of transformants that were selectedin 96-well tissue culture plates. Replicates of these panels werecrossed to wild-type strain CU428 and visualized for GFP-nuclearlocalization using an inverted fluorescence microscope. (B)Representative images of mating cells expressing four different GFPfusions to coding sequences identified as histone H2B, histone H4,micronuclear linker histone (MLH), PDD2 and one novel gene,LIA3, are shown. For each pair or exconjugant, a series of DIC, greenfluorescence and DAPI-stained images is presented. Whitearrowheads indicate the different nuclei for positional referencebetween images.

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specific expression that was induced near the time thatmacronuclear anlagen form (~6.5 hours into conjugation).Transcript sizes of these five genes ranged from 1.1 kb to 3.5kb. For LIA2, a 2.7 kb transcript was initially detected thatdropped to 2.4 kb in size by 9 hours of conjugation, presumablyindicating some delay in the completion of mRNA processing.Transcription of two of the seven candidates, LIA6 and LIA7,was either undetectable at any stage or not restricted to

conjugation (data not shown), and we therefore excluded thesefrom further analysis. As the expression patterns of LIA1-LIA5suggested that they might have important roles in macronucleardifferentiation, we proceeded to more thoroughly examine theirgene structures (Fig. 4B) and localization patterns (Fig. 5).

In the construction of our cDNA library, we did not attemptto generate full-length products, and none of the LIA genes wascloned in its entirety. The sequence of the macronuclear

genome of Tetrahymena (Eisen et al.,2006) has greatly facilitated ourdetermination of the structure ofeach of these genes (Fig. 4B). Eachgene contains three to seven intronswith the exception of the intron-lessLIA4. All introns were verified bytheir absence in our cloned LIA genecDNAs or by comparison of thegenome sequence to GenBankcDNA sequences annotated on theTetrahymena genome browser(www.ciliate.org/cgi-bin/gbrowse/tt-genomic/) (R.S.C., M. Thiagarajanand J. A. Eisen, unpublished results).The LIA1 cDNA we clonedcoincidentally retained one of thetwo introns present within the codingsequence. Most of the introns inthese genes are less than 100 bases,which is typical for Tetrahymenagenes, although introns one and twoof LIA2 are 127 and 1113 bases,respectively. Both LIA1 and LIA3contain introns within their 5� non-translated regions, which are notevident in current gene predictionmodels. Although the currentgene predictions given at theTetrahymena Genome Database(TGD; www.ciliate.org) (Stover etal., 2006) were close to the structureswe determined, we detected missed

Table 1. Identification of nucleus-localized proteins by fusion to GFPA. Wells screened Transformants screened* Nuclear localized Known proteins Novel proteins

Screen #1 ~2000 4700 10 5 5‡

Screen #2 6760 17,826 45 40† 5Total 8760 22,700 55 45† 10

B. Known proteins identified Screen #1 Screen #2 Total

Histone H2B 1 22 23§

Histone H4 1 1Histone H3 1 1Histone H1 (mac) 1 1Mic linker histone 1 1HMG-B 1 1Pdd1p 2 6 8Pdd2p 2 2Polyubiquitin 1 1

*Estimated based on Poisson distribution from the number of transformant wells/plate.†Includes five duplications of proteins identified as novel in screen #1.‡Includes a fragment of the rRNA and the NGOA antisense cDNA (see text).§Biased amplification of this cDNA in the library appears to have occurred prior to the second screen.

Fig. 4. LIA genes encode novel proteins expressed during nuclear differentiation. (A) Each lanecontains 20 �g total RNA isolated from vegetatively growing (gr), starved (st) or conjugatingcells at the indicated time after mixing of complementary mating types. The estimated transcriptsizes are given to the left of each scanned autoradiogram. (B) The gene structures of LIA1 to LIA5are depicted: open boxes, coding regions; gaps spanned with carets, introns; solid black bars,transcribed non-translated regions. The size is given above each intron. The divisions of the scalebar represent 250 bp. All non-translated sequences, introns and the LIA4 polyadenylation site(pA) were verified by our cloned cDNAs or database cDNA sequence. Gray bars underline thesequences found in the original cDNAs recovered in the screen, which were radiolabeled asprobes for the northern blot analysis shown in A.

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or mis-called introns (LIA1 and LIA5) and split coding regions(LIA2 and LIA3) that result in inaccurate predictions of someof these coding sequences.

The LIA genes encode predicted proteins of 233-1048 aminoacids (Fig. 4B, Table 2). To get initial clues to the function ofthese proteins, we analyzed their sequences for conservedprotein domains. The identification of the chromodomains inPdd1p and Pdd3p provided the first mechanistic link betweenDNA rearrangement and heterochromatin formation(Madireddi et al., 1996; Nikiforov et al., 2000), and we hopedsimilar insight might be gleaned. Of these five genes, onlyLIA2 has extensive homology to known proteins. It containsboth DEXDc and HELICc domains found in DEAD box RNAhelicase proteins (Tanner and Linder, 2001) and may be ahomolog of the conserved p68 RNA helicase (Ford et al., 1988;Hirling et al., 1989). This finding intrigued us because of the

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known role of an RNAi-like mechanism in genomereorganization, but in light of more detailed studies discussedbelow, its significance is uncertain.

