Histone Synthesis Research Articles

Evidence for an S-Phase checkpoint regulating DNA replication after heat shock: a review

Exposure of cells to heat inhibits a number of nuclear activities associated with semi-conservative replication of DNA including the incorporation of radiolabelled precursors into acid-insoluble DNA, the initiation of new replicons, the elongation of the DNA fibre at the replication fork, the synthesis and deposition of new histones into chromatin and the reorganization of nascent DNA into mature chromatin. These effects are likely to underlie the heat sensitivity of S-phase cells and may contribute to the radiosensitization observed in this phase of the cell cycle. While some of these effects may be explained as ‘passive’ consequences of heat-induced damage on chromatin structures, experiments reviewed here point to the activation of a checkpoint as a contributing factor to the observed inhibition of DNA replication. Activation of a heat responsive S-phase checkpoint targets the activity of RPA via interaction with nucleolin. Nucleolin, a major nucleolar protein, is found normally sequestered in the nucleolus. Exposure of cells to heat causes a rapid translocation of nucleolin from the nucleolus into the nucleoplasm that enables RPA/nucleolin interaction. This interaction inhibits functions of RPA associated with the initiation of DNA replication and contributes to the immediate inhibition of DNA synthesis observed after heat shock. The results suggest that the nucleolus serves as a sequestration centre for the temporary inactivation of regulatory molecules, such as nucleolin, capable of regulating essential cellular functions after heat shock. It is speculated that this regulatory process is integrated in the network of responses that determine cell sensitivity to heat and that it may be involved in heat radiosensitization to killing as well.

A Degrading Job

Checkpoint responses lead to cell cycle arrest following DNA damage, which allows time for DNA repair. In Saccharomyces cerevisiae , Rad53 kinase is critical to this arrest and also for deoxyribonucleotide triphosphate production during S phase. Most of Rad53's functions depend on its activation by the upstream checkpoint kinase Mec1; however, rad53 deletion mutants show a slow-growth phenotype, whereas mec1 deletion mutants do not. During S phase, histone synthesis is coordinated with DNA replication, and newly synthesized histones are deposited behind the replication fork by chaperones (see Quivy and Almouzni). Gunjan and Verreault investigated the role of Rad3, which can interact with the histone chaperone Asf1, in histone metabolism in wild-type and mutant strains of S. cerevisiae . Yeast lacking--or expressing kinase-deficient--Rad3 were hypersensitive to histone overexpression (which is toxic), whereas yeast lacking Mec1 were not. Western analysis of tagged overexpressed histones or of fractionated or chaperone-bound endogenous histones indicated that degradation of excess histones that were not packaged into chromatin was impaired in rad53 mutants. Therefore, rad53 mutant cells accumulated excess histones. Histone overexpression increased sensitivity to DNA damage, without directly damaging DNA, and reducing histone gene dosage suppressed the rad53 mutant phenotype of sensitivity to DNA damage, slow growth, and chromosome loss. Rad53 formed a complex with histones that did not include Asf1 or any other known histone chaperone proteins; complex formation was affected by Rad53 kinase activity. Thus, Rad53 appears to have a novel function of monitoring and maintaining histone levels that is independent of Mec1 and its activation by DNA damage. A. Gunjan, A. Verreault, A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae . Cell 115 , 537-549 (2003). [Online Journal] J.-P. Quivy, G. Almouzni, Rad53: A controller ensuring the fine-tuning of histone levels. Cell 115 , 508-510 (2003). [Online Journal]

Lymphocytes from bipolar and schizophrenic patients share common biochemical markers related to histone synthesis and histone cell membrane localization characteristic of an activated state

