Identification of novel chromatin-associated proteins involved in ...

JCS ePress online publication date 22 May 2007 1978

Research Article

Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena Meng-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,§ 1

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA Department of Biology, Washington University, St. Louis, MO 63130, USA

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*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: [email protected])

Journal of Cell Science

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

Summary Extensive DNA rearrangements occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome of Tetrahymena thermophila. To identify genes encoding proteins likely to be involved in this process, we devised a cytological screen to find proteins that specifically localize in macronuclear anlagen (Lia proteins) at the stage when rearrangements occur. We compared the localization of these with that of the chromodomain protein, Pdd1p, which is the most abundant known participant in this genome reorganization. We show that in live cells, Pdd1p exhibits dynamic localization, apparently shuttling from the parental to the developing nuclei through cytoplasmic bodies called conjusomes. Visualization of GFP-tagged Pdd1p also highlights the substantial three-dimensional nuclear reorganization in the formation of nuclear foci that Introduction The separation of germ line and the soma allows for both extensive specialization within the current generation of an organism and the propagation of a totipotent genome to its progeny. Although this separation is most commonly described in the context of distinct cell types in multicellular organisms, many single cell organisms maintain their germ line apart from the soma. In the ciliated protozoa, including Tetrahymena thermophila, each cell maintains two distinct genomes within separate types of nuclei (Prescott, 1994). A silent germ line genome is housed in the micronucleus and a transcriptionally active somatic genome in the macronucleus. During each round of sexual development, the old somatic macronucleus is discarded and a new one is generated from the micronucleus. Tetrahymena development involves an intricate series of nuclear events that initiate upon pairing of two cells of compatible mating types (Martindale et al., 1982; Ray, 1956). Within two hours of pairing, the micronucleus of each cell elongates to a crescent shape that extends throughout much of the cytoplasm and demarks prophase of meiosis I (see Fig. 1). After this stage, the meiotic nuclei contract and the elongated chromosomes condense and congress near the nuclear center. Completion of two meiotic divisions in each mating partner gives rise to four haploid nuclei, three of which (the relics) are

occur coincident with DNA rearrangements. We found that late in macronuclear differentiation, four of the newly identified proteins are organized into nuclear foci that also contain Pdd1p. These Lia proteins are encoded by primarily novel genes expressed at the beginning of macronuclear differentiation and have properties or recognizable domains that implicate them in chromatin or nucleic acid binding. Three of the Lia proteins also localize to conjusomes, a result that further implicates this structure in the regulation of DNA rearrangement. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/12/1978/DC1 Key words: DNA rearrangement, RNAi, Ciliate, Heterochromatin, Chromodomain

resorbed. The remaining one serves as the gametic micronucleus and undergoes mitotic division. One division product is then transferred to the other conjugant, where it fuses with its stationary gametic nucleus to generate a zygotic diploid micronucleus within each cell. These zygotic nuclei perform two mitotic divisions prior to the start of nuclear differentiation. The second of these post-zygotic nuclear divisions distributes two undifferentiated nuclei to the anterior of the each cell and two to the posterior. Shortly thereafter, the two anterior products enlarge and initiate their development into the new macronuclei of the final two progeny cells. These new somatic macronuclei finally segregate to daughter cells during the first post-conjugative cell division. Differentiation of the developing somatic macronuclei (historically called macronuclear anlagen) involves extensive programming of the previously silent germ line DNA derived from micronuclei to create the transcriptionally active macronucleus for the next vegetative generation. In addition to the establishment of chromatin modifications associated with gene regulation (Strahl et al., 1999), the development of the macronucleus involves genome-wide DNA rearrangement of two major types, chromosome breakage and DNA elimination (Yao et al., 2002). As a result of the first process, the five distinct germ-line-derived chromosomes are fragmented into


Genome rearrangement genes nearly 250 unique somatic chromosomes by chromosome breakage at sites identified by a highly conserved 15 bp sequence (Fan and Yao, 2000; Hamilton et al., 2006; Yao et al., 1987). In the second process, DNA segments are excised from an estimated 6000 loci dispersed throughout the chromosomes (Yao et al., 1984). These internal eliminated sequences (IESs) consist of both unique and repetitive sequences that comprise 15-20 Mbp of the germ line DNA (Yao and Gorovsky, 1974). Both these DNA rearrangements occur relatively late in conjugation (12-14 hours, see Fig. 1) (Austerberry et al., 1984; Duharcourt and Yao, 2002). After these events, the macronuclear chromosomes are amplified to 45-50 copies each and the progeny begin vegetative growth. Although whole genome rearrangement is rather unconventional, it is mechanistically related to the formation of heterochromatin in other eukaryotes. Prior to the removal of IESs, their associated chromatin is marked by dimethylation (me2) of Lys9 (K9) of histone H3 (Taverna et al., 2002). This heterochromatin-associated chromatin modification is targeted to these loci by an RNA interference (RNAi)-related mechanism (Malone et al., 2005; Meyer and Chalker, 2007; Mochizuki et al., 2002; Mochizuki and Gorovsky, 2004b; Yao and Chao, 2005; Yao et al., 2003) and is required for efficient IES 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-lineenriched small RNAs that are generated by the Dicer-like 1 (Dcl1) ribonuclease (Malone et al., 2005; Mochizuki and Gorovsky, 2005). The small RNAs then associate with the PIWI-family protein, Twi1p, and are carried into macronuclear anlagen (Mochizuki et al., 2002; Mochizuki and Gorovsky, 2004a), where it is believed that they direct the heterochromatin-associated chromatin modifications leading to elimination of the marked sequences. Thus the somatic macronucleus does not just silence its heterochromatin, but eliminates it altogether. Two chromodomain-containing proteins that bind H3K9me2-modified chromatin, encoded by programmed DNA degradation genes, PDD1 and PDD3 (Taverna et al., 2002), are members of only a handful of proteins that are known to be required for these massive DNA rearrangements. These were identified biochemically, along with a novel protein, Pdd2p, as developmentally expressed proteins that specifically localize to developing macronuclei (Madireddi et al., 1996; Madireddi et al., 1994; Nikiforov et al., 2000; Smothers et al., 1997b). Pdd1p is a very abundant protein that appears to play diverse roles during development. This protein begins to accumulate within the first few hours of conjugation, when it localizes within both meiotic micronuclei and parental macronuclei (Coyne et al., 1999). Near the start of post-zygotic stages, Pdd1p has been observed in a transient cytoplasmic structure called the conjusome (Janetopoulos et al., 1999) and in the newly emerging macronuclear anlagen. Late in development, the protein is concentrated in foci in the developing macronuclei where DNA rearrangement is presumed to occur as they also contain IESs and associated H3K9me2-modified chromatin (Madireddi et al., 1996; Taverna et al., 2002). Removal of Pdd1 from parental macronuclei abolishes pre-zygotic expression and leads to the absence of H3K9me2-modified chromatin and failure of DNA rearrangement (Coyne et al., 1999; Taverna et al., 2002).

