Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2205-2209, April 1988 Genetics Cell-cycle regulation as a mechanism for targeting proteins to specific DNA sequences in Tetrahymena thermophila (linker-histone genes/DNA replication/macro- and micronuclei/ciiates/protein localization) MIN WU*, C. DAVID ALLISt, AND MARTIN A. GOROVSKY* *Department of Biology, University of Rochester, Rochester, NY 14627; and tVerna and Marrs Mclean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030 Communicated by Joseph G. Gall, November 16, 1987

ABSTRACT Transcriptionally active macronuclei and each cell also contains a transcriptionally inert germinal mi- transcriptionally inert micronuclei of the ciliated protozoan cronucleus that functions in the transmission of the genome Tetrahymena thermophila contain similar DNA sequences but during conjugation, the sexual stage of the life cycle (13). have very different histones associated with the linker regions Thus, in vegetative cells, the same genes are found in a trans- of chromatin. In situ hybridization showed that a gene coding criptionally active state in macronuclei and in a repressed for micronuclear linker histone is expressed only in association state in micronuclei. Correlated with this (and other) differ- with micronuclear DNA replication, whereas the gene for ence(s) in chromatin function in macro- and micronuclei are macronuclear Hi histone is expressed during macronuclear differences in histone composition between the two nuclei (but not during micronuclear) S phase. These results indicate (14). One of the most striking of these differences concerns that cell-cycle regulation plays an important role in directing histone H1. Macronuclei contain an H1-type histone (Mac proteins to the appropriate nucleus in Tetrahymena and that H1) whose solubility, size, amino acid composition, and the replication-expression model [Gottesfeld, J. & Bloomer, phosphorylation pattern resemble that of higher eukaryotes, L. S. (1982) Cell 28, 781-791; Wormington, W. M., Schlissel, although recent studies indicate that it lacks the globular M. & Brown, D. D. (1983) Cold Spring Harbor Symp. Quant. central region typical of other H1 histones (15). Micronuclei Biol. 47, 879-884] for establishing appropriate transcription- do not contain significant amounts of protein with Hi-like ally active or repressed chromatin complexes during DNA solubility properties. Instead, they contain three still incom- replication is generally applicable. pletely characterized peptides (a, f3, and y; these will be referred to collectively as micronuclear linker histones, or Recent studies on the chromatin structure (1-3) and replica- Mic LH) that are associated with the linker regions of tion (4-6) of the dual 5S rRNA gene system in Xenopus micronuclear chromatin (16). laevis have lent support to a model (7, 8) relating the timing The existence of distinctly different proteins associated of replication of genes to their transcriptional activity. This with the linker regions of two nuclei containing largely iden- "replication-expression" model predicted that, in somatic tical genomes (17) in a single cell raises the interesting cells where they are transcriptionally active, the somatic- question of how these proteins are targeted to their proper type 5S rRNA genes would replicate early, associate with location. The replication-expression model provides a possi- and deplete a limiting supply of the transcription factor ble mechanism. Macro- and micronuclei of T. thermophila TFIIIA, and be assembled into an active chromatin com- have distinctly different periods of DNA replication in the cell plex. The more numerous, oocyte-type 5S rRNA genes cycle. If Mac H1 is synthesized only during macronuclear S would replicate late and, in the absence of TFIIIA, would phase and Mic LH are synthesized only during micronuclear form a transcriptionally inactive form of chromatin. The S, their nuclear-specific localization could be a simple conse- replication pattern of 5S rRNA genes in Xenopus somatic quence of the coincidence of their expression and the repli- cells