LIA1, LIA3 and LIA4 contain no identifiable conservedprotein domains. Nevertheless, it may be of note that bothLia1 (27 kDa) and Lia3 (49 kDa) are relatively small proteinsrich in basic amino acids, which suggests a potential to bindnucleic acids. LIA4 encodes a 115 kDa protein of which thefirst ~200 residues are rich in acidic amino acids (29% Asp andGlu) and the C-terminal 700 amino acids are a neutrallycharged domain that is rich in Gln (16.3%). LIA5 encodes a122 kDa protein that consists of a low-complexity N-terminaldomain (23.8% Gln, 16.7% Glu and 15.1% Asn over its first300 aa) and a C-terminal Zn finger of the FYVE/PHD family.PHD Zn fingers are primarily found in a wide variety of nuclearproteins and form protein interaction interfaces, which have

Table 2. Conjugation-specific LIA genesGene Size of gene (bp)* Hours of expression† Size of RNA (kb)‡ Coding region (aa)§ Protein domains

LIA1 734 6-12+ 1.1 233 BasicLIA2 1730 6-12+ 2.4 713 DEAD box helicaseLIA3 968 6-12+ 2.0 416 BasicLIA4 2428 6-12+ 3.5 991 Acidic, Q-richLIA5 1434 6-12+ 3.1 1048 Q-rich, FVYE/PHD Zn finger

*Size of original cDNA insert cloned and submitted to GenBank. Only 1537 bp of the LIA2 cDNA were derived from the genomic locus.†Indicates period of conjugation that RNA is detected by northern blot analysis; expression not detected in growing cells. ‡Estimated size of RNA transcript from northern blot analysis. §Number of amino acids (aa) within the predicted coding regions of each gene.

Fig. 5. Lia proteins exhibit dynamic localization during conjugation. N-terminal GFP fusion proteins were expressed from the pIGF-LIAexpression cassette shown at the top. Representative DIC, GFP and DAPI-stained image series are shown for LIA1, LIA3, LIA4 and LIA5 asindicated. (A) Early macronuclear development 7-8 hours into conjugation. (B) Late macronuclear development 12-14 hours into conjugation.White arrows indicate conjusome (C) localization. White arrowheads denote representative punctate foci that are similar to Pdd1p-containingfoci.

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been shown in some cases to bind directly to chromatin (Bienz,2006). Thus, we have identified a diverse group of mostly novelproteins that one could predict to associate with nucleic acidsor chromatin.

The LIA proteins exhibit dynamic distribution duringmacronuclear differentiationTo more carefully examine the localization of each Lia proteinduring conjugation, we fused the entire coding region of eachto the C-terminus of GFP in a vector that allowed us to expressthese translational fusions at the appropriate stage ofdevelopment by CdCl2 induction of the upstream MTT1promoter. Transformants containing each GFP-LIA expressionvector were mated to non-transformed cells and CdCl2 wasadded between 4 and 6 hours after mixing. We examined GFPfluorescence at the beginning of macronuclear development (7-8 hours) and later (12-14 hours) when DNA rearrangementoccurs. All but one of the full-length Lia protein fusionslocalized to developing macronuclei, which was expectedgiven the identification of their genes in our screen (Fig. 5A).The lone exception was the GFP fusion to full-length LIA2,which showed no detectable fluorescence in conjugating cells.Concurrently, we had generated LIA2 gene disruption lines, inwhich copies of this locus were knocked out of both micro-and macronuclei. These lines showed no obvious phenotypeduring growth or conjugation (C.H.R., R.J. and D.L.C.,unpublished results), therefore we discontinued furtheranalysis of this candidate and focused on the remaining LIAgenes.

At the beginning of macronuclear differentiation, LIA1,LIA3, LIA4 and LIA5 all localized primarily to macronuclearanlagen. The distribution of each protein was very uniformthroughout these developing nuclei, similar to one another and

to the localization observed for GFP-Pdd1p (Fig. 1). However,the average intensity of the fluorescence of each GFP-Liaprotein is much lower that that observed for GFP-Pdd1p. Thisis not surprising because Pdd1p is a very abundantdevelopmentally expressed protein in developing macronuclei(Madireddi et al., 1994). Tetrahymena cells appear able toeffectively control protein levels post-transcriptionally becausemultiple proteins that we have expressed using the MTT1promoter from high-copy vectors show very different levels ofprotein accumulation as judged by GFP fluorescence intensity(data not shown). This observation gives us higher confidencethat the localization patterns we observe from proteinsexpressed from rDNA-based vectors faithfully recapitulateendogenous protein localization.

In addition to the nuclear localization we observed, eachGFP-Lia transformant line showed some general cytoplasmiclocalization at this stage as well, which was greatest for Lia5pand suggests a slower rate of nuclear import. In addition togeneral cytoplasmic GFP fluorescence, Lia1p, Lia3p and Lia5pall showed apparent localization to the conjusome (indicatedby arrows in Fig. 5A). This finding further supports the ideathat this structure may be involved in trafficking specificproteins to developing macronuclei. Conjusome localizationalso further links these proteins to the DNA rearrangementprocess given that the essential DNA rearrangement protein,Pdd1p, is a major component of this structure (Janetopoulos etal., 1999) (see Fig. 2).