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Although the exact functions of Pdd1p in macronuclear differentiation are yet to be determined, its diverse actions highlight the dynamic nature of this process. Specific chromatin marks must be placed on dispersed IESs, which are then assembled into foci with Pdd1p and the rest of the DNA rearrangement machinery. The composition of this machinery beyond the three Pdd proteins is unknown. This leaves an extensive gap in our understanding of the activities necessary to carry out such dramatic genome reorganization. To identify additional components involved in these events, we developed a cytological screen that allowed us to identify genes encoding proteins that localize specifically in macronuclear anlagen when DNA rearrangements occur. From this screen, we have identified novel genes that are specifically expressed at the beginning of macronuclear differentiation. We provide evidence that at least four of these genes are involved in DNA rearrangement by their specific localization to DNA rearrangement foci and have thus begun to further uncover the repertoire of proteins involved in forming the Tetrahymena somatic nucleus. Results GFP-Pdd1p fusions shows dynamic localization during conjugation Differentiation of the somatic macronucleus from the germ line micronuclear genome involves extensive DNA rearrangements and chromosome reorganization. As Pdd1p is an abundant protein that is essential for these events, we hypothesized that genes whose products exhibit similar localization during conjugation probably also participate in this genome remodeling. Although Pdd1p localization at specific stages of development 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 of its behavior in live cells throughout conjugation has not been performed. To better understand the dynamic nature of nuclear development, we visualized the localization of GFP- or CFPtagged Pdd1p in mating Tetrahymena cells. We examined both an N-terminal GFP fusion expressed from a fragment of the PDD1 promoter and a C-terminal CFP fusion expressed from the CdCl2-inducible MTT1 promoter. Even though these fusion proteins are expressed from high-copy rDNA-based vectors, their abundance is comparable to endogenous Pdd1p levels (supplementary material Fig. S1). Both fusion proteins showed indistinguishable localization patterns; however, the MTT1 promoter allowed us to examine localization during prezygotic development because early conjugation expression from a partial PDD1 promoter fragment in our GFP vector was not robust. Expression of Pdd1p-CFP in cells carrying a defective Pdd1 allele (Pdd1-Ntag) rescued the conjugation defects upon induction of tagged protein expression, which provides functional significance to the localization patterns described (supplementary material Table S1). Prezygotic developmental stages At the beginning of conjugation, Pdd1p was detected in both parental macronuclei and in micronuclei as they enter meiosis (Fig. 1A) (Coyne et al., 1999). In meiotic nuclei, Pdd1p was uniformly distributed in the nucleoplasm and appeared to be excluded from the condensed chromosomes in both prophase (Fig. 1A) and during meiotic division (Fig. 1B, note the


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Journal of Cell Science 120 (12)

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.) Differential interference contrast images (DIC) of cells are paired with GFP or CFP and DAPI fluorescence. The observed nuclear Pdd1p localization is illustrated in the chronological progression through conjugation in the top diagram as gray shading. Image panels corresponding to the illustrations are: (A) prophase Meiosis I; (B) metaphase Meiosis I; (C) nuclear exchange (data not shown for post-zygotic divisions and new macronuclei 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.