is completely consistent with this hypothesis (4, 5). An cation of the appropriate DNA. In this report, we use in situ implied but unproven feature of this model is that, in somatic hybridization to demonstrate that mRNA sequences encoding cells, the accumulation of the 5S rRNA gene-specific tran- Mic LH are present in Tetrahymena only during (or slightly scription factor, TFIIIA, is also cell-cycle-regulated to en- preceding) the period of micronuclear DNA replication, sure its presence at high concentration during replication of whereas the gene coding for Mac H1 is expressed in cells somatic-type 5S rRNA genes and its relative absence during during the macronuclear S phase. These observations provide replication of the oocyte-type genes. Although many details a simple mechanism for nuclear-specific targeting of proteins of the structure and composition of transcriptionally active in Tetrahymena and support the replication-expression hy- and inactive complexes remain to be worked out (9, 10), a pothesis as a general mechanism for creating stable chromatin role has been postulated (11) for histone H1 in establishing complexes (1) on eukaryotic genes. and/or maintaining the repressed state. The abundance of H1 in eukaryotic nuclei, recent discoveries of transcription factors for genes transcribed by RNA polymerase II, and EXPERIMENTAL PROCEDURES numerous observations that actively transcribed genes rep- Analysis of DNA Replication. T. thermophila strain CU 428 licate in early S phase (12) make it possible that some form was grown to a density of about 105 cells per ml at 30'C in of the replication-expression model will be generally appli- enriched medium containing 1% proteose peptone instead of cable. 2% as originally described (18). Cells were pulse-labeled In the ciliated protozoan Tetrahymena thermophila, tran- with [methyl-3H]thymidine (50)mCi/ml; 1 ,tCi = 37 kBq) for scriptionally active genes are found largely if not entirely in either 5 or 15 min (in two separate experiments). After the macronucleus of vegetatively growing cells. However, labeling, cells were fixed and prepared for autoradiography as described (19). Cells were examined under the micro- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: Mac H1, macronuclear Hi-type histone; Mic LH, in accordance with 18 U.S.C. §1734 solely to indicate this fact. micronuclear linker histone(s).

Downloaded by guest on September 29, 2021 2205 2206 Genetics: Wu et al. Proc. Natl. Acad. Sci. USA 85 (1988)

scope at x 400 magnification and scored for the frequency Table 1. Percentages of Tetrahymena cells in various and morphology of cells containing labeled macro- or micro- cytological stages nuclei. % cells In Situ Hybridization. Growing cells were prepared, hybrid- ized, stained, and analyzed as described (20). In brief, cells Stage Mac H1* Mic LHt DNA synthesist were hybridized in situ with RNA transcripts derived from I 4.9 6.5 appropriate cloned probes (see below). Cell-cycle stages were II 2.8 1.8 9.6 determined by a combination of morphological staging III 3.7 3.5 (micronuclear division, macronuclear division, cytokinesis) IV 2.1 2.0 and cell size (for cells having no distinctive morphology). The V 2.4 2.5 6.9 staging presented here differs slightly from that described by VI 1.5 2.2 Yu et al. (20) in that seven distinct morphological stages (I, VII 82.5 81.4 83.5 elongated micronucleus adjacent to macronucleus; II, elon- *Data were taken from slides used to study in situ hybridization of gated micronucleus not adjacent to macronucleus; III, di- Mac Hi; n = 270. vided micronucleus, round macronucleus; IV, divided micro- tData were taken from slides used to study in situ hybridization of nucleus, elongated macronucleus; V, divided micronucleus, Mic LH; n = 324. tData were taken from slides used to study [3H]thymidine incorpo- macronuclear division furrow; VI, divided micronucleus, ration; n = 638. No attempt was made to distinguish among stages divided macronucleus, cell division furrow; VII, interphase I-III or IV-VI in this experiment, since the labeling patterns ofcells cell) were