Micronuclei of Tetrahymena contain two types of histone H3.

Allis, C. David; Glover, Claiborne V.C.; Gorovsky, Martin A.

doi:10.1073/pnas.76.10.4857

Cited by 97 publications

(86 citation statements)

References 19 publications

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“…lb-d). When histone preparations were separated by triton/acid/ urea-PAGE (Allis et al 1979;Allis et al 1980a), which alters the relative mobility of the histones, staining was again specific to the hvl region (data not shown). These immunoblot results indicate all three sera are highly specific to the minor histone variant, hvl.…”

Section: Generation and Characterization Of Hvl Antiseramentioning

confidence: 99%

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Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

Stargell¹,

Bowen²,

Dadd³

et al. 1993

Genes & Development

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120

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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 hvl, 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 hvl protein and synthetic peptides from the amino and carboxyl domains of hvl. 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 hvl early in conjugation, hvl 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 hvl may be related to the elimination of hvl during the dynamic restructuring 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 hvl functions in establishing a transcriptionally competent state of chromatin.

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Section: Generation and Characterization Of Hvl Antiseramentioning

confidence: 99%

“…Histones were extracted from Tetrahymena macronuclei with 0.4 N H2SO 4 (Allis et al 1979) and precipitated with 0.5 N perchloric acid. The acid-insoluble pellet was dissolved in 4 M urea, 5% ~-mercaptoethanol, and centrifuged for 5 rain at 16,000g to remove particulates.…”

Section: Histone Purificationmentioning

confidence: 99%

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

Stargell¹,

Bowen²,

Dadd³

et al. 1993

Genes & Development

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120

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“…Nuclear Isolation and Histone Extraction-Macronuclei were isolated from growing cells as described previously (31) and extracted with 0.2 M H 2 SO 4 for 12-18 h at 4°C, and the acid-soluble material was precipitated with 20% trichloroacetic acid (38).…”

Section: Making Htb1 Somatic Rescue Constructs and Creation Of Htb1 Smentioning

confidence: 99%

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Wang

Cui²,

Gorovsky

2009

Journal of Biological Chemistry

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Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitinconjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the "cross-talk" among post-translational modifications on different histones.In the nuclei of eukaryotic cells, DNA is highly compacted into chromatin, a nucleoprotein complex, whose major protein components are the histones. The basic unit of chromatin is the nucleosome core in which 146 base pairs of double-stranded DNA are wrapped in ϳ1.75 left-handed superhelical turns around an octamer containing two molecules of each of the four conserved core histones H2A, H2B, H3, and H4 (1, 2). A fifth, less conserved linker histone H1, associates with a variable length of linker DNA between nucleosome cores. Site-specific histone post-translational modifications, such as acetylation, methylation, phosphorylation, and ubiquitylation, are correlated with diverse chromatin functions, including DNA replication, DNA repair, chromatin assembly, gene transcription, and other chromatin-based processes (3, 4). It has been suggested that at least some distinct histone modifications act sequentially or in combination to form what has been variously referred to as a histone, epigenetic, or nucleosome "code" that is read by other proteins or protein complexes to alter chromatin structure and regulate distinct downstream events (5, 6). Deciphering this code, which is likely to have complex redundant, combinatorial, and multivalent features, is essential for understanding the in vivo function of the diverse post-translational modifications on histones.Ubiquitin is a 76-amino acid globular protein that is conserved in eukaryotes. Unlike proteins that are targeted by polyubiquitylation to the proteosome for degradation, histones are reversibly modified by covalent attachment of one ubiquitin or a short ubiquitin chain. The bulk of histone ubiquitylation occurs on chromatin by the addition of a single ubiquitin molecule via an isopeptide linkage to a specific lysine residue in the C-terminal tail of histones H2A and H2B (7,8). To a lesser extent, histones H1 (9), H3, and H4 (10, 11) also can be ubiquitylated in vivo. Ubiquitylation on different histones has distinct functions (12).A number of recent studies have focused on ubiquitylation of H2B. In the budding yeast Saccharomyces cerevisiae, H2B is monoubiquitylated at l...

