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B M Turner

Publications and source records attributed to B M Turner.

At least 19 recordsLinked to original sources
PubMed1997

Different strategies of X-inactivation in germinal and somatic cells: histone H4 underacetylation does not mark the inactive X chromosome in the mouse male germline.

It has previously been shown by immunocytochemistry that the inactive X chromosome (Xi) in somatic cells of human and mouse females is marked by underacetylation of histone H4. It has been suggested that this may be important for transcriptional silencing of genes on Xi. We have now investigated X-inactivation in meiotic cells of the male germline. In these cells the single X chromosome is transcriptionally inactive and expresses XIST, a gene that in somatic cells is transcribed only from Xi. By immunostaining with antibodies to H4 acetylated at lysines 5, 8, 12, or 16, we demonstrate that histone H4 on the male X is not underacetylated. We conclude that there is a differential germline strategy for maintenance of X-inactivation and that H4 underacetylation, though associated with the long-term marking of inactive X chromosomes in the female soma, is not always essential for the transcriptional down-regulation of X-linked genes.

Acetylation↗
PubMed1996

X-Inactivation and histone H4 acetylation in embryonic stem cells.

In female mammalian cells, dosage compensation for X-linked genes is achieved by the transcriptional silencing, early in development, of many genes on just one of the two X chromosomes. Several properties distinguish the inactive X (Xi) from its active counterpart (Xa). These include expression of Xist, a gene located in the X-inactivation center (Xic), late replication, differential methylation of selected CpG islands and underacetylation of histone H4. The relationship between these properties and transcriptional silencing remains unclear. Female mouse embryonic stem (ES) cells have two active X chromosomes, one of which is inactivated as cells differentiate in culture. We describe here the use of these cells in studying the sequence of events leading to X-inactivation. By immunofluorescent labeling of metaphase chromosome spreads from ES cells with antibodies to acetylated H4, we show that an underacetylated X chromosome appears only after 4 days of differentiation, and only in female cells. The frequency of cells with an underacetylated X reaches a maximum by Day 6. In undifferentiated cells, H4 in centric heterochromatin is acetylated to the same extent as that in euchromatin but has become relatively underacetylated, as in adult cells, by Day 4 of differentiation (i.e. , when deacetylation of Xi is first seen). The overall deacetylation of Xi follows Xist expression and the first appearance of a single, late-replicating X, both of which occur on Day 2. It also follows the silencing of X-linked genes. Levels of mRNA from four such genes, Hprt, G6pd, Rps4, and Pgk-1, had all fallen by approximately 50% (relative to the autosomal gene Aprt) by Days 2-4. The results show that properties that characterize Xi are put in place in a set order over several days. H4 deacetylation occupies a defined place within this sequence, suggesting that it is an intrinsic part of the X-inactivation process. The stage at which a completely deacetylated Xi is first seen suggests that deacetylation may be necessary for the maintenance of silencing but is not required for its initiation. Nor is it required for, or an immediate consequence of, late replication. However, we note that selective deacetylation of H4 on specific genes would not be detected by the microscopical approach we have used and that such selective deacetylation may still be part of the silencing process.

Acetylation↗
PubMed1996

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription.

Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an approximately 350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an approximately 600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Amino Acid Sequence↗
PubMed1996

Histone H4 acetylation and replication timing in Chinese hamster chromosomes.

The distribution of acetylated isoforms of histone H4 along Chinese hamster chromosomes has been studied by immunostaining with antibodies recognizing H4 acetylated at defined lysines in its N-terminal domain. The heterochromatic long arm of the X chromosome in both female (CHO) and male (DON) cell lines is underacetylated at three out of four lysines (5, 8, and 12). In contrast, the level of acetylation at lysine 16, which is the first to be acetylated in mammals, was similar in X chromosomes and autosomes. Labeling of the cells with bromodeoxyuridine (BrdU) to mark late-replicating chromosome domains, followed by double immunostaining with antibodies to BrdU and acetylated H4, showed a close, though not perfect, correlation between late replication and low levels of H4 acetylation. The results show that levels of histone acetylation are associated with the replication timing of defined domains on both the X chromosome and autosomes, but the exceptions we observe suggest that this link is not absolute or essential.

