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Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity.

This protocol provides a method for purification of Tetrahymena nuclei using sedimentation at unit gravity. It is useful for applications where purity of micro- and macronuclei is required beyond that which is typically achieved by differential sedimentation alone. When differential sedimentation is followed by sedimentation at unit gravity, micro- and macronuclei can be purified to virtual homogeneity. This method also permits the purification of homogeneous populations of developing new macronuclei (anlagen) and distinct classes of micronuclei from various stages of conjugation.

Chromatin is the physiologically relevant substrate for all genetic processes inside the nuclei of eukaryotic cells. Dynamic changes in the local and global organization of chromatin are emerging as key regulators of genomic function. Indeed, a multitude of signals from outside and inside the cell converges on this gigantic signaling platform. Numerous post-translational modifications of histones, the main protein components of chromatin, have been documented and analyzed in detail. These 'marks' appear to crucially mediate the functional activity of the genome in response to upstream signaling pathways. Different layers of cross-talk between several components of this complex regulatory system are emerging, and these epigenetic circuits are the focus of this review.

A clear relationship exists between histone acetylation and transcriptional output, the balance of which is conferred by opposing histone acetyltransferases (HATs) and histone deacetylases (HDACs). To explore the role of HDAC activity in determining the transcriptional competency of chromatin, we have exploited the biological features of Tetrahymena as a model. Each vegetative cell contains two nuclei: a somatic, transcriptionally active macronucleus containing hyperacetylated chromatin and a transcriptionally silent, germ line micronucleus containing hypoacetylated histones. Using a PCR-based strategy, a deacetylase gene (named THD1) encoding a homolog of the yeast HDAC Rpd3p was cloned. Thd1p deacetylates all four core histones in vitro. It resides exclusively in the macronucleus during vegetative growth and is asymmetrically distributed to developing new macronuclei early in their differentiation during the sexual pathway. Together, these data are most consistent with a potential role for Thd1p in transcriptional regulation and suggest that histone deacetylation may be important for the differentiation of micronuclei into macronuclei during development.

Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression. The roles of other covalent histone modifications remain poorly understood. To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena, yeast, and human HeLa cells. In Tetrahymena, transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3. Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast. These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone. In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena, we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined. Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Thousands of DNA elimination events occur during somatic differentiation of many ciliated protozoa. In Tetrahymena, the eliminated DNA aggregates into submacronuclear structures containing the protein Pdd1p, a member of the chromodomain family. We disrupted somatic copies of PDD1, eliminating parental expression of the gene early in the sexual phase of the life cycle. Even though zygotic expression, from the undisrupted germline PDD1 copy, is activated before DNA elimination normally occurs, the somatic knockout cells suffer defects in DNA elimination, genome endoduplication, and nuclear resorption, and eventually die, demonstrating that PDD1 is essential and suggesting Pdd1p is directly involved in establishing a chromatin structure required for DNA elimination.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

Previous studies have suggested that micronuclear linker histones are phosphorylated by cAMP-dependent protein kinase (PKA) in Tetrahymena (Sweet, M.T., and C.D. Allis. 1993. Chromosoma. 102: 637-647). In this study, we report that a rapid and dramatic phosphorylation of the micronuclear linker histone, delta, occurs early in the sexual pathway, conjugation. Phosphorylated isoforms of delta are detected as early as 30 min after mixing cells of different mating types; blocking pair formation abolishes this induction completely. Phosphorylation of delta is stimulated by the addition of N6-benzoyladenosine 3':5' cyclic monophosphate to starved (nonmating) cells, suggesting that a PKA/cAMP signal transduction pathway is involved. Maximal phosphorylation of delta is observed during meiotic prophase, a period when micronuclei become transcriptionally active. In situ staining, using phospho-delta-specific antibodies combined with [3H]uridine autoradiography, shows that decondensed micronuclear chromatin undergoing active transcription is enriched in phosphorylated delta isoforms. In contrast, condensed inactive micronuclear chromatin is enriched in dephosphorylated delta. These results strongly suggest that phosphorylation of linker histone plays an important and previously unsuspected role in establishing transcriptional competence in micronuclei.