absence of GFP fluorescence overlapping with DAPI fluorescence). Exclusion from chromatin was somewhat unexpected as this was not apparent in studies with fixed cells (Coyne et al., 1999); however, this pattern has also been observed with Dcl1p, the DNA rearrangement-associated Dicer-like protein (Malone et al., 2005). Localization within micronuclei continued through meiosis, but was observed to a lesser degree in post-meiotic, gametic micronuclei and in the relics – the three discarded products of meiosis (Fig. 1C). Pdd1p was also found in the post-zygotic micronuclei prior to the start of differentiation of these nuclei into either micro or macronuclei (data not shown). By the second post-zygotic nuclear division (~6 hours into conjugation) that gives rise to the immediate precursors of the new micronuclei and macronuclei (also called macronuclear anlagen), Pdd1p was absent from the micronuclei for the remainder of development. In contrast to the homogeneous localization of Pdd1p in meiotic micronuclei, this protein is found within the old macronuclei concentrated in a large number of discrete foci, the function of which is unknown. However, it is tempting to

speculate that this localization is associated with the genome comparison between the germ line and somatic genomes through which sequences in the old macronucleus can epigenetically alter DNA rearrangement patterns (Chalker et al., 2005; Chalker and Yao, 1996). Pdd1p remained strongly localized within these old macronuclei throughout pre-zygotic development. However, upon the second post-zygotic division, after which new macronuclei emerge, the protein was predominantly lost from old macronuclei and rapidly appeared in the developing macronuclear anlagen as they enlarged in the anterior of the mating pairs (compare Fig. 1C to 1D). This swift transition indicates that that the existing protein was either degraded or that it relocalized to the new developing nuclei. Macronuclear differentiation Upon the emergence of developing macronuclear anlagen, we observed consistent, strong localization of tagged Pdd1p almost exclusively to these nuclei despite the concurrent presence of old macronuclei and developing micronuclei (Fig. 1D,E). Initially, GFP-Pdd1p was rather evenly distributed

Genome rearrangement genes

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(Janetopoulos et al., 1999). Although conjusome-like foci were observed, more often, multiple smaller GFP-Pdd1p cytoplasmic foci were apparent (Fig. 2C,D). These multiple foci were primarily observed just prior to (see Fig. 1B) or during the stage of development when conjusomes have been described. This observation may suggest that the conjusome can exist as a single subcellular structure or as multiple entities. Both the timing and concentration of these cytoplasmic structures near the anterior where anlagen initially begin their development is consistent with a role of these structures as trafficking centers for Pdd1p and other anlagen-localized proteins. This examination of fluorescently tagged Pdd1p reinforces and expands prior descriptions of the dynamic behavior of this protein during Tetrahymena conjugation.

Fig. 2. GFP-Pdd1p localizes to the conjusome and DNA rearrangement foci. GFP-Pdd1p was visualized in live cells between 6 and 7 hours (A-D) or 12 and 14 hours (E-G). DIC and corresponding views of GFP fluorescence are shown. F and G show a threefold enlargement of the region boxed in E. White arrowheads highlight cytoplasmic structures (A-D) or nuclear (E-G) DNA rearrangement foci.

throughout the macronuclear anlagen until just prior to mating pair separation. As macronuclei differentiation continued within each exconjugant, GFP-Pdd1p fluorescence became increasingly more punctate (Fig. 1F,G) until Pdd1p-containing foci eventually decreased in abundance and then disappeared altogether (Fig. 1H) (Madireddi et al., 1996; Madireddi et al., 1994). Closer examination of the late-stage, Pdd1p-containing nuclear foci by differential interference contrast (DIC) microscopy together with GFP fluorescence shows that these foci are spherical structures or doughnut-shaped bodies within developing macronuclei (Fig. 2E-G). The three-dimensional nature of these apparent DNA elimination structures, as visualized in live cells, highlights the extensive nuclear reorganization that occurs during the elimination of 15-20 Mbp of DNA. Cytoplasmic bodies In addition to anlagen localization, we detected transient localization of GFP-Pdd1p in the cytoplasm near the time (67 hours after pairing) that macronuclear anlagen emerged (Fig. 2A-D). In some cases a single cytoplasmic focus was observed near the anterior of the mating pair (see Fig. 1D and the right side partner of mating pair in Fig. 2B) that resembles the conjusome, a large (diameter up to 7 ␮m), anteriorly localized, subcellular structure that is temporally and spacially correlated with the appearance of anlagen and is known to contain Pdd1p

Identification of development-specific nuclear localized proteins Mutant screens have revealed that a number of loci are required for Tetrahymena development (Cole et al., 1997; Cole and Soelter, 1997), yet few proteins and their corresponding genes have been identified that have putative roles in somatic genome development. The Pdd proteins, encoded by PDD1, PDD2 and PDD3, were discovered biochemically by identifying developmentally expressed proteins that were enriched in isolated 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 in RNAi, Twi1 and Dcl1, are currently the only proteins demonstrated to be required for DNA rearrangement (Malone et al., 2005; Mochizuki et al., 2002; Mochizuki and Gorovsky, 2005). Given that the zygotic genome must be rapidly transformed into the new somatic genome once anlagen are formed, we reasoned that a significant fraction of the developmentally expressed proteins that specifically localize to differentiating macronuclei are likely to be important for DNA rearrangements or other aspects of nuclear development. To identify such proteins, we devised a screening strategy (Fig. 3A) in which a library of cDNAs, generated from polyAselected, developmentally expressed RNA, was fused to the Cterminus of GFP in vector pCGF-1, such that the resulting fusion proteins could be expressed in conjugating Tetrahymena. This GFP-tagged cDNA library was transformed into Tetrahymena to produce a panel of transformants that were arrayed in 96-well plates and examined for specific protein localization. Once the transformants matured to mating competence, replicates of the panels were mated to wild-type cells, and wells were surveyed using an inverted fluorescence microscope for mating pairs showing nuclear GFP localization. The initial transformant panels were plated at a density at which most of the 96 wells contained drug-resistant cells; thus, based on a Poisson distribution, each well contained, on average, four or more independent transformants. This allowed us to screen fewer plates; however, once GFP localization was observed, it was necessary to subclone transformants from the positive wells and perform secondary screens to identify the specific transformant responsible for the desired localization. We screened two transformant panels arrayed in a total of 8760 wells – an estimated 22,700 transformants (Table 1A). Screen 1 differed from Screen 2 in that the library used for the second panel was amplified by growth in E. coli prior to introduction into Tetrahymena. Overall, an estimated one-