Acetylation↗
PubMed1996

Evolution of dosage compensation.

In polytene chromosome squashes from the fruit fly Drosophila melanogaster, the single, dosage-compensated X chromosome in males can be distinguished from the autosomes by the presence of an isoform of histone H4 acetylated at lysine 16, H4.Ac16. We have used H4.Ac16 as a marker to examine the evolving relationship between dosage compensation and sex chromosome composition in species of Drosophila with one (D. melanogaster), two (D. pseudoobscura) or three (D. miranda) identifiable X chromosome arms. In each case, we find that H4.Ac16 is distributed as discrete, closely spaced bands along the entire length of each X chromosome, the only exception being the X2 chromosome of D. miranda in which a terminal region constituting about 10% of the chromosome by length is not labelled with anti-H4.Ac16 antibodies. We conclude that, with this exception, dosage compensation extends along the X chromosomes of all three species. As D. pseudoobscura and D. miranda diverged only about 2 Mya, the spread of dosage-compensated loci along X2 has been rapid, suggesting that regional changes rather than piecemeal, gene-by-gene, changes may have been involved.

Acetylation↗
PubMed1996

Differential immunostaining of plant chromosomes by antibodies recognizing acetylated histone H4 variants.

Metaphase chromosomes of Vicia faba were exposed to antibodies recognizing defined acetylated isoforms of histone H4. After indirect immunostaining with antibodies directed against H4 acetylated on lysines 5, 8 and 12 respectively, the entire chromosome complement was labelled. The brightest signal appeared at the nucleolus organizing region (NOR). The large genetically inert heterochromatic regions, which are composed of late replicating tandemly repetitive DNA sequences, remained unlabelled. Thus, the chromosomal distribution of histones H4 acetylated at positions of lysine 5, 8 and 12 is broadly correlated with the intensity of transcription and the sequence of replication of the field bean chromatin during interphase. Antibodies against H4 acetylated at lysine 16 also caused a strong signal at the NOR but otherwise a uniform fluorescence along the chromosome.

Acetylation↗
PubMed1996

Differential underacetylation of histones H2A, H3 and H4 on the inactive X chromosome in human female cells.

It has previously been shown that the acetylated forms of histone H4 are depleted or absent in both constitutive, centric heterochromatin and in the facultative heterochromatin of the inactive X chromosome (Xi) in female cells. By immunostaining of metaphase chromosomes from human lymphocytes with antibodies to the acetylated isoforms of histones H2A and H3, we now show that these histones too are underacetylated in both Xi and centric heterochromatin. Xi shows two prominent regions of residual H3 acetylation, one encompassing the pseudoautosomal region at the end of the short arm and one at about Xq22. Both these regions have been shown previously to be sites of residual H4 acetylation. H2A acetylation on Xi is higher overall than that of H3 or H4 and is particularly high around the pseudoautosomal region, but not at Xq22. The results suggest that the acetylated isoforms of H3 and H4 have at least some effects on chromosomal structure and function that are not shared by acetylated H2A.

Acetylation↗
PubMed1996

Tomographic distribution of acetylated histone H4 in plant chromosomes, nuclei and nucleoli.

Root tip cells of broad bean (Vicia faba L. cv. 'Wase soramame') and barley (Hordeum vulgare L. cv. 'Minorimugi') were immunostained with antibodies specific for acetylated histone H4. With an antiserum that recognizes histone H4 acetylated at lysine-5, the nucleolar organizing region (NOR) in mitotic chromosomes was strongly labeled in both species. The broad bean had two signals in the metaphase and telophase chromosome complements and four signals in the prophase and anaphase chromosome complements, while the barley had four signals in the metaphase and telophase chromosome complements and eight signals in the prophase and anaphase complements. Five different patterns of signals were observed at interphase: in type I only nucleoli were wholly stained; in type II perinucleolar knob-like signals and/or fiber-like signals emanated from the nucleus; in type III aggregate signals appeared in the nucleolus; in type IV many small dot-like signals were distributed throughout the nucleus, except nucleoli; and in type V string-like or some granule-like signals appeared in the nucleoli. Type II was very similar to previous results by in situ hybridization with sense rDNA probes. Type III was similar to the patterns of DNA synthesis recognized as chromatin domains by anti-BrdU antibodies. Type V was very similar to the results of in situ hybridization with pTa71, rDNA probes and the appearance of the dense fibrillar components of the nucleolus.

