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C D Allis

Publications and source records attributed to C D Allis.

At least 127 records · Page 7Linked to original sources
PubMed1993

Analysis of nucleosome assembly and histone exchange using antibodies specific for acetylated H4.

Using antibodies that specifically recognize the acetylated forms of histone H4, we show that it is possible to immunoprecipitate newly assembled (acetylated) nucleosomes. Newly replicated HeLa cell chromatin was labeled for 5-30 min with [3H]thymidine in the presence of sodium butyrate (thus inhibiting the deacetylation of newly deposited H4); bulk chromatin DNA was labeled for 24 h with [14C]thymidine. When soluble nucleosomes were incubated with immobilized antibodies, a comparison of the bound and unbound fractions showed up to a 65-fold enrichment for new chromatin DNA in the immunoprecipitate (bound), relative to the supernatant (unbound). No enrichment for new DNA was observed when preimmune control serum was used in a similar fashion. The enrichment for new DNA in the immunopellet was paralleled by a similar enrichment for all four newly synthesized histones. Acetylation was required for antibody recognition: When chromatin was replicated in the absence of butyrate (permitting histone deacetylation and chromatin maturation), equally low levels of new and old chromatin were immunoprecipitated, and no enrichment for new DNA was observed. Competition experiments confirmed these results. Analyses of histone deposition during the inhibition of DNA replication established that acetylated chromatin is the preferential target for H2A/H2B exchange. These experiments provide evidence for the highly selective assembly of newly synthesized H3, H2A, and H2B with acetylated H4, and for the involvement of histone acetylation in dynamic chromatin remodeling. In addition, immunoprecipitations of radiolabeled cytosolic extracts identified a possible somatic chromatin preassembly complex, containing newly synthesized H3 and new (acetylated) H4.

Acetylation↗
PubMed1993

Parental nucleosomes segregated to newly replicated chromatin are underacetylated relative to those assembled de novo.

Antibodies specific for acetylated histone H4 were used to examine the acetylation state of parental histones that segregate to newly replicated DNA. To generate newly replicated chromatin containing only segregated parental nucleosomes, isolated nuclei were labeled with [3H]TTP in vitro; alternatively, whole cells were labeled with [3H]thymidine in the presence of cycloheximide. Soluble chromatin was prepared by micrococcal nuclease digestion, and subjected to immunoprecipitation with "penta" antibodies (Lin et al., 1989). In sharp contrast to nucleosomes containing newly synthesized, diacetylated H4 (Perry et al., 1993), chromatin replicated in vitro was only marginally susceptible to immunoprecipitation. Control experiments established that bona fide acetylated chromatin was selectively immunoprecipitated by the same techniques and that segregated nucleosomes were not disassembled during treatment with "penta" antibodies. When replication was coupled to an in vitro histone acetylation system, the enrichment for segregated nucleosomes in the immunopellet increased approximately 3-fold, demonstrating that changes in the acetylation state of segregated histones can be detected immunologically and that parental histones on new DNA are accessible to acetyltransferases during, or immediately after, DNA replication. In vivo pulse-chase experiments, performed in the presence of cycloheximide, confirmed these results. Uptake experiments further established that concurrent histone acetylation did not alter the rate of DNA synthesis in vitro. Our results provide evidence that replication-competent chromatin is not obligatorily acetylated, and indicate that the acetylation status of segregated histones may be maintained during chromatin replication. The possible significance of this, with respect to the regulation of chromatin higher order structures during DNA replication, and the propagation of transcriptionally active vs inactive chromatin structures, is discussed.

Acetylation↗
PubMed1993

Phosphorylation of linker histones by cAMP-dependent protein kinase in mitotic micronuclei of Tetrahymena.