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Fig. 3. GFP localization identifies candidate genes involved in nuclear differentiation. (A) A developmental cDNA library, inserted into GFP expression vector pCGF1, was transformed into Tetrahymena to generate panels of transformants that were selected in 96-well tissue culture plates. Replicates of these panels were crossed to wild-type strain CU428 and visualized for GFP-nuclear localization using an inverted fluorescence microscope. (B) Representative images of mating cells expressing four different GFP fusions 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, green fluorescence and DAPI-stained images is presented. White arrowheads indicate the different nuclei for positional reference between images.

quarter to one-third of the transformant wells showed some GFP fluorescence. Fifty-five of the ~22,000 transformants showed nuclear localization in one or multiple types of nuclei that we could later recover in secondary screens after subcloning cells. We initially did not limit our screens to anlagen localization, but rather looked for any nuclear localization. One important feature of this screen is that we could rapidly

identify the gene fragments that directed GFP nuclear localization by PCR amplification of the cDNA from the transformants. After amplifying and sequencing the cDNAs from the positive subclones, 45 of the 55 transformants were found to contain coding sequence of previously identified proteins. Most of these proteins were histones or other chromatin proteins, which are probably highly expressed during nuclear development. Over half of these transformants (23) contained GFP fusions to one of two different cDNAs of histone H2B. As most of these histone H2B cDNAs were identified in Screen 2, biased amplification of these particular clones appeared to have occurred during growth and isolation of the library DNA. Future screens using unamplified, normalized libraries would overcome these biases. Closer examination of these transformants revealed expected localization 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 fusion protein was targeted specifically to micronuclei (Fig. 3B). We also recovered cDNAs of PDD1 and PDD2 from eight and two transformants, respectively, which verifies that our screen can identify proteins specifically involved in macronuclear genome differentiation (Table 1B). We did not recover PDD3, thus our screen is clearly not saturated in terms of what can be identified. From the two screens, we identified ten novel sequences. Nine of these ten localized specifically to developing macronuclei and we named these localized in macronuclear anlagen (LIA) genes (see representative GFP localization for the LIA3 cDNA fusion in Fig. 3B). The tenth localized to micronuclei and its coding sequence had similarity to importin ␣ proteins (Goldfarb et al., 2004). We have subsequently found that this gene, which we have named IMA10, is essential for micronuclear division (C. D. Malone and D.L.C., unpublished results). Two of the anlagenlocalizing cDNAs are unlikely to encode actual proteins. One was a segment of ribosomal RNA, and the other was homologous to the starvation-induced NgoA gene (Shen and Gorovsky, 1996), but was fused to GFP in the anti-sense orientation relative to NgoA translation.

LIA genes encode primarily novel proteins with common expression patterns To further assess which of these LIA genes were specifically involved in macronuclear differentiation, we examined their expression by northern blot analysis (Fig. 4A and summarized in Table 2). LIA1-LIA5 showed very strong development-


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Table 1. Identification of nucleus-localized proteins by fusion to GFP A.

Wells screened

Transformants screened*

Nuclear localized

Known proteins

Novel proteins

~2000 6760 8760

4700 17,826 22,700

10 45 55

5 40† 45†

5‡ 5 10

Screen #1

Screen #2

Total

1

22 1

23§ 1 1 1 1 1 8 2 1

Screen #1 Screen #2 Total

B. Known proteins identified Histone H2B Histone H4 Histone H3 Histone H1 (mac) Mic linker histone HMG-B Pdd1p Pdd2p Polyubiquitin

1

2

1 1 1 6 2

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.

specific expression that was induced near the time that macronuclear anlagen form (~6.5 hours into conjugation). Transcript sizes of these five genes ranged from 1.1 kb to 3.5 kb. For LIA2, a 2.7 kb transcript was initially detected that dropped to 2.4 kb in size by 9 hours of conjugation, presumably indicating 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 these from further analysis. As the expression patterns of LIA1-LIA5 suggested that they might have important roles in macronuclear differentiation, we proceeded to more thoroughly examine their gene structures (Fig. 4B) and localization patterns (Fig. 5). In the construction of our cDNA library, we did not attempt to generate full-length products, and none of the LIA genes was cloned in its entirety. The sequence of the macronuclear genome of Tetrahymena (Eisen et al., 2006) has greatly facilitated our determination of the structure of each of these genes (Fig. 4B). Each gene contains three to seven introns with the exception of the intron-less LIA4. All introns were verified by their absence in our cloned LIA gene cDNAs or by comparison of the genome sequence to GenBank cDNA sequences annotated on the Tetrahymena genome browser (www.ciliate.org/cgi-bin/gbrowse/ttgenomic/) (R.S.C., M. Thiagarajan and J. A. Eisen, unpublished results). The LIA1 cDNA we cloned coincidentally retained one of the two introns present within the coding sequence. Most of the introns in these genes are less than 100 bases, which is typical for Tetrahymena genes, although introns one and two of LIA2 are 127 and 1113 bases, respectively. Both LIA1 and LIA3 Fig. 4. LIA genes encode novel proteins expressed during nuclear differentiation. (A) Each lane contain introns within their 5⬘ noncontains 20 ␮g total RNA isolated from vegetatively growing (gr), starved (st) or conjugating translated regions, which are not cells at the indicated time after mixing of complementary mating types. The estimated transcript evident in current gene prediction sizes are given to the left of each scanned autoradiogram. (B) The gene structures of LIA1 to LIA5 models. Although the current are depicted: open boxes, coding regions; gaps spanned with carets, introns; solid black bars, gene predictions given at the transcribed non-translated regions. The size is given above each intron. The divisions of the scale Tetrahymena Genome Database bar represent 250 bp. All non-translated sequences, introns and the LIA4 polyadenylation site (TGD; www.ciliate.org) (Stover et (pA) were verified by our cloned cDNAs or database cDNA sequence. Gray bars underline the sequences found in the original cDNAs recovered in the screen, which were radiolabeled as al., 2006) were close to the structures probes for the northern blot analysis shown in A. we determined, we detected missed

