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Cell-Cycle Regulation As a Mechanism for Targeting Proteins to Specific 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.
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