recognized instead of five. Examples of these within these groups were indistinguishable (no labeling in stages stages are shown in Fig. 3 and values for the percentages of I-III, micronuclear labeling in stages IV-VI). cells in each stage are listed in Table 1. We have arbitrarily started the cell cycle just preceding stage I. Background synthesized and used to probe a genomic library (22) con- hybridization using a message strand as probe was less than taining macronuclear EcoRI fragments in bacteriophage 10 grains per cell and was ignored. AgtWES*Ab. A number of positive clones were selected, one Cloned Probes. Isolation and characterization of the single of which contained a sequence homologous to both /3- gene for Mac H1 were described previously (15). An Eco- derived and y-derived synthetic oligonucleotide probes. Por- RI-Taq I fragment containing the entire coding region, the tions of the derived protein sequence of this clone were intron, and small amounts of 3' and 5' flanking sequence was identical to all of the available partial protein sequences of/3 subcloned in both orientations in plasmids pIBI 20 and 21 and y. Probes derived from this clone hybridize to a single (International Biotechnologies, New Haven, CT). Tritium- size class of poly(A)+ RNA on a gel blot. Based on these labeled message and anti-message transcripts were synthe- observations and the likely relationship between peptides a sized using bacteriophage T7 RNA polymerase (Amersham) and y (21), we believe the cloned sequence contains much if according to the supplier's instructions. not all of the gene(s) coding for the micronuclear linker- A detailed description ofthe isolation and characterization associated histones a, /3, and Ry. A 700-base-pair EcoRI of the gene coding for the Mic LH will be published fragment derived from this clone was subcloned in both elsewhere (M.W., C.D.A., R. G. Cook, and M.A.G., unpub- orientations in plasmid pIBI 76 to yield clones that were lished work). Previous studies showed that a, /3, and 'y transcribed with T7 polymerase to yield message or antimes- peptides are all associated with the linker region of micro- sage probes for Mic LH. nuclear chromatin (16) and suggested that y was derived RNA Gel Blots. Total RNA was isolated from exponen- from a by proteolytic processing (21). In brief, the strategy tially growing cells of T. thermophila by a slight modification employed to clone this sequence was similar to that used to (D. Shapiro, personal communication) of the method of isolate and characterize the gene for Mac H1 (15). Partial Chirgwin et al. (23). RNAs were glyoxylated, electropho- amino acid sequences were obtained from the P3 and y resed in 1.2% agarose gels, and blotted onto nylon mem- peptides (the a peptide has a blocked amino terminus and branes (24). Hybridizations were done according to instruc- was not sequenced). Mixed oligodeoxynucleotides were tions provided by the 1986 Promega Biotec (Madison, WI) Mic Mac catalog. Each probe hybridized to a different size class of LH HI mRNA (Fig. 1). RESULTS Tetrahymena Cell Cycle. Previous studies (25, 26) indicated that micronuclear DNA replication in Tetrahymena thermo- phila (formerly Tetrahymena pyriformis, syngen I) occurs shortly after micronuclear division, which immediately pre- 2.4 kb cedes macronuclear division and cytokinesis. Macronuclear DNA synthesis occupies a significant portion of the inter- phase period. However, those earlier studies were not per- 1.4 kb formed on the same extensively inbred and genetically marked strain used here. To establish precise cell-cycle parameters for strain CU 428, cells were incubated for 5 or 15 min with [3H]thymidine and the labeling pattern was ana- lyzed. No labeling was observed in cells containing dividing micronuclei or in cells whose micronuclei had divided but whose macronuclei showed no signs of dividing (stages I-II). Cells containing dividing macronuclei or in cytokinesis (stages FIG. 1. RNA gel blot of whole cell RNA demonstrating the IV-VI) invariably had labeled micronuclei, allowing the start specificity of probes for Mac Hi