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Journal of Cell Science 120 (12) Table 2. Conjugation-specific LIA genes Gene

Size of gene (bp)*

Hours of expression†

Size of RNA (kb)‡

Coding region (aa)§

Protein domains

LIA1 LIA2 LIA3 LIA4 LIA5

734 1730 968 2428 1434

6-12+ 6-12+ 6-12+ 6-12+ 6-12+

1.1 2.4 2.0 3.5 3.1

233 713 416 991 1048

Basic DEAD box helicase Basic Acidic, Q-rich 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.


†

or mis-called introns (LIA1 and LIA5) and split coding regions (LIA2 and LIA3) that result in inaccurate predictions of some of these coding sequences. The LIA genes encode predicted proteins of 233-1048 amino acids (Fig. 4B, Table 2). To get initial clues to the function of these proteins, we analyzed their sequences for conserved protein domains. The identification of the chromodomains in Pdd1p and Pdd3p provided the first mechanistic link between DNA rearrangement and heterochromatin formation (Madireddi et al., 1996; Nikiforov et al., 2000), and we hoped similar insight might be gleaned. Of these five genes, only LIA2 has extensive homology to known proteins. It contains both DEXDc and HELICc domains found in DEAD box RNA helicase proteins (Tanner and Linder, 2001) and may be a homolog of the conserved p68 RNA helicase (Ford et al., 1988; Hirling et al., 1989). This finding intrigued us because of the

known role of an RNAi-like mechanism in genome reorganization, but in light of more detailed studies discussed below, its significance is uncertain. LIA1, LIA3 and LIA4 contain no identifiable conserved protein domains. Nevertheless, it may be of note that both Lia1 (27 kDa) and Lia3 (49 kDa) are relatively small proteins rich in basic amino acids, which suggests a potential to bind nucleic acids. LIA4 encodes a 115 kDa protein of which the first ~200 residues are rich in acidic amino acids (29% Asp and Glu) and the C-terminal 700 amino acids are a neutrally charged domain that is rich in Gln (16.3%). LIA5 encodes a 122 kDa protein that consists of a low-complexity N-terminal domain (23.8% Gln, 16.7% Glu and 15.1% Asn over its first 300 aa) and a C-terminal Zn finger of the FYVE/PHD family. PHD Zn fingers are primarily found in a wide variety of nuclear proteins and form protein interaction interfaces, which have

Fig. 5. Lia proteins exhibit dynamic localization during conjugation. N-terminal GFP fusion proteins were expressed from the pIGF-LIA expression cassette shown at the top. Representative DIC, GFP and DAPI-stained image series are shown for LIA1, LIA3, LIA4 and LIA5 as indicated. (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-containing foci.



Fig. 6. Lia proteins co-localize with Pdd1 in DNA rearrangement foci. Conjugating cells expressing the indicated GFP-Lia fusion proteins were fixed with paraformaldehyde to preserve GFP fluorescence and incubated with Pdd1p-specific antibodies, detected with Rhodamine-conjugated anti-rabbit secondary antibodies. Images of GFP or immunofluorescence are shown separately and merged with the corresponding DAPI-stained images and pseudocolored (GFP-Lia, green; Pdd1p, red; DAPI, blue) to highlight overlapping localization (yellow). White arrows are given for positional reference between corresponding images.

been shown in some cases to bind directly to chromatin (Bienz, 2006). Thus, we have identified a diverse group of mostly novel proteins that one could predict to associate with nucleic acids or chromatin. The LIA proteins exhibit dynamic distribution during macronuclear differentiation To more carefully examine the localization of each Lia protein during conjugation, we fused the entire coding region of each to the C-terminus of GFP in a vector that allowed us to express these translational fusions at the appropriate stage of development by CdCl2 induction of the upstream MTT1 promoter. Transformants containing each GFP-LIA expression vector were mated to non-transformed cells and CdCl2 was added between 4 and 6 hours after mixing. We examined GFP fluorescence at the beginning of macronuclear development (78 hours) and later (12-14 hours) when DNA rearrangement occurs. All but one of the full-length Lia protein fusions localized to developing macronuclei, which was expected given 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, in which copies of this locus were knocked out of both microand macronuclei. These lines showed no obvious phenotype during growth or conjugation (C.H.R., R.J. and D.L.C., unpublished results), therefore we discontinued further analysis of this candidate and focused on the remaining LIA genes. At the beginning of macronuclear differentiation, LIA1, LIA3, LIA4 and LIA5 all localized primarily to macronuclear anlagen. The distribution of each protein was very uniform throughout these developing nuclei, similar to one another and