Phosphorylated and dephosphorylated isoforms of Tetrahymena macronuclear H1 were separated from each other by cation-exchange high performance liquid chromatography and used to generate a pairwise set of antisera that discriminate the phosphorylation state of this linker histone. Affinity-purified antibodies from each sera recognize appropriate H1 isoforms and stain macronuclei under appropriate physiological conditions. Immunogold localizations demonstrate that phosphorylated and dephosphorylated H1 localize nonrandomly in distinct subdomains of macronuclear chromatin. Dephosphorylated H1 is strongly enriched in the electron-dense chromatin bodies that punctuate macronuclear chromatin. In contrast, phosphorylated H1 isoforms, as well as an evolutionarily conserved H2A.F/Z-like variant (hv1) believed to function in the establishment of transcriptionally competent chromatin, are modestly enriched at the periphery of chromatin bodies and in the surrounding euchromatin. Using antibodies against TATA-binding protein, we show that transcriptionally active chromatin lies outside of the chromatin bodies in an area relatively devoid of H1. Antibodies against general core histones are more or less evenly distributed across these domains. Together, these data are consistent with a model in which phosphorylation of H1, perhaps in association with hv1, loosens the binding of H1 in chromatin leading to chromatin decondensation as part of a first-step mechanism in gene activation. In contrast, our data support the view that dephosphorylation of this linker histone facilitates or stabilizes condensed, transcriptionally silent chromatin.

Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation. Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source. An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena. This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates [3H]acetate from [3H]acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels. p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC. Gel-filtration chromatography of this activity indicates a molecular mass of 220 kDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa. Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A--i.e., chromatin associated.

Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies. Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis). Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die. Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration. Some deficiencies cause aberrant anlage DNA loss. Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes. The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed. Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage. The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate.

An abundant phosphoprotein with an apparent molecular mass of 65 kDa (p65) has been identified that is enriched in developing new macronuclei (or anlagen) isolated from the holotrichous ciliate, Tetrahymena thermophila. During early stages of macronuclear development, p65 is actively synthesized and deposited into young (4C) anlagen and is not found in micronuclei or parental macronuclei. p65 is not detected in older (8C) anlagen or in vegetatively growing or starved cells, and thus p65 is under stringent developmental control. In situ analyses, using polyclonal antibodies generated against p65, demonstrate that p65 undergoes a pronounced change in distribution during anlagen differentiation. Initially, anlagen are uniformly stained with these antibodies. However, following separation of conjugants, this staining pattern converts to one that is punctate and fragmented. As development proceeds, most, if not all, of these p65-based particles become peripherally located in anlagen and appear as well-defined vesicles surrounding a discrete central core of DNA of yet undetermined origin. This remarkable cytological distribution suggests an involvement of p65 in the elimination or processing of DNA during anlagen differentiation. If the above is correct, p65 provides the first inroad into the protein machinery involved in the well-known DNA rearrangements that characterize ciliate macronuclear development.

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1. The transcriptionally active macronuclei stained at all stages of the life cycle. Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation. hv1 staining disappeared from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin.