Late in conjugation after mating pairs have separated, eachof the GFP-Lia fusion proteins no longer shows the uniformmacronuclear localization initially observed. At thisdevelopmental stage, each protein is found in discrete foci (Fig.5B) within macronuclei that are similar in appearance to thePdd1p-containing foci (Fig. 1G,H), which further implicatesthese proteins in genome rearrangement. While not examinedhere, we have found that Lia1p co-immunoprecipitates withPdd1p (C.H.R. and D.L.C., unpublished), which indicates thatthis protein is part of the DNA rearrangement machinery aswell. To directly link these other three Lia proteins to DNArearrangement, we asked whether these GFP foci arecoincident with the Pdd1p-containing foci. To this end, wefixed conjugating cells expressing the GFP fusion proteins withparaformaldehyde to preserve the green fluorescence anddetected Pdd1p localization with specific antisera (Madireddiet al., 1994), visualized with Rhodamine-conjugated secondaryantibodies. We found that Lia3p, 4p, and 5p GFP-foci all co-localized with Pdd1p foci (Fig. 6). Thus we have identified atleast four novel proteins that probably participate in the DNArearrangements that lead to the formation of the new somaticmacronucleus. Our screen has begun to reveal the complexityof the DNA rearrangement machinery that remodels themacronuclear genome during development.

DiscussionTetrahymena conjugation is an intriguing context within whichto examine events of nuclear differentiation. Within a commoncytoplasm, five individual nuclei are directed to undergo threevery different fates: the parental somatic macronucleus issilenced, becomes pycnotic and goes through an apoptotic-likedestruction (Davis et al., 1992); two micronuclear precursorsare preserved as the germ line and their chromatin remainsunmodified and silent; and two macronuclear anlagen are

Fig. 6. Lia proteins co-localize with Pdd1 in DNA rearrangementfoci. Conjugating cells expressing the indicated GFP-Lia fusionproteins were fixed with paraformaldehyde to preserve GFPfluorescence and incubated with Pdd1p-specific antibodies, detectedwith Rhodamine-conjugated anti-rabbit secondary antibodies. Imagesof GFP or immunofluorescence are shown separately and mergedwith the corresponding DAPI-stained images and pseudocolored(GFP-Lia, green; Pdd1p, red; DAPI, blue) to highlight overlappinglocalization (yellow). White arrows are given for positional referencebetween corresponding images.

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transformed into the somatic macronuclei and becometranscriptionally active. Our examination of Pdd1p localizationin live cells throughout conjugation confirms the finding ofprevious studies performed with fixed cells (Coyne et al., 1999;Janetopoulos et al., 1999; Madireddi et al., 1996; Madireddi etal., 1994; Smothers et al., 1997a) and highlights thecompartmentalization of these events (Figs 1 and 2). Duringprezygotic development, Pdd1p was observed in both theparental micro- and macronucleus, but was found later indevelopment uniformly distributed in the macronuclearanlagen before it assembled into foci that are visible as large,three-dimensional structures where DNA rearrangementpresumably occurs (Fig. 2E). The features of Pdd1p focisuggest that the DNA rearrangement machinery is concentratedin developing macronuclei because these structures typicallynumber in the tens at the stage when thousands of IESs areexcised from dispersed loci. The formation of these structuresprobably necessitates large-scale chromosome reorganizationprior to the actual DNA rearrangement events. Both theestablishment of the H3K9me2 chromatin modification(Taverna et al., 2002) and the partitioning of these structuresnear the nuclear periphery are properties shared witheukaryotic heterochromatin (Grewal and Jia, 2007). The abilityto follow the modification of targeted chromosomal regions,starting from a naive chromatin landscape, provides aremarkable system with which to investigate mechanisms thatestablish chromatin domains.

If the marking of IESs for elimination from the somaticgenome is analogous to the establishment of heterochromatin,further identification of the proteins within the DNArearrangement foci should reveal unique insights into thisevolutionarily conserved process.

We reasoned that developmentally expressed proteins thatlocalize primarily to macronuclear anlagen were strongcandidates to carry out DNA rearrangement and the associatedevents. By expressing a GFP-tagged cDNA library inconjugating cells, we identified five candidates (LIA1 to LIA5)that may be important for this process. They represent a set ofTetrahymena genes that are exclusively expressed startingbetween 6 and 6.5 hours into conjugation, peak in theirexpression around 9 hours, and are rapidly repressed at about12 hours (Fig. 4). Their expression patterns do differ subtly.For instance, LIA2 is induced more slowly and appears to besomewhat delayed in its intron removal as two differenttranscripts are observed.

When we more carefully examined the localization patternsof Lia1, Lia3, Lia4 and Lia5 proteins, we found that eachshows the same dynamic behavior as Pdd1p. Although eachappears to be less abundant than Pdd1p, all can be found infoci in late stage anlagen differentiation (Fig. 5B). We showthat these GFP foci for the tagged Lia3, Lia4 and Lia5 proteinsare the same as the Pdd1p foci (Fig. 6). Thus our screeneffectively identified novel components of the DNArearrangement machinery.

Examination of the features of the LIA-encoded proteinsprovided some additional insight into their involvement inDNA rearrangement. Lia5 contains a probable Zn finger of thePHD family (Bienz, 2006). Proteins containing this conservedfeature have been implicated in a wide variety of chromatin-associated processes. PHD domains facilitate proteininteractions, and therefore we are interested to determine the

Journal of Cell Science 120 (12)

binding partners of this Lia5 domain. One substrate of PHDbinding is the nucleosome itself. The highly basic compositionof the predicted proteins encoded by LIA1 and LIA3 indicatesa potential to bind directly to DNA or RNA. Both LIA4 andLIA5 encode regions that are high in Gln codons. Whether thispattern is shared with other proteins involved in DNArearrangement and its structural function will be important toelucidate.