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to the localization observed for GFP-Pdd1p (Fig. 1). However, the average intensity of the fluorescence of each GFP-Lia protein is much lower that that observed for GFP-Pdd1p. This is not surprising because Pdd1p is a very abundant developmentally expressed protein in developing macronuclei (Madireddi et al., 1994). Tetrahymena cells appear able to effectively control protein levels post-transcriptionally because multiple proteins that we have expressed using the MTT1 promoter from high-copy vectors show very different levels of protein accumulation as judged by GFP fluorescence intensity (data not shown). This observation gives us higher confidence that the localization patterns we observe from proteins expressed from rDNA-based vectors faithfully recapitulate endogenous protein localization. In addition to the nuclear localization we observed, each GFP-Lia transformant line showed some general cytoplasmic localization at this stage as well, which was greatest for Lia5p and suggests a slower rate of nuclear import. In addition to general cytoplasmic GFP fluorescence, Lia1p, Lia3p and Lia5p all showed apparent localization to the conjusome (indicated by arrows in Fig. 5A). This finding further supports the idea that this structure may be involved in trafficking specific proteins to developing macronuclei. Conjusome localization also further links these proteins to the DNA rearrangement process given that the essential DNA rearrangement protein, Pdd1p, is a major component of this structure (Janetopoulos et al., 1999) (see Fig. 2). Late in conjugation after mating pairs have separated, each of the GFP-Lia fusion proteins no longer shows the uniform macronuclear localization initially observed. At this developmental stage, each protein is found in discrete foci (Fig. 5B) within macronuclei that are similar in appearance to the Pdd1p-containing foci (Fig. 1G,H), which further implicates these proteins in genome rearrangement. While not examined here, we have found that Lia1p co-immunoprecipitates with Pdd1p (C.H.R. and D.L.C., unpublished), which indicates that this protein is part of the DNA rearrangement machinery as well. To directly link these other three Lia proteins to DNA rearrangement, we asked whether these GFP foci are coincident with the Pdd1p-containing foci. To this end, we fixed conjugating cells expressing the GFP fusion proteins with paraformaldehyde to preserve the green fluorescence and detected Pdd1p localization with specific antisera (Madireddi et al., 1994), visualized with Rhodamine-conjugated secondary antibodies. We found that Lia3p, 4p, and 5p GFP-foci all colocalized with Pdd1p foci (Fig. 6). Thus we have identified at least four novel proteins that probably participate in the DNA rearrangements that lead to the formation of the new somatic macronucleus. Our screen has begun to reveal the complexity of the DNA rearrangement machinery that remodels the macronuclear genome during development. Discussion Tetrahymena conjugation is an intriguing context within which to examine events of nuclear differentiation. Within a common cytoplasm, five individual nuclei are directed to undergo three very different fates: the parental somatic macronucleus is silenced, becomes pycnotic and goes through an apoptotic-like destruction (Davis et al., 1992); two micronuclear precursors are preserved as the germ line and their chromatin remains unmodified and silent; and two macronuclear anlagen are


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transformed into the somatic macronuclei and become transcriptionally active. Our examination of Pdd1p localization in live cells throughout conjugation confirms the finding of previous studies performed with fixed cells (Coyne et al., 1999; Janetopoulos et al., 1999; Madireddi et al., 1996; Madireddi et al., 1994; Smothers et al., 1997a) and highlights the compartmentalization of these events (Figs 1 and 2). During prezygotic development, Pdd1p was observed in both the parental micro- and macronucleus, but was found later in development uniformly distributed in the macronuclear anlagen before it assembled into foci that are visible as large, three-dimensional structures where DNA rearrangement presumably occurs (Fig. 2E). The features of Pdd1p foci suggest that the DNA rearrangement machinery is concentrated in developing macronuclei because these structures typically number in the tens at the stage when thousands of IESs are excised from dispersed loci. The formation of these structures probably necessitates large-scale chromosome reorganization prior to the actual DNA rearrangement events. Both the establishment of the H3K9me2 chromatin modification (Taverna et al., 2002) and the partitioning of these structures near the nuclear periphery are properties shared with eukaryotic heterochromatin (Grewal and Jia, 2007). The ability to follow the modification of targeted chromosomal regions, starting from a naive chromatin landscape, provides a remarkable system with which to investigate mechanisms that establish chromatin domains. If the marking of IESs for elimination from the somatic genome is analogous to the establishment of heterochromatin, further identification of the proteins within the DNA rearrangement foci should reveal unique insights into this evolutionarily conserved process. We reasoned that developmentally expressed proteins that localize primarily to macronuclear anlagen were strong candidates to carry out DNA rearrangement and the associated events. By expressing a GFP-tagged cDNA library in conjugating cells, we identified five candidates (LIA1 to LIA5) that may be important for this process. They represent a set of Tetrahymena genes that are exclusively expressed starting between 6 and 6.5 hours into conjugation, peak in their expression around 9 hours, and are rapidly repressed at about 12 hours (Fig. 4). Their expression patterns do differ subtly. For instance, LIA2 is induced more slowly and appears to be somewhat delayed in its intron removal as two different transcripts are observed. When we more carefully examined the localization patterns of Lia1, Lia3, Lia4 and Lia5 proteins, we found that each shows the same dynamic behavior as Pdd1p. Although each appears to be less abundant than Pdd1p, all can be found in foci in late stage anlagen differentiation (Fig. 5B). We show that these GFP foci for the tagged Lia3, Lia4 and Lia5 proteins are the same as the Pdd1p foci (Fig. 6). Thus our screen effectively identified novel components of the DNA rearrangement machinery. Examination of the features of the LIA-encoded proteins provided some additional insight into their involvement in DNA rearrangement. Lia5 contains a probable Zn finger of the PHD family (Bienz, 2006). Proteins containing this conserved feature have been implicated in a wide variety of chromatinassociated processes. PHD domains facilitate protein interactions, and therefore we are interested to determine the