Linker histones (LHs) in transcriptionally inactive, mitotically dividing micronuclei of Tetrahymena thermophila, alpha, beta, gamma and delta, are highly phosphorylated in vivo. Analysis of the derived sequences of these LHs suggests that none of these polypeptides contain sites of phosphorylation by p34cdc2, the kinase thought to play an essential role governing the entry of all cells into mitosis. Surprisingly alpha, beta, gamma and delta each contain sites for phosphorylation by cyclic AMP-dependent kinase (PKA). p34cdc2 kinase phosphorylases H1 in vitro but fails to phosphorylate alpha, beta, gamma and delta. Conversely, PKA phosphorylates each of the micronuclear LHs but is unable to phosphorylate macronuclear H1. Micronuclear LHs labeled in vivo with [32P]phosphate were purified by reverse phase HPLC. Phosphoamino acid analysis showed that all four micronuclear LHs are phosphorylated exclusively on serine residues in vitro. Cyanogen bromide mapping of alpha, beta, gamma and delta labeled in vivo or in vitro by PKA indicates that each LH is phosphorylated only on peptides that contain either optimum (RR/KXS) or less optimum (RXXS) PKA sequences. This study suggests that PKA or a PKA-like activity(ies), but not p34cdc2 kinase, is(are) responsible for the in vivo phosphorylation of LHs in the mitotic micronucleus of Tetrahymena. We suggest that, at least in Tetrahymena, PKA-driven phosphorylation or dephosphorylation plays a significant role in the control of mitotic processes such as chromosome condensation.

Amino Acid Sequence↗
PubMed1993

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

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.

Amino Acid Sequence↗
PubMed1993

Transcriptional silencing in yeast is associated with reduced nucleosome acetylation.

Two classes of sequences in the yeast Saccharomyces cerevisiae are subject to transcriptional silencing: the silent mating-type cassettes and telomeres. In this report we demonstrate that the silencing of these regions is strictly associated with acetylation of the epsilon-amino groups of lysines in the amino-terminal domains of three of the four core histones. Both the silent mating-type cassettes and the Y domains of telomeres are packaged in nucleosomes in vivo that are hypoacetylated relative to those packaging active genes. This difference in acetylation is eliminated by genetic inactivation of silencing: The silent cassettes from sir2, sir3, or sir4 cells show the same level of acetylation as other active genes. The correspondence of silencing and hypoacetylation of the mating-type cassettes is observed even for an allele lacking a promoter, indicating that silencing per se, rather than the absence of transcription, is correlated with hypoacetylation. Finally, overexpression of Sir2p, a protein required for transcriptional silencing in yeast, yields substantial histone deacetylation in vivo. These studies fortify the hypothesis that silencing in yeast results from heterochromatin formation and argue that the silencing proteins participate in this formation.

Acetylation↗
PubMed1993

An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila.

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.

Animals↗
PubMed1993

Fractionation of small tryptic phosphopeptides by alkaline PAGE followed by amino acid sequencing.

A novel two-step approach for localizing the site(s) of phosphorylation within intact proteins is described. Phosphorylated (32P-labeled) tryptic peptides are first resolved in a high-percentage polyacrylamide gel that has been optimized for the enrichment and separation of small, negatively charged peptides. Then the resolved peptides are located by autoradiography, excised, eluted and immobilized on a positively charged membrane, Immobilon -N, where they can be sequenced directly. The methods have been developed using a small, basic phosphoprotein (histone H1 from Tetrahymena); however, the approach is probably applicable to a wide variety of phosphoproteins.

Amino Acid Sequence↗
PubMed1992

Replication-dependent and independent regulation of HMG expression during the cell cycle and conjugation in Tetrahymena.

Two abundant high-mobility-group (HMG)-like proteins, HMG B and HMG C, exist in the ciliated protozoan, Tetrahymena thermophila. Of these, HMG C is specific to transcriptionally active macronuclei, while HMG B is found in macronuclei and in transcriptionally inactive micronuclei [1]. Using Northern and in situ analyses, we show that the genes encoding HMG B and HMG C are not expressed uniformly throughout the vegetative cycle or during the sexual process, conjugation. Elevated expression of both genes is observed during macronuclear S phase of the vegetative cycle and during endoreplication of developing new macronuclei in later stages of conjugation. Interruption of any of these macronuclear DNA replications by aphidicolin leads to a rapid drop in the message levels of HMG B and HMG C. These results resemble what is typically observed for replication-dependent nucleosomal histones and differ from the apparent lack of cell cycle regulation observed for HMG genes in vertebrates. A specific-induction of HMG B mRNA is also observed early in conjugation and during this interval, inhibition of micronuclear DNA synthesis by aphidicolin does not affect the message level of HMG B. Thus, during conjugation, expression of HMG B shows both replication-dependent and independent regulation. Results similar to these with HMG B are obtained with histone H4II gene, a gene which is also expressed during micro- and macronuclear S phases during the vegetative cycle. These results demonstrate surprising complexity in the expression of HMG genes in Tetrahymena and lend support to the hypothesis that cell cycle regulation plays an important role in directing HMG-like proteins to the appropriate nucleus [2]. Interestingly, expression of neither HMG gene is perfectly synchronized with that of histone H4II gene during the developmental program suggesting that important differences exist between vegetatively growing (cell cycle control) and conjugating (developmental control) cells.