Acetylation↗
PubMed1996

Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern.

Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.

Acetylation↗
PubMed1996

The pattern of histone H4 acetylation on the X chromosome during spermatogenesis of the desert locust Schistocerca gregaria.

We have used antibodies directed against histone H4 acetylated at lysine residue 5, 8, 12, or 16 and indirect immunofluorescence microscopy to probe chromosomes from spermatogonia and spermatocytes of the desert locust, Schistocerca gregaria. The autosomes showed bright overall fluorescence, indicative of high levels of H4 acetylation. In contrast, the X chromosome, which is facultatively heterochromatic during spermatogenesis of the locust, remained completely unstained in spermatogonia and secondary spermatocytes and showed only a small terminal fluorescent band in primary spermatocytes. This band probably corresponds to centromere associated constitutive heterochromatin. Thus, underacetylation is a cytogenetic marker for facultative heterochromatin, but not necessarily constitutive heterochromatin, during spermatogenesis of the locust. Scanning electron microscopy of chromosomes from prophase spermatogonia and prophase I spermatocytes revealed that underacetylation of histone H4 in the X chromosome was not accompanied by a chromatin organization visibly different from that of the autosomes. Transmission electron microscopy of mitotic spermatogonia showed that the X chromosome is separated from the autosomes in a small nuclear compartment of its own in prophase and telophase and associated with membranes in metaphase. In prophase I spermatocytes, autosomes and the sex univalent were in the same compartment. This compartmentalization may be responsible for the underacetylation and (or) transcriptional silencing of the X chromosome in spermatogonia mitosis.

Acetylation↗
PubMed1996

Preparation of insect chromosomes for immunolabeling.

We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of histone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins.

Animals↗
PubMed1995

Histone H4 acetylation distinguishes coding regions of the human genome from heterochromatin in a differentiation-dependent but transcription-independent manner.

By immunoprecipitation of chromatin fragments from cultured human HL-60 cells with antibodies specific for H4 acetylated at specific lysine residues we have defined the level of H4 acetylation within transcriptionally active and inactive regions of the genome. H4 within or adjacent to coding regions had a similar level of overall acetylation to input (bulk) chromatin and a similar pattern of acetylation of individual lysines (i.e. 16 > 8, 12 > 5). The acetylation of H4 in coding (and adjacent) regions was not correlated with transcriptional activity and did not vary with position along the constitutively active c-myc gene. Turnover of H4 acetates was not selectively increased in transcriptionally active chromatin. H4 associated with centric heterochromatin or with the CCCTAA repeat of telomeric heterochromatin was infrequently acetylated (< 1%) at all lysines. We conclude that nucleosomes containing acetylated H4 are scattered infrequently and possibly randomly through coding and adjacent regions and are essentially absent from heterochromatin. Induction of differentiation of HL-60 cells by exposure to dimethylsulfoxide or 12-o-tetradecanoylphorbol 13-acetate (TPA) did not alter the level of H4 acetylation within either the c-myc or c-fos genes or other coding regions, but did induce a transient increase in H4 acetylation within centric heterochromatin.

Acetylation↗
PubMed1995

Histone H4, the cell cycle and a question of integrity.

The N-terminal domain of histone H4 has been implicated in various nuclear functions, including gene silencing and activation and replication-linked chromatin assembly. Many of these have been identified by using h4 mutants in the yeast S. cerevisiae. In a recent paper, Megee et al. use this approach to show that mutants in which all four N-terminal H4 lysines are substituted with glutamines accumulate increased levels of DNA damage. A single lysine, but not an arginine, anywhere in the N-terminal domain suppresses this phenotype. It is suggested that histone H4 plays a role in maintaining genome integrity through the cell cycle, possibly by a mechanism involving lysine acetylation.

Amino Acid Sequence↗
PubMed1995

Histone acetylation in chromatin and chromosomes.