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

During conjugation in the ciliated protozoan Tetrahymena, new macronuclei differentiate from germinal zygotic micronuclei while parental (old) macronuclei are eliminated in two stages, condensation or pycnosis coincident with cessation of transcription followed by resorption. We show that pycnosis is accompanied by degradation of old macronuclear DNA into oligonucleosome-sized fragments, a hallmark of programmed cell death, or apoptosis, in a variety of eukaryotic systems. As expected, oligonucleosome formation does not occur in the new micro- and macronuclei, confirming the coordination of different developmental fates for different nuclei in a common cytoplasm. NULLI 3 conjugants have wild-type old macronuclei but lack chromosome 3 germinally and hence in the new macronucleus. In NULLI 3 conjugants, old macronuclear pycnosis and oligonucleosome fragmentation occur normally but the resorption step fails, and the pycnotic old macronucleus is retained, demonstrating that the two steps are genetically separable and thus distinct and implying that genes on chromosome 3 in the new macronucleus are required for the resorption step. Comparison of whole cell polypeptides synthesized during stages of macronuclear development in both wild-type and NULLI 3 crosses reveal similar profiles. However, a polypeptide (apparent M(r) of 53 kDa) synthesized during old macronuclear elimination is not observed in NULLI 3 conjugants; its role, if any, in elimination of the old macronucleus is unknown. The results show that the old macronucleus is selectively destroyed by a mechanism which is remarkably similar to apoptosis in other eukaryotes and that the zygotic genome is required for the resorption step.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inert micronucleus. During vegetative growth, macronuclear histones H2A and H2B and micronuclear H2A are ubiquitinated. Despite differences in function, macro- and micro-nuclei are related. During conjugation (the sexual phase of the life cycle in Tetrahymena), postzygotic division products of micronuclei give rise to new micro- and macro-nuclei. Using an anti-ubiquitin antibody in Western blotting experiments, we determined the levels of ubiquitinated histones in new macro- and micro-nuclei at various times during conjugation. Very soon after the second postzygotic division (approximately 8 h) when new macronuclei begin to synthesize RNA, ubiquitinated H2B and polyubiquitinated H2A are present. At this time micronuclei have only low levels of ubiquitinated H2A. During later stages of conjugation (15 h), the level of polyubiquitinated H2A decreases, while ubiquitinated H2B increases in developing new macronuclei, attaining levels of ubiquitinated H2B approaching that of parental macronuclei. Ubiquitinated histones are not detectable in the 15-h micronuclei. These results show that ubiquitination of H2B coincides with the transformation of an inert germinal nucleus into that of a transcriptionally active somatic nucleus, suggesting that ubiquitinated H2B has a role in maintaining the transcriptionally active chromatin state.

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Earlier studies ( Allis , C. D., C. V. C. Glover , J. K. Bowen, and M. A. Gorovsky , 1980, Cell, 20:609-617; and Allis , C. D., Y. S. Ziegler , M. A. Gorovsky , and J. B. Olmsted, 1982, Cell, 31:131-136) demonstrated the existence of a macronuclear-specific histone variant, hv1 , which is enriched in small punctate regions in nucleoli of several mammalian cell lines. These observations suggest that this histone variant is highly conserved in evolution and may be associated with actively transcribed sequences. Despite large differences in structure and function during vegetative growth, macro- and micronuclei are related. During conjugation, the sexual phase of the life cycle in Tetrahymena, postzygotic division products of micronuclei give rise to new micro- and macronuclei, while the old macronucleus moves to the posterior of each cell and is eliminated. In this study using antiserum specific for hv1 , we determined by indirect immunofluorescence the time during conjugation at which hv1 first appears in the developing new macronuclei. In growing, starved, and young mating cells (2-5 h after mixing opposite mating types), only macronuclei are detected with affinity-purified antibodies against hv1 . Newly formed macronuclei are either not stained or only weakly stained in cells in which the old macronucleus is located in the center of the cell. However, new macronuclei are clearly observed in cells in which the old macronucleus has moved to the posterior of the cell (approximately 8 h). During later stages of conjugation (10-16 h), the intensity of hv1 staining in new macronuclei increases with time corresponding to the increasing DNA content of these nuclei. Disappearance of detectable hv1 from old macronuclei begins nearly 1 h after these nuclei reach the posterior cytoplasm (approximately 9-10 h) and is sometimes complete before these nuclei are eliminated from the cells. Autoradiography of cells labeled for brief periods with [3H]uridine shows that new macronuclei begin to synthesize RNA very soon after the second postzygotic division (approximately 8 h). During stages when hv1 is clearly detected in new macronuclei, anlagen are active in RNA synthesis. RNA synthesis in old macronuclei ceases very close to the time when RNA synthesis begins in new macronuclei. Thus, the addition of hv1 coincides closely with the transformation of a transcriptionally inactive germinal nucleus into that of a transcriptionally active somatic nucleus. We suspect that addition of hv1 plays a fundamental role in

