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C D Allis

Publications and source records attributed to C D Allis.

At least 145 records · Page 8Linked to original sources
PubMed1991

Timing of the appearance of ubiquitinated histones in developing new macronuclei of Tetrahymena thermophila.

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.

Animals↗
PubMed1989

Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin.

Using an anti-ubiquitin antibody in Western blotting experiments, we detected polyubiquitinated species of histones H2A, H2A.Z, and H2B in histone preparations of bovine thymus, chicken erythrocyte, and Tetrahymena macro- and micronuclei. Histone H2A had the greatest level of polyubiquitinated species, with tetra- to hexaubiquitinated forms of this histone being observed. The fraction of bovine thymus and chicken erythrocyte chromatin enriched in transcriptionally active gene sequences was enriched in mono- and polyubiquitinated species of histones H2A, H2B, and H2A.Z, especially in the ubiquitinated forms of histone H2B. Histones H2A and H2B were ubiquitinated in the transcriptionally active Tetrahymena macronucleus, with monoubiquitinated (u) H2B being the predominant ubiquitinated histone species. Ubiquitinated forms of histones H2A and H2B were found in transcriptionally inert micronuclei, but at lower levels than seen in macronuclear histones. Also, the level of micronuclear uH2A was greater than that of uH2B which may be from macronuclei that contaminate the preparation. These results indicate that the mono- and polyubiquitinated species of histone H2B are preferentially located in transcriptionally active chromatin regions. Ubiquitinated histone H2A is located in both expressed and repressed chromatin domains, but expressed chromatin is enriched in mono- and polyubiquitinated forms of this histone. These observations are consistent with the hypothesis that ubiquitinated histones have a role maintaining the structure of transcriptionally active chromatin.

Animals↗
PubMed1989

Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation 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.

Acetylation↗
PubMed1989

Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei.

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.

Animals↗
PubMed1988

Sequence and properties of the message encoding Tetrahymena hv1, a highly evolutionarily conserved histone H2A variant that is associated with active genes.

hv1 is a histone H2A variant found in the transcriptionally active Tetrahymena macronucleus, but not in the transcriptionally inert micronucleus. hv1 also contains antigenic determinants conserved in the histone complements of representatives of all four eukaryotic kingdoms. A cDNA clone encoding hv1 has been isolated and sequenced. Comparison of the derived protein sequence of hv1 with that of the chicken variant H2A.F and the sea urchin variant H2A.F/Z reveals remarkable homology in all but the extreme amino- and carboxy-termini and a small region in the conserved core. Putative regions of conserved antigenicity are discussed. Evidence is presented that suggests that hv1 is a single-copy, intron-containing gene that encodes a polyadenylated message. Unusual features in the 3' flanking sequence and in codon usage are also described. Evidence is also presented showing that hv1 message amounts are ten-fold greater in growing cells than in starved cells.

Amino Acid Sequence↗
PubMed1988

Cell-cycle regulation as a mechanism for targeting proteins to specific DNA sequences in Tetrahymena thermophila.

Transcriptionally active macronuclei and transcriptionally inert micronuclei of the ciliated protozoan Tetrahymena thermophila contain similar DNA sequences but have very different histones associated with the linker regions of chromatin. In situ hybridization showed that a gene coding for micronuclear linker histone is expressed only in association with micronuclear DNA replication, whereas the gene for macronuclear H1 histone is expressed during macronuclear (but not during micronuclear) S phase. These results indicate that cell-cycle regulation plays an important role in directing proteins to the appropriate nucleus in Tetrahymena and that the replication-expression model [Gottesfeld, J. & Bloomer, L. S. (1982) Cell 28, 781-791; Wormington, W. M., Schlissel, M. & Brown, D. D. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 879-884] for establishing appropriate transcriptionally active or repressed chromatin complexes during DNA replication is generally applicable.

Animals↗
PubMed1988

Micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase activity which is highly specific for free histone H4.

Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free histone. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form. These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro- or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles which are different from that of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.