and Mic LH. The probe for Mac Hi of the micronuclear S period to be staged with relative hybridizes to an mRNA of about 1.4 kilobases (kb). The probe for precision to cells initiating macronuclear division. Micronu- Mic LH hybridizes to a message of about 2.4 kb. clear labeling was also observed in 23% ofthe interphase cells Downloaded by guest on September 29, 2021 Genetics: Wu et al. Pror. Natl. Acad. Sci. USA 85 (1988) 2207 labeled for 5 min. Typically, these cells are small and often Stage contain small DNA bodies eliminated from the recently Mic LH Mac HI divided macronucleus (Fig. 2). Thus, micronuclear S phase

occupies about 30%o of the cell cycle (23% plus the frequency . of cells in stages IV-VI). Labeled macronuclei were observed 'O.I "j.: -VdmbwLl. ;:..-.0. in 24% of the interphase cells labeled for 5 min. Interestingly, I i..:.-:f.l-.l-.A d in cells labeled for 5 min, no cells were observed with both ,MP macro- and micronuclei labeled, but in cells labeled for 15 min, about 14% of the cells had both nuclei labeled. There- fore, the start of macronuclear DNA replication must occur within about 10 min of the end of micronuclear S. Cells grown .-m-iwWIWL`:'2ill under the conditions used here have a generation time of II about 150 min, allowing placement of the beginning of macro- nuclear S about 7% ofthe cell cycle (10 min/150 min) after the end of micronuclear S. These approximations of the macro- and micronuclear S periods in the Tetrahymena cell cycle are indicated in Fig. 4 and confirm the earlier studies indicating III that, in Tetrahymena, macro- and micronuclear S are nono- verlapping (25, 26). Cell-Cycle Expression of the Mic LH Gene. Fig. 3 demon- I % .s;.w strates the hybridization of the Mic LH probe to selected li, IW. cytological stages. The results of quantitative analyses of .* .-,o Mic LH labeling are shown in Fig. 4A. Only cells in stages IV 11 III-VI and small (presumably recently divided) interphase *!Alia cells are significantly labeled. Accumulation of Mic LH %b .": message occurs just before the onset of micronuclear DNA ..fALS., replication (stage III cells are unlabeled with [3H]thymidine V ' ^'.I _ but contain detectable amounts of Mic LH mRNA). The U. amount of Mic LH mRNA declines rapidly just before the - end of micronuclear replication. Little or no Mic LH mes- sage is detectable in cells during macronuclear S period. Thus, the cell-cycle expression of the Mic LH gene is precisely timed to allow synthesis of the Mic LH proteins to VI , ' coincide exclusively with micronuclear DNA replication. Cell-Cycle Expression ofthe Mac H1 Gene. Fig. 3 illustrates selected stages labeled with a probe for the mRNA coding for Mac H1, and Fig. 4B shows a quantitative analysis of the VII hybridization. Most strikingly, little or no accumulation of a Mac H1 message occurs in stages of the cell cycle containing replicating micronuclei or mRNA for Mic LH. Accumulation I'i of Mac H1 message roughly coincides with macronuclear S phase, but the correspondence is much less precise than that between Mic LH gene expression and micronuclear S. How- FIG. 3. In situ hybridization of probes for Mac H1 and Mic LH to stages ofthe Tetrahymena cell cycle. Saturating concentrations of probes for the messages coding for Mac H1 (10- cpm/,l) or the Mic LH (1.7 x 10' cpm/,ul) were hybridized to cells from a logarithmi- cally growing culture of T. thermophila. Slides were exposed for 2 (Mac H1) or 4 (Mic LH) days. Examples are presented of all of the stages analyzed. Note that there are both heavily labeled and s~~~~~~~~~~~~~~~Va9 f s.