The packaging of DNA into chromatin is an important regulator of transcription. This regulation may operate either by short-term switching of the transcription of specific genes or by packaging chromosome domains into structures that either facilitate or repress the potential for gene expression. Such packaging may occur during chromatin assembly through S-phase of the cell cycle. Recent evidence shows that the post-translational acetylation of histones of the nucleosome core particle is intimately involved in all these processes. New approaches allowing exploration of the molecular details, the functional effects and the regulation of histone acetylation promise to reveal new mechanisms of genomic regulation.

Acetylation↗
PubMed1995

Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo.

Using immunofluorescent labeling and laser-scanning confocal microscopy, we show that isoforms of histone H4 acetylated on lysine 5, 8 and/or 12 (H4.Ac5-12), as well as RNA polymerase II, become enriched at the nuclear periphery around the time of zygotic gene activation, i.e., the 2-cell stage, in the preimplantation mouse embryo. In contrast, DNA and H4 acetylated on lysine 16 are uniformly distributed throughout the cytoplasm. Culture of embryos with inhibitors of histone deacetylase trichostatin A and trapoxin results in an increase in the (1) amount of acetylated histone H4 detected by immunoblotting, (2) intensity and sharpness of the peripheral staining for H4.Ac5-12, and (3) relative rate of synthesis of proteins that are markers for zygotic gene activation. The enhanced staining for H4.Ac5-12 at the nuclear periphery seems to require DNA replication, but appears independent of cytokinesis or transcription, since its development is inhibited by aphidicolin but not by either cytochalasin D or alpha-amanitin. Lastly, the restricted localization of H4.Ac 5-12 is not observed in the 4-cell embryo or at later stages of preimplantation development. These results suggest that changes in chromatin structure underlie, at least in part, zygotic gene activation in the mouse.

Acetylation↗
PubMed1994

Suppression of heat-shock protein synthesis by short-chain fatty acids and alcohols.

We have shown that ethanol, propanol and butanol (at 0.5-2%) and salts of butyric and propionic acids (at 8-40 mM) all cause a major reduction in heat-shock protein (hsp) synthesis when present in the growth medium of Drosophila cultured cells (Kc and SL2) subjected to either increased temperature or chemical stressors. Inhibition of normal protein synthesis in unstressed cells was comparatively slight, and the usual suppression of synthesis of non-heat-shock proteins in stressed cells was unaffected. Maximum suppression of hsp synthesis occurred only if inhibitors were added before initiation of the stress response, an observation that eliminates the possibility that these findings are due to non-specific, toxic effects. Suppression was accompanied by severely reduced levels of both hsp70 mRNA and active heat-shock factor (HSF). We conclude that the inhibitors act by suppressing the initiation of transcription of heat-shock genes.

Alcohols↗
PubMed1994

Histone H4 acetylated at lysine 16 and proteins of the Drosophila dosage compensation pathway co-localize on the male X chromosome through mitosis.

In the fruit fly Drosophila, dosage compensation involves several proteins acting in concert to double the transcriptional activity of genes on the single male X chromosome. Three of these proteins, MLE, MSL-1 and histone H4 acetylated at lysine 16 (H4Ac16), have recently been shown to be located almost exclusively on the male X chromosome in interphase (polytene) cells. We show here that in neuroblasts from third instar Drosophila larvae antisera to H4Ac16, MLE and MSL-1 uniquely label the distal, euchromatic region of the male X chromosome through mitosis. The centromere-proximal, heterochromatic region of the male X is not labelled with these antisera, nor are male autosomes or any chromosomes in female cells. That the association of H4Ac16 with the male X chromosome persists, even when the chromosome is maximally compacted and transcriptionally quiescent, argues that this modified histone is an integral component of the dosage compensation pathway. In the nuclei of interphase neuroblasts from male (but never female) larvae, antibodies to H4Ac16 revealed a small, brightly labelled patch against a background of generally weak nuclear staining. In double-labelling experiments, this patch was also labelled, albeit comparatively weakly, with antibodies to MSL-1. These results strongly suggest that the distal, euchromatic region of the X chromosome in male cells occupies a limited and relatively compact nuclear domain.

Acetylation↗