Acetylation↗
PubMed1988

Characterization of phosphorylation sites in histone H1 in the amitotic macronucleus of Tetrahymena during different physiological states.

Histone H1 is highly phosphorylated in transcriptionally active, amitotic macronuclei of Tetrahymena during vegetative growth. However, the level of H1 phosphorylation changes dramatically in response to different physiological conditions. H1 is hyperphosphorylated in response to heat shock and during prezygotic stages of conjugation. Conversely, H1 is largely dephosphorylated during prolonged starvation and during elimination of parental macronuclei during conjugation. Mapping of phosphorylation sites within H1 indicates that phosphorylation occurs at multiple sites in the amino-terminal portion of the molecule, predominantly at threonine residues. Two of these sites have been identified by compositional analyses and microsequencing of tryptic peptides. Interestingly, two major sites contain the sequence Thr-Pro-Val-Lys similar to that contained in the sites recognized by growth-associated histone kinase in other organisms. No new sites are detected during the hyperphosphorylation of H1 which occurs during heat shock or in early stages of conjugation, and no sites are preferentially dephosphorylated during starvation or later stages of conjugation. Therefore, changes in the overall level of H1 phosphorylation, as opposed to phosphorylation or dephosphorylation at particular sites, appear to be important in the regulation of chromatin structure under these physiological conditions. Further, since no cell division or DNA replication occurs under these conditions, changes in the level of H1 phosphorylation are best correlated to changes in gene expression during heat shock, starvation, and conjugation. We suggest that, at least in Tetrahymena, H1 hyperphosphorylation is used as a rapid and transient mechanism for the cessation of transcription under conditions of cellular stress.

Amino Acid Sequence↗
PubMed1987

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis.

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.

Aging↗
PubMed1987

Scheduled and unscheduled DNA synthesis during development in conjugating Tetrahymena.

Autoradiography has been used to confirm and to extend previous microspectrophotometric studies (Doerder and DeBault, 1975) on the timing of DNA synthesis during conjugation in Tetrahymena thermophila. The majority of DNA synthesis occurs at the expected periods preceding gamete formation and the two postzygotic divisions and during macronuclear development. DNA in new macronuclei is endoreplicated in an extremely discontinuous fashion. Under starvation conditions, the first endoreplication (2C to 4C) occurs immediately after the second postzygotic division when both new macronuclei and new micronuclei replicate. The second endoreplication (4C to 8C) does not occur until after separation of conjugants. If mating cells are kept under prolonged starvation conditions (20-24 hr), refeeding induces a partially synchronous division, after which an unexpectedly high percentage of cells incorporate tritiated thymidine into both macro- and micronuclei. Two previously undescribed periods of DNA synthesis were observed in the micronuclei of conjugating Tetrahymena. The first occurs during the early stages of meiotic prophase, before full crescent elongation. The second takes place in an extended period corresponding to macronuclear anlagen development, before conjugants have separated. CsCl gradient analyses indicate that, in micronuclear fractions, only main band DNA is being synthesized in both of these periods. However, in macronuclear fractions from both stages, a significant fraction (approximately 20%) of the DNA being synthesized has the buoyant density of ribosomal DNA. The finding that macro- and micronuclear DNA can be synthesized simultaneously in a single cell, both during conjugation and after refeeding starved exconjugants, raises interesting questions of how macro- or micronuclear-specific histones are targeted to the appropriate nuclei.

Animals↗
PubMed1987

Tetrahymena contain two distinct and unusual high mobility group (HMG)-like proteins.

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG-1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG-1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schröter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1987

A single histone acetyltransferase from Tetrahymena macronuclei catalyzes deposition-related acetylation of free histones and transcription-related acetylation of nucleosomal histones.