The role of LIA2 in development is less clear. We wereinitially attracted to Lia2 because it has strong similarity to thep68 DEAD box helicase. The Drosophila p68 homologue,Dmp68, is associated with DmFMR1, the fragile X mentalretardation protein, and components of the miRNA-containingRISC complex (Ishizuka et al., 2002). In addition, the DEADbox helicase protein Spindle-E has been implicated inheterochromatin formation (Pal-Bhadra et al., 2004) and thuswe could envision obvious connections with RNAi-directedDNA rearrangement. However, our GFP fusion to the fullgenomic coding sequence did not produce detectable fusionprotein, so we do not know whether it is localized within Pdd1pfoci. It is possible that detection of the fusion protein in theoriginal screen was facilitated by it being a cDNA fusion,whereas our full-length LIA2-GFP construct was a genomicDNA fusion. The poor expression might be related with theapparent delay in processing of the LIA2 mRNA observed innorthern blot analysis (Fig. 4). Furthermore, when wedisrupted the LIA2 gene from both the micro- andmacronucleus, we did not observe obvious defects inconjugation (R.J., C.H.R. and D.L.C., unpublished results).The Tetrahymena genome encodes a large number of DEAD-box-helicase-containing proteins including one other (TGDgene TTHERM_00190830) that shares strong similarity withp68 and Lia2. Thus we cannot yet conclude that Lia2 does notparticipate in DNA rearrangement as the lack of a knockoutphenotype may be due to functional genetic redundancy.

The presence of Lia1, Lia3 and Lia5 in the conjusomeprovides further evidence that this structure plays an importantrole in genome reorganization. We speculate that theconjusome could play a similar role to other cytoplasmicstructures that function as centers of assembly or trafficking ofRNA-protein complexes. These include the chromatoid bodyof mammals, perinuclear entities that are important duringdevelopment of the male germ line. Recently RNAicomponents have been observed to localize to the chromatoidbody and similar structures in other cell types (Kotaja et al.,2006a; Kotaja et al., 2006b; Kotaja and Sassone-Corsi, 2007).Given the role of small RNAs in directing DNArearrangements, it is compelling that an analogous structurethat participates in RNAi-regulated events may exist in ciliates,as these organisms are evolutionarily distant from mammals.

One role of the conjusome may be to facilitatecommunication between the parental and germ line genome.We have shown that the abnormal presence of an IES sequencein the parental macronucleus can block the efficientelimination of the corresponding IES from the developingmacronucleus (Chalker and Yao, 1996). This inhibition isestablished through the action of cytoplasmically diffusiblefactors between 5 and 7 hours of conjugation (Chalker et al.,2005). Thus the period in which this regulation is enforcedoverlaps with the formation of the conjusome and the apparentshuttling of Pdd1p from the parental macronucleus to this

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structure prior to its anlagen localization, which leads us tospeculate that this genome comparison may be through theaction of homologous RNAs in these cytoplasmic bodies. Theputative Paramecium RNA binding protein, NOWA1p, isrequired for the elimination of IESs that exhibit similarepigenetic regulation by the content of the parentalmacronucleus and has been demonstrated to relocate betweenthe maternal macronucleus and the anlagen (Nowacki et al.,2005). It is not known whether a conjusome-like structureexists in Paramecium, but the regulation of DNArearrangement by the parental somatic genome appears to be aconserved feature of this process in ciliates.

Materials and MethodsStrainsConventional lab strains CU427 [chx1-1/chx1-1 (VI, cy-s)] and CU428 [mpr1-1/mpr1-1 (VII, mp-s)], B2086 (II) (obtained originally from P. J. Bruns, CornellUniversity) or transgenic strains derived from these were used in the course of thisstudy. Tetrahymena growth and manipulations were as previously described(Gorovsky et al., 1975; Orias et al., 2000). FH200 (Anrev::Neo/Anrev::Neo [An-s])and FH203 (Chx1-1/Chx1-1 Anrev::Neo/Anrev::Neo [An-r]) are F1 lines of atransgenic strain derived from conjugating CU427 and CU428 transformed with ananisomycin-resistant rRNA gene allele (J. Ward and M.-C.Y., unpublished). Strainscontaining a poorly expressed epitope-tagged PDD1 allele resulted from the partialdisruption of the promoter by the insertion of the neo3 selection cassette (Shang etal., 2002) 571 bp upstream of the PDD1 transcription start site. These strains wereused to examine tagged Pdd1p localization and the functionality of the fusionproteins. This allele was crossed into lines B*(VI) and B*(VII) to produceheterokaryon lines in which this is the only PDD1 allele in the germ line (D.L.C,unpublished). These strains were transformed with pCGF-PDD1 as we obtainedmore consistent fluorescence in mating pairs expressing low levels of endogenousPdd1p, presumably because the vector-expressed copies contributed a greaterfraction of the Pdd1p pool. Strains containing this allele as the only PDD1 gene intheir parental macronuclei behave similar to macronuclear PDD1-knockout strainsand fail to survive conjugation due to insufficient Pdd1p expression.