binding partners of this Lia5 domain. One substrate of PHD binding is the nucleosome itself. The highly basic composition of the predicted proteins encoded by LIA1 and LIA3 indicates a potential to bind directly to DNA or RNA. Both LIA4 and LIA5 encode regions that are high in Gln codons. Whether this pattern is shared with other proteins involved in DNA rearrangement and its structural function will be important to elucidate. The role of LIA2 in development is less clear. We were initially attracted to Lia2 because it has strong similarity to the p68 DEAD box helicase. The Drosophila p68 homologue, Dmp68, is associated with DmFMR1, the fragile X mental retardation protein, and components of the miRNA-containing RISC complex (Ishizuka et al., 2002). In addition, the DEAD box helicase protein Spindle-E has been implicated in heterochromatin formation (Pal-Bhadra et al., 2004) and thus we could envision obvious connections with RNAi-directed DNA rearrangement. However, our GFP fusion to the full genomic coding sequence did not produce detectable fusion protein, so we do not know whether it is localized within Pdd1p foci. It is possible that detection of the fusion protein in the original screen was facilitated by it being a cDNA fusion, whereas our full-length LIA2-GFP construct was a genomic DNA fusion. The poor expression might be related with the apparent delay in processing of the LIA2 mRNA observed in northern blot analysis (Fig. 4). Furthermore, when we disrupted the LIA2 gene from both the micro- and macronucleus, we did not observe obvious defects in conjugation (R.J., C.H.R. and D.L.C., unpublished results). The Tetrahymena genome encodes a large number of DEADbox-helicase-containing proteins including one other (TGD gene TTHERM_00190830) that shares strong similarity with p68 and Lia2. Thus we cannot yet conclude that Lia2 does not participate in DNA rearrangement as the lack of a knockout phenotype may be due to functional genetic redundancy. The presence of Lia1, Lia3 and Lia5 in the conjusome provides further evidence that this structure plays an important role in genome reorganization. We speculate that the conjusome could play a similar role to other cytoplasmic structures that function as centers of assembly or trafficking of RNA-protein complexes. These include the chromatoid body of mammals, perinuclear entities that are important during development of the male germ line. Recently RNAi components have been observed to localize to the chromatoid body 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 DNA rearrangements, it is compelling that an analogous structure that 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 facilitate communication between the parental and germ line genome. We have shown that the abnormal presence of an IES sequence in the parental macronucleus can block the efficient elimination of the corresponding IES from the developing macronucleus (Chalker and Yao, 1996). This inhibition is established through the action of cytoplasmically diffusible factors between 5 and 7 hours of conjugation (Chalker et al., 2005). Thus the period in which this regulation is enforced overlaps with the formation of the conjusome and the apparent shuttling of Pdd1p from the parental macronucleus to this

Genome rearrangement genes structure prior to its anlagen localization, which leads us to speculate that this genome comparison may be through the action of homologous RNAs in these cytoplasmic bodies. The putative Paramecium RNA binding protein, NOWA1p, is required for the elimination of IESs that exhibit similar epigenetic regulation by the content of the parental macronucleus and has been demonstrated to relocate between the maternal macronucleus and the anlagen (Nowacki et al., 2005). It is not known whether a conjusome-like structure exists in Paramecium, but the regulation of DNA rearrangement by the parental somatic genome appears to be a conserved feature of this process in ciliates. Materials and Methods


Strains Conventional lab strains CU427 [chx1-1/chx1-1 (VI, cy-s)] and CU428 [mpr11/mpr1-1 (VII, mp-s)], B2086 (II) (obtained originally from P. J. Bruns, Cornell University) or transgenic strains derived from these were used in the course of this study. 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 a transgenic strain derived from conjugating CU427 and CU428 transformed with an anisomycin-resistant rRNA gene allele (J. Ward and M.-C.Y., unpublished). Strains containing a poorly expressed epitope-tagged PDD1 allele resulted from the partial disruption of the promoter by the insertion of the neo3 selection cassette (Shang et al., 2002) 571 bp upstream of the PDD1 transcription start site. These strains were used to examine tagged Pdd1p localization and the functionality of the fusion proteins. This allele was crossed into lines B*(VI) and B*(VII) to produce heterokaryon 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 obtained more consistent fluorescence in mating pairs expressing low levels of endogenous Pdd1p, presumably because the vector-expressed copies contributed a greater fraction of the Pdd1p pool. Strains containing this allele as the only PDD1 gene in their parental macronuclei behave similar to macronuclear PDD1-knockout strains and fail to survive conjugation due to insufficient Pdd1p expression.