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~;i unlabeled stage VII cells hybridized to the Mac H1 probe, indicating that Mac H1 mRNA accumulation probably does not encompass the mac entire postdivision period. ( x 720-840.) IV'S~Lv ever, as pointed out previously (20), the staging of interphase cells by cell size in these cytological preparations is much less precise than the morphological criteria used to stage those portions of the cell cycle during which Mic LH gene expres- sion occurs. Thus, even though we cannot pinpoint the initiation of accumulation and the disappearance of mRNA coding for Mac H1 with great precision, it is clearly correlated with macronuclear S, and Mac H1 message (and therefore H1 protein synthesis) is reduced or absent in cells in micronuclear S phase. FIG. 2. [3H]Thymidine incorporation into Tetrahymena illus- trates distinctly different periods of DNA replication in macro- and DISCUSSION micronuclei. Cells were incubated with [3Hithymidine (50 ACi/ml) The results in for 15 min, fixed, prepared for autoradiography and stained with presented Figs. 3 and 4 demonstrate that the 4',6-diamidino-2-phenylindole (DAPI) as described (19). The small accumulation of Mac H1 and Mic LH messages are periodic DAPI-stained body to the right of the macronucleus in the cell with and probably nonoverlapping during the Tetrahymena cell the labeled micronucleus is probably a DNA elimination body. cycle. Periodic accumulation of mRNAs for so-called "repli- (x650.) cation-dependent" histones in the cell cycle is a well-known Downloaded by guest on September 29, 2021 2208 Genetics: Wu et al. Proc. Natl. Acad. Sci. USA 85 (1988) A macronuclear-specific proteins are synthesized and depos- ited during conjugation when macronuclei (or developing macronuclei) are not synthesizing DNA (19). Clearly, in these and similar cases in other organisms, temporal regula- tion, passive diffusion, and binding to newly replicated DNA are inadequate to explain histone localization to nuclei. We proposed a mechanism for targeting different linker- associated histones to macro- and micronuclei based on the replication-expression model proposed for establishing sta- ble alternate transcriptional states of 5S rRNA genes in 0 Xenopus (7, 8). Our results are completely consistent with a) that model and provide evidence that synthesis of proteins cn 0 20 40 60 80 100 120 that will become associated with specific DNA sequences is . correlated with the replication of those sequences. It could be argued that this mechanism is peculiar to ciliates, neces- 0 6 B sitated by their unusual nuclear dimorphism. We believe, on z the contrary, that the observations described here indicate 0 that the replication-expression mechanism is evolutionarily M. \ancient and is widely used for establishing and maintaining alternate compositional and functional states of chromatin. 4uu \ Periodic, precisely timed expression of histone genes and of genes for other proteins acting on DNA (28, 34-36) has been described in diverse eukaryotic cell types, as have distinct periods of replication of transcriptionally active and inactive genes (12). It is our contention that the partition of transcrip- tionally active and inactive states of chromatin between macro- and micronuclei and the alternate forms of linker histones associated with them simply provide a unique oppor- 0 20 40 60 80 100 120 tunity to visualize this general phenomenon. % cell cycle This work was supported by research grants from the National Institutes of Health to M.A.G. (GM21793) and to C.D.A. FIG. 4. Quantitation of Mic LH (A) and Mac H1 (B) expression (HD16259). in the Tetrahymena cell cycle. Horizontal bars indicate the micro- nuclear and macronuclear S periods determined by pulse labeling 1. Brown, D. D. (1984) Cell 37, 359-365. with [3H]thymidine (see text for details). 2. Kmiec, E. B., Razvi, F. & Worcel, A. (1986) Cell 45, 209-218. 3. Wolffe, A. P. & Brown, D. D. (1986) Cell 47, 217-227. phenomenon (27). However, to our knowledge, this is the first 4. Gilbert, D. M. (1986) Proc. Natl. Acad. Sci. USA 83, 2924- case where histone genes in the same cell have been shown to 2928. have distinctly different of results 5. Guinta, D. R. & Korn, L. J. (1986) Mol. Cell Biol. 6, 2536- periods expression. Our 2542. shed little light on the mechanisms regulating accumulation or 6. Guinta, D. R., Tso, J. Y., Narayanswami, S., Hamkalo, B. A. disappearance of these messages, although the precise mor- & Korn, L. J. (1986) Proc. Natl. Acad. Sci. USA 83, 5150- phological staging based on nuclear divisions and cytokinesis 5154. allowed a demonstration of the striking rapidity with which 7. Gottesfeld, J. & Bloomer, L. S. (1982) Cell 28, 781-791. they can affect the Mic LH messages. 