Generation of GFP expression vectors and a GFP-cDNAlibraryThe S65T GFP variant was PCR amplified and cloned in place of the rpL29 codingsequence in a vector containing a 3.4 kbp HindIII genomic fragment containing thisgene (Yao and Yao, 1991). The GFP sequence replaced rpL29 nucleotides 341-2214of the published sequence (Acc. no. M76719) with a PmeI restriction site placed atthe upstream rpL29/GFP junction. An additional linker sequence (5�-AACC -TCGAGTGAATGATTTGAGGGCCC-3�) was added to the last codon of GFP thatcontains one leucine codon attached to an in-frame XhoI site that is followed bythree out-of-frame stop codons and terminating with an ApaI site that allows fusionto coding sequences or random cDNAs. This entire rpL29-GFP cassette wassequentially inserted into the HindIII site of pHSS6 and then as a NotI fragmentinto rRNA gene transformation vector pD5H8 (Godiska and Yao, 1990) to createpVGF-1 that allows GFP expression in growing cells. The rpL29 promoter was thendisplaced with a 0.85 kbp fragment of the PDD1 promoter inserted into the PmeIsite at the 5� end of GFP (the fusion point was nucleotide 215 of the publishedPDD1 sequence, Acc. no. U66364) to create the conjugation expressed fusionvector, pCGF-1. Subsequently, the rpL29 promoter sequence was completelyreplaced with a 1.3 kbp fragment containing the MTT1 promoter (nt 1263 to 2538of the published sequence; Acc. no. AY061892) to generate the inducible GFPfusion vector pIGF-1 (Malone et al., 2005). To facilitate cloning of coding sequencesinto this large expression vector, a Gateway recombination cassette (Invitrogen)containing attL sites, a chloramphenicol-resistance gene, and ccdB gene for negativeselection was added in frame with GFP between the XhoI and ApaI sites to generatepIGF-gtw. A vector, pICC-gtw, that allows inducible expression of C-terminal CFPfusions was created by amplifying the gateway-CFP fusion cassette frompEarleyGate102 (Earley et al., 2006) and fusing it to the MTT1 promoter fragmentfrom pIGF-1.

The PDD1 coding sequence was amplified using Tetrahymena genomic DNA asa template and then cloned into pCR2.1 using the TopoTA cloning kit (Invitrogen).Using SalI restriction sites added to the ends of the PDD1 sequence, the genomicsequence was fused downstream of GFP after XhoI digestion of pCGF-1 to createpCGF-PDD1. PDD1 and LIA gene coding sequences were amplified fromTetrahymena genomic DNA and the PCR generated fragments were cloned intopENTR-Topo/D (Invitrogen). LR clonase II reactions were used to recombine thesesequences into either pICC-gtw or pIGF-gtw, respectively.

The conjugation-specific cDNA library was created by converting polyA-subtracted RNA to cDNA by reverse transcription using random hexamers

containing BamHI restriction site extensions on their 5� ends. The resulting cDNAswere digested with BamHI, partially end-filled by addition of DNA polymerase(Klenow fragment) in the presence dATP and dGTP. These products were ligatedto vector pCGF-1 digested with XhoI, partial end-filled with TTP and dCTP to createtwo base overhangs that could anneal precisely with these cDNAs. This library wastransformed into E. coli and transformants were selected on L-agar plates containingampicillin. Transformants were washed from the plates and DNA was isolated afterlimited growth (screen 1) or inoculated into fresh medium containing ampicillin andgrown additional generations before plasmid DNA isolation (screen 2).

Tetrahymena transformations and GFP library screensTetrahymena rDNA-based vectors were introduced into cells by conjugativeelectrotransformation (CET) (Gaertig and Gorovsky, 1992; Gaertig et al., 1994;Gaertig and Kapler, 2000). For each transformation, 5-25 �g of plasmid DNA wasmixed with 5�106 mating pairs prior to conjugative electroporation and cells wereselected in 1� SPP medium containing 100 �g/ml paromomycin. To introduce thepCGF-cDNA library, 25 �g of the library and 30 �g yeast tRNA were mixed witha conjugating population of FH200 and FH203 just prior to CET. Cells wereresuspended in 150 ml 1� SPP medium, distributed into fifteen 96-well TC plates(100 �l/well) and allowed to recover for 16-20 hours at 30°C prior to selection.Selected transformants were replicated to fresh medium daily for 7-10 days untilcells reached sexual maturity. The panels of mature transformants were replicatedinto wells of round-bottom plates containing 10 �l of 0.5� NEFF medium andgrown to saturation for 2 days at 30°C. An equal number of prestarved CU428 cellsin 150 �l of 10 mM Tris-HCl (pH 7.4) was added to each well to initiate matingand each well was examined for GFP fluorescence on a Zeiss IM35 invertedmicroscope using a 20� long working distance lens. The cDNAs fused to GFPgiving desired localization were recovered by amplification of DNA extracted fromthe transformant subclones using oligos GFP5 (5�-ACCACATGGTCCTTC -TTGAG-3�) and CHX22 (5�-TTG TAT GAT ATA TGA GCA TAT-3�) that flank thecDNA. The amplified products were cloned into pCR2.1 using the TopoTA cloningkit and cloned inserts were characterized by DNA sequence analysis. Sequenceswere submitted to GenBank and are available under the following accessionnumbers: LIA1, EF219411; LIA2, EF219412; LIA3, EF219413; LIA4, EF219414;LIA5, EF219415; LIA6, EF219416; LIA7, EF219417; IMA10, EF219418.

Fluorescence microscopyTo image GFP localization, 0.5-1.5 ml of conjugating cells (~105 cells/ml) wereconcentrated 100- to 200-fold by centrifugation at 1000 g for 2-3 minutes. DAPIwas added to cells to 1 �g/ml. Cells were immobilized by mounting 1 �l ofconcentrated cells in 5-6 �l of 2% methylcellulose under 22�22 mm coverslips.Fluorescence was then visualized using a Nikon model E600 microscope equippedwith a Qimaging Retiga EX CCD camera. Images were captured using Openlabsoftware (Improvision).