Macronuclei of Tetrahymena thermophila contain a typical H1 which has been shown to be missing from micronuclei. Instead, micronuclei contain three unique polypeptides, alpha, beta, and gamma, which are associated with linker regions of micronuclear chromatin. In this report polyclonal antibodies raised against macronuclear H1 are shown to react with alpha, beta, and gamma by immunoblotting analyses. This result suggests that these polypeptides share some common structural feature(s). Also consistent with this result is the finding that both macro- and micronuclei in growing and mating cells stain positively with H1 antibodies by in situ indirect immunofluorescence. However, these analyses demonstrate that the level of linker histone is greatly reduced in the micronucleus of starved cells and in young macronuclear anlagen. These results are in agreement with earlier biochemical studies and together provide strong evidence that dramatic changes in linker histone accompany nuclear differentiation (and dedifferentiation) in Tetrahymena.

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones alpha, beta, gamma, and H3F. Of these histones, only alpha appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, alpha is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14-16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14-16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides beta and gamma in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that beta and gamma are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10-16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3S into H3F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena.

In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly.

Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila. Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating. The way in which histone H4 localized to either the macronucleus or the micronucleus suggested that it accumulates in whichever nucleus is replicating. The inability of the micronucleus to accumulate Tetrahymena H1 or heterologous nuclear proteins, even at a period in the cell cycle when it is accumulating H4, suggests that it has a specialized transport system. These studies demonstrate that although the mechanism for localizing proteins to nuclei is highly conserved among eukaryotes, it can differ between two porecontaining nuclei lying in the same cytoplasm.

During vegetative growth, micronuclei of the ciliated protozoan Tetrahymena thermophila contain two electrophoretically distinct forms of H3, H3S and H3F [4, 5]. Of these two forms, H3F is unique to micronuclear chromatin and is derived from H3S by a physiologically regulated proteolytic processing event [5]. While the function of this processing event is not clear, several lines of evidence [2, 5] suggest that it may be related to chromatin condensation during mitosis. In this report pulse-chase experiments have been used to study the processing of H3S into H3F during the sexual phase of the life cycle, conjugation. Our results demonstrate that even though micronuclei divide mitotically (and meiotically) several times during the mating process, processing of H3S into H3F does not occur. Failure of H3S to be converted into H3F during these divisions causes a significant increase in the amount of H3S (relative to H3F) as conjugation proceeds. By 10 h of conjugation, essentially all of the micronuclear H3 is in the form of H3S (also see [3]). As long as mating cells are maintained under starvation conditions, processing of H3S into H3F does not occur. However, if exconjugants are returned to food and allowed to proceed through the first true cell division following exconjugation, processing of H3S into H3F occurs. Thus, the return of the processing of H3(3) into H3F following conjugation seems to be tightly coupled to a division which is part of a cell division cycle (as appears to be the case with vegetatively growing cells). The relevancy of these results to the differentiation of new macro- and micronuclei is discussed. H3F is specifically phosphorylated in growing cells, and it has been suggested that this phosphorylation event may be related to chromatin condensation during mitosis [2]. Since in mating cells H3S becomes the more predominant form of H3, the pattern of histone phosphorylation was examined during stages of conjugation where micronuclei are active in mitotic division (6-7 h). While a low level of phosphate label is observed over H3S in mating cells, more phosphate label is associated with the small amount of H3F which remains in micronuclei at this stage of conjugation. We also observe significant amounts of phosphate label associated with micronuclear H2A, H2B, and H4 and each of the micronuclear H1-like molecules, alpha, beta and gamma.