A salt-extracted histone acetyltransferase activity from Tetrahymena macronuclei acetylates mostly histone H3 and H4 when free histones are used as substrate. Free histone H4 is acetylated first at position 11 (monoacetylated) or positions 11 and 4 (diacetylated). This activity strongly resembles in vivo, deposition-related acetylation of newly synthesized histones. When acetylase-free mononucleosomes are used as substrate, all four core histones are acetylated by the same extract, and H4 is acetylated first at position 7 (monoacetylated) or positions 7 and 4 (diacetylated). In this respect, the activity of the extract is indistinguishable from postsynthetic, transcription-related histone acetylation that occurs in vivo or in isolated nuclei. Heat inactivation curves with both substrates are indistinguishable, and free histones compete with chromatin for limiting amounts of enzyme activity. These results argue strongly that two distinct, biologically important histone acetylations, one deposition related and one transcription related, are carried out by a single acetyltransferase.

Acetylation↗
PubMed1986

Enzyme activity dot blots: a rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose.

Methods are described for assaying (Tetrahymena) histone acetyltransferase activity and (Drosophila) casein kinase II activity by spotting extracts on nitrocellulose filters. The methods are quantitative over a wide range of enzyme concentrations and are almost as sensitive as liquid assays. Examples are presented for illustrating the use of these methods for enzyme purification, concentration, and desalting, as well as for electrophoretic blotting from agarose gels. A simple method for autoradiographic enhancement of nitrocellulose filters is also described.

Acetyltransferases↗
PubMed1986

hv1 is an evolutionarily conserved H2A variant that is preferentially associated with active genes.

Polyclonal antibodies to the Tetrahymena macronuclear-specific histone variant hv1 cross-react with histone-like molecules from yeast, wheat, and mouse. A novel purification scheme has allowed isolation of sufficient hv1 to enable determination of the sequence of 61 amino-terminal residues as well as 27 additional internal residues. These data clearly demonstrate that hv1 shares a number of conserved sequence elements with the H2A family of histones. Comparison of hv1 with H2A.F (= H2A.Z = M1), another evolutionarily conserved H2A variant whose sequence is known, reveals that they share an unblocked amino-terminal alanine (instead of acetylserine) and a distinctive structure in a "variant box" region that distinguishes them from major H2As. In addition, 10 residues have been identified which are identical (or highly similar) in hv1 and H2A.F, but are different from residues conserved in the major H2As. Therefore, in many ways hv1 resembles chick H2A.F more than the major Tetrahymena H2A. The sites of acetylation of hv1 also differ from those of the major Tetrahymena H2As. In spite of their similarities, hv1 and H2A.Z differ significantly in their amino termini, and antibodies against hv1 do not react with H2A.Z. Interestingly, the nucleolar staining pattern reported with anti-hv1 serum is similar to that reported for an antiserum to another H2A variant, mouse testes-enriched H2A.X. Since both H2A.Z and hv1 appear to be enriched in transcriptionally active chromatin, these results suggest that there may be a number of different, functionally distinct, nonallelic variants in the H2A family of histones and that hv1 is a hybrid H2A variant with properties of both vertebrate H2A.Z and H2A.X.

Acetylation↗
PubMed1986

Nonrandom utilization of acetylation sites in histones isolated from Tetrahymena. Evidence for functionally distinct H4 acetylation sites.