Generation of GFP expression vectors and a GFP-cDNA library The S65T GFP variant was PCR amplified and cloned in place of the rpL29 coding sequence in a vector containing a 3.4 kbp HindIII genomic fragment containing this gene (Yao and Yao, 1991). The GFP sequence replaced rpL29 nucleotides 341-2214 of the published sequence (Acc. no. M76719) with a PmeI restriction site placed at the upstream rpL29/GFP junction. An additional linker sequence (5⬘-AACCTCGAGTGAATGATTTGAGGGCCC-3⬘) was added to the last codon of GFP that contains one leucine codon attached to an in-frame XhoI site that is followed by three out-of-frame stop codons and terminating with an ApaI site that allows fusion to coding sequences or random cDNAs. This entire rpL29-GFP cassette was sequentially inserted into the HindIII site of pHSS6 and then as a NotI fragment into rRNA gene transformation vector pD5H8 (Godiska and Yao, 1990) to create pVGF-1 that allows GFP expression in growing cells. The rpL29 promoter was then displaced with a 0.85 kbp fragment of the PDD1 promoter inserted into the PmeI site at the 5⬘ end of GFP (the fusion point was nucleotide 215 of the published PDD1 sequence, Acc. no. U66364) to create the conjugation expressed fusion vector, pCGF-1. Subsequently, the rpL29 promoter sequence was completely replaced with a 1.3 kbp fragment containing the MTT1 promoter (nt 1263 to 2538 of the published sequence; Acc. no. AY061892) to generate the inducible GFP fusion vector pIGF-1 (Malone et al., 2005). To facilitate cloning of coding sequences into this large expression vector, a Gateway recombination cassette (Invitrogen) containing attL sites, a chloramphenicol-resistance gene, and ccdB gene for negative selection was added in frame with GFP between the XhoI and ApaI sites to generate pIGF-gtw. A vector, pICC-gtw, that allows inducible expression of C-terminal CFP fusions was created by amplifying the gateway-CFP fusion cassette from pEarleyGate102 (Earley et al., 2006) and fusing it to the MTT1 promoter fragment from pIGF-1. The PDD1 coding sequence was amplified using Tetrahymena genomic DNA as a 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 genomic sequence was fused downstream of GFP after XhoI digestion of pCGF-1 to create pCGF-PDD1. PDD1 and LIA gene coding sequences were amplified from Tetrahymena genomic DNA and the PCR generated fragments were cloned into pENTR-Topo/D (Invitrogen). LR clonase II reactions were used to recombine these sequences into either pICC-gtw or pIGF-gtw, respectively. The conjugation-specific cDNA library was created by converting polyAsubtracted RNA to cDNA by reverse transcription using random hexamers

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

Tetrahymena transformations and GFP library screens Tetrahymena rDNA-based vectors were introduced into cells by conjugative electrotransformation (CET) (Gaertig and Gorovsky, 1992; Gaertig et al., 1994; Gaertig and Kapler, 2000). For each transformation, 5-25 ␮g of plasmid DNA was mixed with 5⫻106 mating pairs prior to conjugative electroporation and cells were selected in 1⫻ SPP medium containing 100 ␮g/ml paromomycin. To introduce the pCGF-cDNA library, 25 ␮g of the library and 30 ␮g yeast tRNA were mixed with a conjugating population of FH200 and FH203 just prior to CET. Cells were resuspended 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 until cells reached sexual maturity. The panels of mature transformants were replicated into wells of round-bottom plates containing 10 ␮l of 0.5⫻ NEFF medium and grown to saturation for 2 days at 30°C. An equal number of prestarved CU428 cells in 150 ␮l of 10 mM Tris-HCl (pH 7.4) was added to each well to initiate mating and each well was examined for GFP fluorescence on a Zeiss IM35 inverted microscope using a 20⫻ long working distance lens. The cDNAs fused to GFP giving desired localization were recovered by amplification of DNA extracted from the transformant subclones using oligos GFP5 (5⬘-ACCACATGGTCCTTCTTGAG-3⬘) and CHX22 (5⬘-TTG TAT GAT ATA TGA GCA TAT-3⬘) that flank the cDNA. The amplified products were cloned into pCR2.1 using the TopoTA cloning kit and cloned inserts were characterized by DNA sequence analysis. Sequences were submitted to GenBank and are available under the following accession numbers: LIA1, EF219411; LIA2, EF219412; LIA3, EF219413; LIA4, EF219414; LIA5, EF219415; LIA6, EF219416; LIA7, EF219417; IMA10, EF219418.

Fluorescence microscopy To image GFP localization, 0.5-1.5 ml of conjugating cells (~105 cells/ml) were concentrated 100- to 200-fold by centrifugation at 1000 g for 2-3 minutes. DAPI was added to cells to 1 ␮g/ml. Cells were immobilized by mounting 1 ␮l of concentrated cells in 5-6 ␮l of 2% methylcellulose under 22⫻22 mm coverslips. Fluorescence was then visualized using a Nikon model E600 microscope equipped with a Qimaging Retiga EX CCD camera. Images were captured using Openlab software (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 2 mM 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 incubated overnight at 4°C in rabbit polyclonal antibodies specific for Pdd1p (Madireddi et al., 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 with goat 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 analysis Total 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 as described (Chalker and Yao, 2001). Each cloned cDNA fragment was removed from pCR2.1 by restriction enzyme digestion followed by recovery of the DNA fragment after separation in low gelling temperature agarose. These were used for hybridization probes following random hexamer-primed radiolabeleing with [32P]dATP using the Klenow fragment of DNA polymerase I (Feinberg and Vogelstein, 1983; Feinberg and Vogelstein, 1984).

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

The authors would like to thank Caterina Randolph, Scott Kalishman, Colin Malone and Jason Motl for technical assistance with 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. and GM26210 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 to D.L.C. R.J. was supported in part by a WU/HHMI Summer Undergraduate Research Fellowship funded by an Undergraduate Biological Sciences Education Program grant from the Howard Hughes Medical Institute to Washington University.


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