8. Wormington, W. M., Schlissel, M. & Brown, D. D. (1983) Histones are small proteins and are known to accumulate Cold Spring Harbor Symp. Quant. Biol. 47, 879-884. in nuclei after microinjection into cells (28-30). However, 9. Wolffe, A. P., Andrews, M. T., Crawford, E., Losa, R. & the precise mechanism of transport and accumulation Brown, D. D. (1987) Cell 49, 301-302. (pas- 10. Worcel, A. (1987) Cell 49, 302-303. sive diffusion, nucleoplasmin-mediated, target-sequence- 11. Schlissel, M. S. & Brown, D. D. (1984) Cell 37, 903-913. directed) of histones into nuclei is not clear. Based on the 12. Goldman, M. A., Holmquist, G. P., Gray, M. C., Caston, studies described here it is tempting to suggest that temporal L. A. & Nag, A. (1984) Science 224, 686-692. regulation of synthesis coupled with passive diffusion and 13. Gorovsky, M. A. (1980) Annu. Rev. Genet. 14, 203-239. binding to newly replicated DNA is sufficient to explain the 14. Gorovsky, M. A. (1986) in The Molecular Biology of Ciliated targeting of different linker-associated proteins to macro- Protozoa, ed. Gall, J. G. (Academic, Orlando, FL), pp. 227-261. and micronuclei. It should be noted, however, that while we 15. Wu, M., Allis, C. D., Richman, R., Cook, R. G. & Gorovsky, think it likely that Tetrahymena linker-histone deposition M. A. (1986) Proc. Natl. Acad. Sci. USA 83, 8674-8678. occurs after our studies have not 16. Allis, C. D., Glover, C. V. C. & Gorovsky, M. A. (1979) Proc. immediately synthesis, Natl. Acad. Sci. USA 76, 4857-4861. directly addressed this point. Also, while we believe that 17. Yao, M.-C. & Gorovsky, M. A. (1974) Chromosoma 48, 1-18. cell-cycle regulation plays an important role, other lines of 18. Gorovsky, M. A., Yao, M.-C., Keevert, J. B. & Pleger, G. L. evidence suggest that accumulation and deposition of his- (1975) Methods Cell Biol. 9, 311-327. tones into nuclei may be considerably more complicated. 19. Allis, C. D., Colavito-Shepanski, M. & Gorovsky, M. A. For example, Jackson and Chalkley (31) showed that, in (1987) Dev. Biol. 124, 469-480. hepatoma cells, histone Hi is deposited randomly over 20. Yu, S.-M., Horowitz, S. & Gorovsky, M. A. (1987) Genes chromatin (i.e., on old as well as on newly replicated DNA) Dev. 1, 683-692. even though Hi synthesis is tightly coupled to DNA repli- 21. Allis, C. D., Allen, R. L., Wiggins, J. C., Chicoine, L. G. & cation. It has been known for some time that a class of Richman, R. (1984) J. Cell Biol. 99, 1669-1677. 22. Horowitz, S. & Gorovsky, M. A. (1985) Proc. Natl. Acad. Sci. histones, the so-called "replacement histones," are synthe- USA 82, 2452-2455. sized and deposited in nuclei in the absence of DNA synthe- 23. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J. & Rutter, sis (32). In Tetrahymena, for instance, newly synthesized W. J. (1979) Biochemistry 18, 5294-5299. hv2, an H3 histone variant, is deposited in macronuclei but 24. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201- not in micronuclei of non-growing (starved) cells (33). Other 5205. Downloaded by guest on September 29, 2021 Genetics: Wu et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2209

25. McDonald, B. B. (1%2) J. Cell Biol. 13, 193-203. 31. Jackson, V. & Chalkley, R. (1985) Biochemistry 24, 6921-6930. 26. Woodard, J., Kaneshiro, E. & Gorovsky, M. A. (1972) Genet- 32. Old, R. W. & Woodland, H. R. (1984) Cell 38, 624-626. ics 70, 251-260. 33. Bannon, G. A., Calzone, F. J., Bowen, J. K., Allis, C. D. & 27. Schumperli, D. (1986) Cell 45, 471-472. Gorovsky, M. A. (1983) Nucleic Acids Res. 11, 3903-3917. 28. DeRobertis, E. M. (1983) Cell 32, 1021-1025. 34. Nasmyth, K. (1985) Cell 42, 225-235. 29. Dingwall, C. (1985) Trends Biochem. Sci. 10, 64-66. 35. White, J. H. M., Barker, D. G., Nurse, P. & Johnston, L. H. 30. Wu, L. H., Kuehl, R. & Rechsteiner, M. (1986) J. Cell Biol. (1986) EMBO J. 5, 1705-1709. 103, 465-474. 36. Kaczmarek, L. (1976) Lab. Invest. 54, 365-376. Downloaded by guest on September 29, 2021