Macro- and micronuclei of the ciliated protozoan Tetrahymena thermophila afford a unique opportunity to study histone acetylation under conditions where postsynthetic "transcription"-related acetylation and synthetic "deposition"-related acetylation are nonoverlapping. Recent studies have demonstrated that at least two general systems of acetylation operate in Tetrahymena. One is postsynthetic, macronuclear specific, and may be related to gene expression in that nucleus (Vavra, K. J., Allis, C. D., and Gorovsky, M. A. (1982) J. Biol. Chem. 257, 2591-2598). The other is synthetic, common to macro- and micronuclei, and is likely related to histone deposition during replication (Allis, C. D., Chicoine, L. G., Richman, R., and Schulman, I. G. (1985a) Proc. Natl. Acad. Sci. U. S. A., 82, 8048-8052). A unique feature of H3 and H4 in Tetrahymena is that neither are blocked at their amino termini. We have exploited this fact as well as the resolving capability of acid-urea gel electrophoresis and current microsequencing techniques to examine whether utilization of different NH2-terminal acetylation sites is random or nonrandom during the progression toward high acetylation states. Of the four acetylation sites which have been identified in H4 (in Tetrahymena these are lysines at positions 4, 7, 11, and 15), we find that lysine 7 is the exclusive site of postsynthetic acetylation in populations of monoacetylated H4 isolated from macronuclei. This site is retained in populations of diacetylated H4, which are acetylated exclusively at lysines 4 and 7. Our data also suggest that there is some preference for using lysine 11 (as compared to 15) as the third site of acetylation in triacetylated molecules. The data demonstrate that the postsynthetic acetylation-deacetylation process is surprisingly nonrandom for H4 in Tetrahymena macronuclei. We have also investigated the same question with macronuclear H3 (which contains acetylation sites at lysines 9, 14, 18, and 23). Our data demonstrate that unlike H4, lysines at position 9 or 14 are likely to be utilized as sites of acetylation within a population of monoacetylated H3. Both of these acetylation sites are retained in diacetylated H3 which suggests that if site 9 is used initially as the site of monoacetylation, 14 is used secondarily (and vice versa). Our data show, moreover, that there is a preference for utilizing lysine 18 as the third acetylation site (in triacetylated H3). Thus, these data show that H3 is also acetylated in a nonrandom fashion in macronuclei. Finally, we have determined which acetylation sites are utilized in macro- or micronuclear H4 when it is undergoing active synthesis and deposition.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗
PubMed1986

Regulation of histone acetylation during macronuclear differentiation in Tetrahymena: evidence for control at the level of acetylation and deacetylation.

During the postzygotic period of the sexual cycle (conjugation) in the ciliated protozoan, Tetrahymena, daughter products from a single micronuclear mitotic division develop into new macronuclei (anlagen) or new micronuclei depending upon their cytoplasmic location. In this study we have monitored the status of histone acetylation in synchronous populations of developing nuclei isolated from conjugating cells. Particular attention has been paid to the level of histone acetylation in new macronuclei following their differentiation from micronuclei. Like micronuclei isolated from vegetative cells (Vavra et al., 1982), micronuclei from conjugating cells (5 hr, 10-12 hr, and 15-16 hr) contain little if any acetylated histone and incorporate little postsynthetic acetate under any of our experimental conditions. In contrast, young new macronuclei (4C, 10-12 hr) incorporate significant amounts of acetate in vitro and in vivo provided that sodium butyrate is included during the labeling period. These results suggest that 4C anlagen contain both active acetylase and deacetylase activities even though the actual steady state level of acetylation found in these nuclei is low, more like that of micronuclei. At later stages of macronuclear maturation (8C, 15-16 hr), inner histones are hyperacetylated in a manner similar to parental, fully differentiated macronuclei. Furthermore, 8C anlagen incorporate acetate well even in the absence of sodium butyrate. Taken together these results suggest that endogenous deacetylase enzymes become either down-regulated and/or the rate of histone acetylases increases markedly during macronuclear differentiation.

Acetylation↗
PubMed1986

An intervening sequence in an unusual histone H1 gene of Tetrahymena thermophila.

An intervening sequence of 254 base pairs interrupts the coding region of the single gene for macronuclear histone H1 of the ciliated protozoan, Tetrahymena thermophila. The intervening sequence has splice junctions similar to those found in RNA polymerase II genes of other organisms. No obvious similarities are observed between this intron and the self-splicing intervening sequence of the Tetrahymena ribosomal gene. The derived amino acid sequence describes a small extremely basic H1 protein missing most of the central hydrophobic domain that is conserved in all other H1 proteins. Macronuclei divide amitotically, without chromosome condensation, suggesting the conserved globular domain of H1 plays a role in higher-order chromatin structure.

Amino Acid Sequence↗