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“…Cells were grown in 1% tryptone/0.1% (wt/vol) yeast extract/0.001% (wt/vol) EDTA at 29°C in flasks 10× larger than the culture volume without shaking to a tiler of 4-5 × 105 cells/ml. Nuclei were isolated and histones were extracted after the procedure described by Allis et al (5), except that 0.25 M HCI was used instead of H2SO4 and that histones were precipitated with acetone instead of TCA. Histones were separated either on 12.5% acrylamide slab gels containing Na-dodecylsulfate (26) and electrophoretically transferred to nitrocellulose sheets (36) or on Triton/urea/acetic acid gels and blotted by diffusion as described (30).…”

Section: Methodsmentioning

confidence: 99%

Histone acetylation in conjugating Tetrahymena thermophila [published erratum appears in J Cell Biol 1989 Dec;109(6 Pt 1):3214-7]

Pfeffer¹

1989

The Journal of Cell Biology

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Abstract. We have monitored histone acetylation during conjugation of the ciliated protozoan Tetrahymena thermophila using antibodies against the tetraacetylated form of H4 histone (Pfeffer, U., N. Ferrari, and G. Vidali. 1986. J. Biol. Chem. 261:2496-2498. During meiosis, the three prezygotic divisions, fertilization, and the first postzygotic division, micronuclei, do not contain highly acetylated forms of H4 histone. However, after the second postzygotic division, when anteriorly located micronuclei begin to develop into new macronuclei, they are strongly stained by the anti-tetraacetylated H4 histone antibody. In the old macronucleus, histones are actively deacetylated when it has ceased to transcribe but before it is eliminated. Histone acetylation processes analyzed here appear to be correlated to the commitment to transcription rather than to the transcription process itself. This is in good correlation with evidence we have obtained in chick erythrocyte nuclei during reactivation upon fusion with mammalian cells (Pfeffer, U., N. Ferrari, E Tosetti, and G. Vidali. 1988. Exp. Cell Res. 178:25-30). Furthermore, it becomes clear from our data that histone acetylation occurs in close correlation to the position of nuclei within the cytoplasm of T. thermophila.Mechanisms that control differential histone acetylation and deacetylation are discussed.S INCE the original observation, histone acetylation has been correlated to transcription and considerable evidence has accumulated indicating that this reversible postsynthetic modification of lysine residues in the aminoterminal portion of core histones plays a key role in the regulation of gene expression (for a review see reference 2).Different methods have been developed to fractionate chromatin depending on its transcriptional activity and accordingly, it has been found that fractions containing actively transcribed genes were enriched in highly acetylated histones (1,17,33). Recent analyses using anti-acetyl-lysine antibodies have established that a given gene is associated with highly acetylated histones when it is transcribed (23), although it cannot be ruled out that genes competent to be transcribed, but inactive at the moment of analysis, may also contain acetylated histones.Nucleosomes undergo reversible conformational changes during transcriptional activation (10, 32), histones are absent from promoter sequences of active genes (25), nucleosomes inhibit initiation of transcription but do not inhibit elongation of initiated transcripts (27, 28) and at least H4 histone is retained on a highly transcribed gene (35). The contact points between nucleosomal DNA and H4 histone have been analyzed and a highly basic cluster in the amino-terminal portion has been shown to be involved in sharp bending of the DNA double helix (18). Interestingly, this cluster encompasses lysine at position 16 which is the first to be acetylated in vivo in calf thymus (15) and, moreover, this region is in close contact to a site 55-65 bp from the end of the nucleosomal DNA, which ...

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Micronuclei of Tetrahymena contain two types of histone H3.

Cited by 97 publications

References 19 publications

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Histone acetylation in conjugating Tetrahymena thermophila [published erratum appears in J Cell Biol 1989 Dec;109(6 Pt 1):3214-7]

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