Evidence is presented that micronuclei of Tetrahymena thermophila contain significant amounts of two types of histone H3. One is indistinguishable from that found in macronuclei and the other is unique to micronuclei. The micronucleus-specific H3 has a slightly faster mobility than the common H3 in three different gel systems (both of these species were artifactually lost during procedures for histone preparation in previous studies). Both micronuclear H3s appear to contain a single cysteine residue and are present in sucrose gradient-purified nucleosomes. Acid extracts from micronuclei also contain three prominent high molecular weight proteins that also were lost during previous procedures. These proteins are present in extracts from oligomers but are not observed in extracts from mononucleosomes, suggesting that they may be associated with linker regions between nucleosomes.

Specific rearrangement of internal chromosomal regions occurs during development of the somatic macronucleus in Tetrahymena thermophila and results in elimination of germ-line (micronuclear) DNA sequences. The timing and mechanism of genome rearrangement within one particular 9.3-kilobase region, which contains three distinct eliminated sequences, were investigated. Portions of this cloned region were used as probes in Southern hybridization experiments to analyze DNA from developing macronuclei (anlagen). All three deletions were found to occur predominantly within a 2-hr time period in which the nuclear DNA contents increased from 4C to 8C (1C represents the amount of DNA present in a haploid genome). The three deletion events can occur independently because intermediate forms, having sustained one or two deletions, were detected. One of the deletions occurs in two alternative ways, resulting in two equally abundant products of different size. Because reciprocal products expected from unequal sister chromatid exchange were not detected, an intramolecular DNA splicing mechanism is suggested.

The patterns of histone phosphorylation in amitotically dividing, transcriptionally active macronuclei and in mitotically dividing, transcriptionally inert micronuclei of the ciliated protozoan Tetrahymena thermophila have been analyzed. Taken together, the major phosphorylation events in these two nuclei and their dependence on cell growth and/or division are remarkably similar to those in mammalian cells. Phosphorylation of H1-type proteins occurs in both nuclei and is positively correlated with growth and/or division. Phosphorylation of histone H3 is also positively correlated with growth and/or division but occurs only in micronuclei. Phosphorylation of histone H2A is relatively independent of growth state and occurs largely, if not exclusively, in macronuclei. Given the unique partition of nuclear functions between macro- and micronuclei, these results, coupled with previously reported temporal correlations between specific histone phosphorylations and cell cycle events in mammalian cells [Gurley, L. R., Tobey, R. A., Walters, R. A., Hildebrand, C. E., Hohmann, P. G., D'Anna, J. A., Barham, S. S., & Deaven, L. L. (1978a) in Cell Cycle Regulation (Jeter, J. R., Cameron, J. L., Padilla, G. M. & Zimmerman, A. M. Eds.) pp 37-60, Academic Press, New York], allow insights into the functions of histone phosphorylations. Specifically, a nonmitotic function for extensive H1 phosphorylation and a unique mitotic function for H3 phosphorylation are clearly indicated. A new role for H2A phosphorylation in the regulation of transcriptional activity is also proposed.

Histone acetylation in Tetrahymena macro- and micronuclei has been studied utilizing a combination of electrophoretic and autoradiographic techniques. Histones H2A, H2B, H3, and H4 are acetylated to varying extents in the transcriptionally active macronucleus. There are few, if any, acetylated subspecies of these histones in the transcriptionally inert micronucleus, and micronuclei incorporate little radioactive acetate, either in vivo or in isolated nuclei. Butyrate is shown to inhibit histone deacetylation in Tetrahymena, both in vivo and in isolated nuclei. Incorporation of acetate into micronuclei is unaffected by high concentrations of this inhibitor, indicating that the extremely low levels of histone acetylation observed in micronuclei are not due to rapid deacylation but probably result from the absence of histone acetylation. We also present evidence that macronuclear core histones are composed of at least two classes of molecules distinguishable on the basis of their acetate turnover rates and that individual histone species differ in the distribution of their populations between these classes.