For co-localization of GFP-tagged proteins and Pdd1p, cells were fixed in 2%paraformaldehyde (EM sciences), 25 mM HEPES (pH 6.8), 10 mM EGTA and 2mM MgC12 for 20-30 minutes at 30°C in a protocol adapted from (Guerra et al.,2003). Cells were blocked in 1% bovine serum albumin (BSA) and incubatedovernight at 4°C in rabbit polyclonal antibodies specific for Pdd1p (Madireddi etal., 1994) diluted 1:1000 in Tris-buffered saline (TBS), 1% BSA, 0.1% Tween20.Cells were washed to remove excess primary antibodies and then incubated withgoat anti-rabbit Rhodamine-conjugated antibodies (Pierce) for 1-2 hours at 25°C.Excess secondary antibodies were removed, nuclei were stained with DAPI (0.1�g/�l) for 10 minutes at 25°C and wet mounted on slides in TBS plus 1% BSA.

RNA analysisTotal RNA was extracted from Tetrahymena by RNAsol extraction (Fan et al., 2000).For northern blot analysis, 20 �g total RNA per well was electrophoresed in 1.2%agarose/1� MOPS/1% formaldehyde gels and transferred to nylon membranes asdescribed (Chalker and Yao, 2001). Each cloned cDNA fragment was removed frompCR2.1 by restriction enzyme digestion followed by recovery of the DNA fragmentafter separation in low gelling temperature agarose. These were used forhybridization probes following random hexamer-primed radiolabeleing with[32P]dATP using the Klenow fragment of DNA polymerase I (Feinberg andVogelstein, 1983; Feinberg and Vogelstein, 1984).

Data presentationOpenlab TIFF format files were imported into Adobe Photoshop CS and imagebrightness uniformly adjusted if necessary. Northern blot data was either capturedon X-ray film or digitally by phosphorimager analysis. TIFF files created byQuantity One software or autoradiography films captured on a flatbed scanner(Epson) were cropped and scaled using Adobe Photoshop CS. Graphics weregenerated using Adobe Illustrator 10 and combined with digital data.

The authors would like to thank Caterina Randolph, ScottKalishman, Colin Malone and Jason Motl for technical assistancewith aspects of this work and Deborah Frank for comments on the

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manuscript. This effort was supported by the following sources:National Institutes of Health (NIH) grants GM069593 to D.L.C. andGM26210 to M.-C.Y.; NIH NSRA fellowship GM16129 to R.S.C.;and by a grant from the American Cancer Society, IRG-58-010-45 toD.L.C. R.J. was supported in part by a WU/HHMI SummerUndergraduate Research Fellowship funded by an UndergraduateBiological Sciences Education Program grant from the HowardHughes Medical Institute to Washington University.

ReferencesAusterberry, C. F., Allis, C. D. and Yao, M. C. (1984). Specific DNA rearrangements

in synchronously developing nuclei of Tetrahymena. Proc. Natl. Acad. Sci. USA 81,7383-7387.

Bienz, M. (2006). The PHD finger, a nuclear protein-interaction domain. Trends Biochem.Sci. 31, 35-40.

Chalker, D. L. and Yao, M.-C. (1996). Non-Mendelian, heritable blocks to DNArearrangement are induced by loading the somatic nucleus of Tetrahymena thermophilawith germ line limited DNA. Mol. Cell. Biol. 16, 3658-3667.

Chalker, D. L. and Yao, M. C. (2001). Nongenic, bidirectional transcription precedesand may promote developmental DNA deletion in Tetrahymena thermophila. GenesDev. 15, 1287-1298.

Chalker, D. L., Fuller, P. and Yao, M. C. (2005). Communication between parental anddeveloping genomes during Tetrahymena nuclear differentiation is likely mediated byhomologous RNAs. Genetics 169, 149-160.

Cole, E. S. and Soelter, T. A. (1997). A mutational analysis of conjugation inTetrahymena thermophila 2. Phenotypes affecting middle and late development: thirdprezygotic nuclear division, pronuclear exchange, pronuclear fusion, and postzygoticdevelopment. Dev. Biol. 189, 233-245.

Cole, E. S., Cassidy-Hanley, D., Hemish, J., Tuan, J. and Bruns, P. J. (1997). Amutational analysis of conjugation in Tetrahymena thermophila 1. Phenotypes affectingearly development: meiosis to nuclear selection. Dev. Biol. 189, 215-232.

Coyne, R. S., Nikiforov, M. A., Smothers, J. F., Allis, C. D. and Yao, M. C.(1999). Parental expression of the chromodomain protein Pdd1p is required forcompletion of programmed DNA elimination and nuclear differentiation. Mol. Cell4, 865-872.

Davis, M. C., Ward, J. G., Herrick, G. and Allis, C. D. (1992). Programmed nucleardeath: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena. Dev.Biol. 154, 419-432.

Duharcourt, S. and Yao, M. C. (2002). Role of histone deacetylation in developmentallyprogrammed DNA rearrangements in Tetrahymena thermophila. Eukaryotic Cell 1,293-303.

Earley, K. W., Haag, J. R., Pontes, O., Opper, K., Juehne, T., Song, K. and Pikaard,C. S. (2006). Gateway-compatible vectors for plant functional genomics andproteomics. Plant J. 45, 616-629.

Eisen, J. A., Coyne, R. S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J. R., Badger,J. H., Ren, Q., Amedeo, P., Jones, K. M. et al. (2006). Macronuclear genomesequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol. 4,e286.

Fan, Q. and Yao, M.-C. (2000). A long stringent sequence for programmed chromosomebreakage in Tetrahymena thermophila. Nucleic Acids Res. 28, 895-900.

Fan, Q., Sweeney, R. and Yao, M. C. (2000). Creation and use of antisense ribosomesin Tetrahymena thermophila. Methods Cell Biol. 62, 533-547.