Animals↗
PubMed1992

Highly acetylated H4 is associated with histone displacement in rat spermatids.

The presence of highly acetylated histone H4 during spermatogenesis was studied to evaluate its correlation with the events of gene transcription, histone deposition, and histone displacement. We utilized an antibody raised to a pentaacetylated synthetic peptide that preferentially recognizes highly (tetra- and tri-) acetylated forms of rat testis H4. Electrophoretic separation of histones from enriched fractions of spermatogenic cells followed by detection of these forms by staining and by immunoblotting using this antibody showed that the highly acetylated forms were limited almost exclusively to spermatids beginning at step 11 of development. Immunoflurescence also revealed a striking polarity in the progression of histone from the spermatid nucleus. Highly acetylated H4 was displaced from the anterior to the caudal portion of the spermatid nucleus during steps 11 and 12, along with other histones, prior to their displacement by transition proteins. Thus, while monoacetylated and low levels of diacetylated forms of H4 were associated with stages at which histone deposition and transcription occur, the more highly acetylated forms appeared in high levels only at the stage at which histone displacement occurs.

Acetylation↗
PubMed1992

Programmed nuclear death: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena.

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.

Animals↗
PubMed1992

Chromatin condensation: does histone H1 dephosphorylation play a role?

In this article we describe three distinct biological systems where histone H1 phosphorylation is uncoupled from mitosis and highly condensed chromatin is enriched in dephosphorylated forms of H1: the amitotic macronucleus of Tetrahymena, terminally differentiated avian erythrocytes and sea urchin sperm. Each system offers informative contrasts to the idea that H1 hyperphosphorylation is causally related to mitotic chromosome condensation. Assuming that higher order chromatin folding is primarily driven by electrostatic interactions between H1 and DNA, an alternative model is presented for the role of H1 phosphorylation in chromatin condensation.

Animals↗
PubMed1991

A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions.

Using the single cell eukaryote Tetrahymena thermophila, a simple method was developed for studying protein-DNA associations by cross-linking proteins to DNA with formaldehyde and immunoprecipitating the solubilized chromatin fragments with a specific antiserum. The protocol uses crude antiserum and involves only three steps: cross-linking, shearing to solubilize the chromatin, and immunoprecipitation. Methods for optimizing certain critical parameters, such as fixation time and NaCl concentration, are described. The method is likely to be generally useful for a variety of nuclear antigens.

Animals↗
PubMed1991

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

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.

Animals↗
PubMed1991

Cell-cell interactions trigger the rapid induction of a specific high mobility group-like protein during early stages of conjugation in Tetrahymena.

Conjugation in Tetrahymena represents an ordered developmental pathway which represents the sexual phase of the ciliate life cycle. This pathway is initiated when starved cells of opposite mating types are mixed and are allowed to make a series of cell-cell contacts (a period termed costimulation) which lead to the formation of mating pairs. Here, we demonstrate that two previously described abundant high mobility group (HMG)-like proteins, HMG B and HMG C, whose synthesis appeared to be coordinately regulated in vegetative cells, are not required during the same stages of conjugation. The level of mRNA for both HMG B and HMG C is high during vegetative growth and during the development of new macronuclei. However, specific induction of HMG B mRNA is observed soon after cells of opposite mating types are mixed. Thus, the genes which encode HMG B and HMG C in Tetrahymena can be controlled independently or coordinately. Nuclear run-on experiments show that a significant factor underlying the rapid induction of HMG B message early in the sexual cycle is an increase in the transcriptional activity of the HMG B gene. Experiments are presented which show that this induction of HMG B message requires protein synthesis and is dependent upon the cell-cell contacts made during costimulation. Essentially all of the HMG B protein, which is newly synthesized during this period, is targeted to parental macronuclei where it serves an as yet undetermined function(s).

Animals↗
PubMed1991

Proteolytic removal of core histone amino termini and dephosphorylation of histone H1 correlate with the formation of condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

During the sexual cycle in Tetrahymena, the germ-line micronucleus gives rise to new macro- and micronuclei, whereas the former somatic macronucleus ceases transcription, becomes highly condensed, and is eventually eliminated from the cell. With polyclonal antibodies specific for acetylated forms of histone H4, immunofluorescent analyses have demonstrated that transcriptionally active macronuclei stain positively at all stages of the life cycle except during conjugation, when parental macronuclei become inactive and are eliminated from the cell. In this report using affinity-purified antibodies to either the acetylated or unacetylated amino-terminal domain of H4, immunofluorescent analyses suggest that the acetylated amino-terminal tails of H4 are proteolytically removed in "old" macronuclei during this period. This suggestion was further confirmed by biochemical analysis of purified old macronuclei that revealed several polypeptides with molecular mass 1-2 kD less than that of intact core histones. These species, which are unique to old macronuclei, are not newly synthesized and fail to stain with either acetylated or unacetylated H4 antibodies. Microsequence analysis clearly shows that these polypeptides are proteolytically processed forms of core histones whose amino-terminal "tails" (varying from 13 to 21 residues) have been removed. During the same developmental period, histone H1 is dephosphorylated rapidly and completely in old macronuclei. These results strongly suggest that the developmentally regulated proteolysis of core histones and dephosphorylation of histone H1 participate in a novel pathway leading to the formation of highly condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

Acetylation↗
PubMed1991

Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence.

HMG (high-mobility-group protein) B and HMG C are abundant nonhistone chromosomal proteins isolated from Tetrahymena thermophila macronuclei with solubilities, molecular weights, and amino acid compositions like those of vertebrate HMG proteins. Genomic clones encoding each of these proteins have been sequenced. Both are single-copy genes that encode single polyadenylated messages whose amounts are 10 to 15 times greater in growing cells than in starved, nongrowing cells. The derived amino acid sequences of HMG B and HMG C contain a highly conserved sequence, the HMG 1 box, found in vertebrate HMGs 1 and 2, and we speculate that this sequence may represent a novel, previously unrecognized DNA-binding motif in this class of chromosomal proteins. Like HMGs 1 and 2, HMGs B and C contain a high percentage of aromatic amino acids. However, the Tetrahymena HMGs are small, are associated with nucleosome core particles, and can be specifically extracted from macronuclei by elutive intercalation, properties associated with vertebrate HMGs 14 and 17, not HMGs 1 and 2. Thus, it appears that these Tetrahymena proteins have features in common with both of the major subgroups of higher eucaryotic HMG proteins. Surprisingly, a linker histone found exclusively in transcriptionally inactive micronuclei also has several HMG-like characteristics, including the ability to be specifically extracted from nuclei by elutive intercalation and the presence of the HMG 1 box. This finding suggests that at least in T. thermophila, proteins with HMG-like properties are not restricted to regions of transcriptionally active chromatin.

Amino Acid Sequence↗
PubMed1991

Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes.

The in vivo association of histone H1 with specific genes in Tetrahymena thermophila was studied by using a simplified cross-linking and immunoprecipitation technique. Four genes were analyzed whose activities vary in three different developmental states (logarithmic growth, starvation, and conjugation). Hybridization of the immunoprecipitated DNA to cloned probes showed an inverse correlation between the level of immunoprecipitation with H1 antiserum and transcriptional activity. This represents the first demonstration of an alteration in histone H1-DNA interaction associated with developmental changes in transcriptional activity.

Actins↗