Feinberg, A. P. and Vogelstein, B. (1983). A technique for radiolabeling DNA restrictionendonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13.

Feinberg, A. P. and Vogelstein, B. (1984). Addendum – a technique for radiolabelingDNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137,266-267.

Ford, M. J., Anton, I. A. and Lane, D. P. (1988). Nuclear protein with sequencehomology to translation initiation factor eIF-4A. Nature 332, 736-738.

Gaertig, J. and Gorovsky, M. A. (1992). Efficient mass transformation of Tetrahymenathermophila by electroporation of conjugants. Proc. Natl. Acad. Sci. USA 89, 9196-9200.

Gaertig, J. and Kapler, G. (2000). Transient and stable DNA transformation ofTetrahymena thermophila by electroporation. Methods Cell Biol. 62, 485-500.

Gaertig, J., Gu, L., Hai, B. and Gorovsky, M. A. (1994). High frequency vector-mediated transformation and gene replacement in Tetrahymena. Nucleic Acids Res. 22,5391-5398.

Godiska, R. and Yao, M. C. (1990). A programmed site-specific DNA rearrangement inTetrahymena thermophila requires flanking polypurine tracts. Cell 61, 1237-1246.

Goldfarb, D. S., Corbett, A. H., Mason, D. A., Harreman, M. T. and Adam, S. A.(2004). Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol. 14,505-514.

Gorovsky, M. A., Yao, M. C., Keevert, J. B. and Pleger, G. L. (1975). Isolationof micro- and macronuclei of Tetrahymena pyriformis. Methods Cell Biol. 9, 311-327.

Grewal, S. I. and Jia, S. (2007). Heterochromatin revisited. Nat. Rev. Genet. 8, 35-46.

Guerra, C., Wada, Y., Leick, V., Bell, A. and Satir, P. (2003). Cloning, localization,and axonemal function of Tetrahymena centrin. Mol. Biol. Cell 14, 251-261.

Hamilton, E. P., Williamson, S., Dunn, S., Merriam, V., Lin, C., Vong, L., Russell-Colantonio, J. and Orias, E. (2006). The highly conserved family of Tetrahymena

thermophila chromosome breakage elements contains an invariant 10-base-pair core.Eukaryotic Cell 5, 771-780.

Hirling, H., Scheffner, M., Restle, T. and Stahl, H. (1989). RNA helicase activityassociated with the human p68 protein. Nature 339, 562-564.

Ishizuka, A., Siomi, M. C. and Siomi, H. (2002). A Drosophila fragile X protein interactswith components of RNAi and ribosomal proteins. Genes Dev. 16, 2497-2508.

Janetopoulos, C., Cole, E., Smothers, J. F., Allis, C. D. and Aufderheide, K. J. (1999).The conjusome: a novel structure in Tetrahymena found only during sexualreorganization. J. Cell Sci. 112, 1003-1011.

Kotaja, N. and Sassone-Corsi, P. (2007). The chromatoid body: a germ-cell-specificRNA-processing centre. Nat. Rev. Mol. Cell Biol. 8, 85-90.

Kotaja, N., Bhattacharyya, S. N., Jaskiewicz, L., Kimmins, S., Parvinen, M.,Filipowicz, W. and Sassone-Corsi, P. (2006a). The chromatoid body of male germcells: similarity with processing bodies and presence of Dicer and microRNA pathwaycomponents. Proc. Natl. Acad. Sci. USA 103, 2647-2652.

Kotaja, N., Lin, H., Parvinen, M. and Sassone-Corsi, P. (2006b). Interplay ofPIWI/Argonaute protein MIWI and kinesin KIF17b in chromatoid bodies of male germcells. J. Cell Sci. 119, 2819-2825.

Liu, Y., Mochizuki, K. and Gorovsky, M. A. (2004). Histone H3 lysine 9 methylationis required for DNA elimination in developing macronuclei in Tetrahymena. Proc. Natl.Acad. Sci. USA 101, 1679-1684.

Madireddi, M. T., Davis, M. and Allis, D. (1994). Identification of a novel polypepetideinvolved in the formation of DNA-containing vesicles during macronucleardevelopment in Tetrahymena. Dev. Biol. 165, 418-431.

Madireddi, M. T., Coyne, R. S., Smothers, J. F., Mickey, K. M., Yao, M.-C. and Allis,C. D. (1996). Pdd1p, a novel chromodomain-containing protein, links heterochromatinassembly and DNA elimination in Tetrahymena. Cell 87, 75-84.

Malone, C. D., Anderson, A. M., Motl, J. A., Rexer, C. H. and Chalker, D. L. (2005).Germ line transcripts are processed by a Dicer-like protein that is essential fordevelopmentally programmed genome rearrangements of Tetrahymena thermophila.Mol. Cell. Biol. 25, 9151-9164.

Martindale, D. W., Allis, C. D. and Bruns, P. (1982). Conjugation in Tetrahymenathermophila: a temporal analysis of cytological stages. Exp. Cell Res. 140, 227-236.

Meyer, E. and Chalker, D. L. (2007). Epigenetics of Ciliates. In Epigenetics (ed. C. D.Allis, T. Jenuwein, D. Reinberg and M.-L. Caparros), pp. 127-150. Cold Spring Harbor:Cold Spring Harbor Laboratory Press.

Mochizuki, K. and Gorovsky, M. A. (2004a). Conjugation-specific small RNAs inTetrahymena have predicted properties of scan (scn) RNAs involved in genomerearrangement. Genes Dev. 18, 2068-2073.

Mochizuki, K. and Gorovsky, M. A. (2004b). Small RNAs in genome rearrangement inTetrahymena. Curr. Opin. Genet. Dev. 14, 181-187.

Mochizuki, K. and Gorovsky, M. A. (2005). A Dicer-like protein in Tetrahymena hasdistinct functions in genome rearrangement, chromosome segregation, and meioticprophase. Genes Dev. 19, 77-89.

Mochizuki, K., Fine, N. A., Fujisawa, T. and Gorovsky, M. A. (2002). Analysis of apiwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena.Cell 110, 689-699.

Nikiforov, M., Gorovsky, M. and Allis, C. (2000). A novel chromodomain protein,Pdd3p, associates with internal eliminated sequences during macronucleardevelopment in Tetrahymena thermophila. Mol. Cell. Biol. 20, 4128-4134.

Nowacki, M., Zagorski-Ostoja, W. and Meyer, E. (2005). Nowa1p and Nowa2p: novelputative RNA binding proteins involved in trans-nuclear crosstalk in Parameciumtetraurelia. Curr. Biol. 15, 1616-1628.

Orias, E., Hamilton, E. P. and Orias, J. D. (2000). Tetrahymena as a laboratoryorganism: useful strains, cell culture, and cell line maintenance. Methods Cell Biol. 62,189-211.

Pal-Bhadra, M., Leibovitch, B. A., Gandhi, S. G., Rao, M., Bhadra, U., Birchler, J.A. and Elgin, S. C. (2004). Heterochromatic silencing and HP1 localization inDrosophila are dependent on the RNAi machinery. Science 303, 669-672.

Prescott, D. M. (1994). The DNA of ciliated protozoa. Microbiol. Rev. 58, 233-267.Ray, C. J. (1956). Meiosis and nuclear behavior in Tetrahymena pyriformis. J. Protozool.

3, 88-96.Shang, Y., Song, X., Bowen, J., Corstanje, R., Gao, Y., Gaertig, J. and Gorovsky, M.

A. (2002). A robust inducible-repressible promoter greatly facilitates gene knockouts,conditional expression, and overexpression of homologous and heterologous genes inTetrahymena thermophila. Proc. Natl. Acad. Sci. USA 99, 3734-3739.

Shen, X. and Gorovsky, M. A. (1996). Linker histone H1 regulates specific geneexpression but not global transcription in vivo. Cell 86, 475-483.

Smothers, J. F., Madireddi, M. T., Warner, F. D. and Allis, C. D. (1997a). ProgrammedDNA degradation and nucleolar biogenesis occur in distinct organelles duringmacronuclear development in Tetrahymena. J. Eukaryot. Microbiol. 44, 79-88.

Smothers, J. F., Mizzen, C. A., Tubbert, M. M., Cook, R. G. and Allis, C. D. (1997b).Pdd1p associates with germline-restricted chromatin and a second novel anlagen-enriched protein in developmentally programmed DNA elimination structures.Development 124, 4537-4545.

Stover, N. A., Krieger, C. J., Binkley, G., Dong, Q., Fisk, D. G., Nash, R., Sethuraman,A., Weng, S. and Cherry, J. M. (2006). Tetrahymena Genome Database (TGD): anew genomic resource for Tetrahymena thermophila research. Nucleic Acids Res. 34,D500-D503.

Strahl, B. D., Ohba, R., Cook, R. G. and Allis, C. D. (1999). Methylation of histoneH3 at lysine 4 is highly conserved and correlates with transcriptionally active nucleiin Tetrahymena. Proc. Natl. Acad. Sci. USA 96, 14967-14972.

Tanner, N. K. and Linder, P. (2001). DExD/H box RNA helicases: from generic motorsto specific dissociation functions. Mol. Cell 8, 251-262.

Jour

nal o

f Cel

l Sci

ence

1989Genome rearrangement genes

Taverna, S. D., Coyne, R. S. and Allis, C. D. (2002). Methylation of histoneh3 at lysine 9 targets programmed DNA elimination in Tetrahymena. Cell 110, 701-711.

Yao, M. C. and Gorovsky, M. A. (1974). Comparison of the sequences of macro- andmicronuclear DNA of Tetrahymena pyriformis. Chromosoma 48, 1-18.

Yao, M. C. and Yao, C. H. (1991). Transformation of Tetrahymena to cycloheximideresistance with a ribosomal protein gene through sequence replacement. Proc. Natl.Acad. Sci. USA 88, 9493-9497.

Yao, M. C. and Chao, J. L. (2005). RNA-guided DNa Deletion In Tetrahymena: anRNAi-based mechanism for programmed genome rearrangements. Annu. Rev. Genet.39, 537-559.

Yao, M. C., Choi, J., Yokoyama, S., Austerberry, C. F. and Yao, C. H. (1984). DNAelimination in Tetrahymena: a developmental process involving extensive breakage andrejoining of DNA at defined sites. Cell 36, 433-440.

Yao, M. C., Zheng, K. and Yao, C. H. (1987). A conserved nucleotide sequence at thesites of developmentally regulated chromosomal breakage in Tetrahymena. Cell 48,779-788.

Yao, M. C., Duharcourt, S. and Chalker, D. L. (2002). Genome-wide rearrangementsof DNA in Ciliates. In Mobile DNA II (ed. N. Craig, R. Craigie, M. Gellert and A.Lambowitz), pp. 730-758. New York: Academic Press.

Yao, M. C., Fuller, P. and Xi, X. (2003). Programmed DNA deletion as an RNA-guidedsystem of genome defense. Science 300, 1581-1584.

Jour

nal o

f Cel

l Sci

ence