In a previous communication, based on the total histone and histone variants’ synthesis rates, biochemical parameters used for the characterization of the activation state of lymphocytes, we showed that a portion of the lymphocyte population obtained from peripheral blood of patients with bipolar disorder in the manic and/or depressed phases of the illness were in an activated state as opposed to normothymic patients and control subjects whose lymphocytes are in a resting, Go, state. In light of these previous findings, in the present investigation, we have analyzed total histone synthesis rates and the H2A and H3 histone variants’ synthesis pattern of acid-extracted histones from the lymphocytes’ nuclear fraction obtained from control subjects, patients with bipolar disorder in all phases of the illness, and patients with schizophrenia. Additional biochemical parameters, such as total cellular protein and DNA synthesis rates, were also studied. Moreover, recent findings from other investigators showed the association of histones on the plasma membrane fraction of PHA-activated, but not Go resting lymphocytes. Based on these results, acid-extracted proteins from the plasma membrane fraction obtained from control, bipolar patients in all phases of the illness, and schizophrenic patients were analyzed by immunoblotting using a polyclonal histone antibody, anti-H2B. All biochemical parameters tested show that a portion of the lymphocyte population from bipolar, i.e. manic and depressive, as well as schizophrenic patients are in an activated state and clearly indicate that the unusual for lymphocytes cell cycle-related histone biochemical properties are common to both disorders.

Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo.

The stem-loop binding protein (SLBP) binds to the 3' end of histone mRNA and participates in 3'-processing of the newly synthesized transcripts, which protects them from degradation, and probably also promotes their translation. In proliferating cells, translation of SLBP mRNA begins at G1/S and the protein is degraded following DNA replication. These post-transcriptional mechanisms closely couple SLBP expression to S-phase of the cell cycle, and play a key role in restricting synthesis of replication-dependent histones to S-phase. In contrast to somatic cells, replication-dependent histone mRNAs accumulate and are translated independently of DNA replication in oocytes and early embryos. We report here that SLBP expression and activity also differ in mouse oocytes and early embryos compared with somatic cells. SLBP is present in oocytes that are arrested at prophase of G2/M, where it is concentrated in the nucleus. Upon entry into M-phase of meiotic maturation, SLBP begins to accumulate rapidly, reaching a very high level in mature oocytes arrested at metaphase II. Following fertilization, SLBP remains abundant in the nucleus and the cytoplasm throughout the first cell cycle, including both G1 and G2 phases. It declines during the second and third cell cycles, reaching a relatively low level by the late 4-cell stage. SLBP can bind the histone mRNA-stem-loop at all stages of the cell cycle in oocytes and early embryos, and it is the only stem-loop binding activity detectable in these cells. We also report that SLBP becomes phosphorylated rapidly following entry into M-phase of meiotic maturation through a mechanism that is sensitive to roscovitine, an inhibitor of cyclin-dependent kinases. SLBP is rapidly dephosphorylated following fertilization or parthenogenetic activation, and becomes newly phosphorylated at M-phase of mitosis. Phosphorylation does not affect its stem-loop binding activity. These results establish that, in contrast to Xenopus, mouse oocytes and embryos contain a single SLBP. Expression of SLBP is uncoupled from S-phase in oocytes and early embryos, which indicates that the mechanisms that impose cell-cycle-regulated expression of SLBP in somatic cells do not operate in oocytes or during the first embryonic cell cycle. This distinctive pattern of SLBP expression may be required for accumulation of histone proteins required for sperm chromatin remodelling and assembly of newly synthesized embryonic DNA into chromatin.

Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity.

DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.

Pathways Mediating the Nuclear Import of Histones H3 and H4 in Yeast

The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells. A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p. Cytosolic H4 is found associated with Kap123p and H3. Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor. We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4. H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains. These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters. Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains. H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.

The effect of the histone deacetylase inhibitor, trichostatin A, on total histone synthesis, H1 0 synthesis and histone H4 acetylation in peripheral blood lymphocytes increases as a function of increasing age: a model study

A pilot study was initiated in order to ascertain whether the age of the donor might affect either the induction of the expression of H1 0 or histone H4 acetylation by the very specific histone deacetylase inhibitor, trichostatin A. This was investigated in a cell system which normally does not express this linker histone variant, i.e. peripheral blood lymphocytes (PBL), which were obtained from donors of different ages (25–95 years). Forty-eight hours after activation by the mitogen phytohemaglutinin (PHA), 250 ng of trichostatin A per 10 6 cells per ml culture medium was added and cultured for an additional 24 h. Assays were performed 72 h after initiation of cultures, i.e. during the S phase. It was found that in PBL, trichostatin A induced the expression of the linker histone variant, H1 0 as well as histone H4 acetylation, and, more importantly, that these effects were enhanced with increasing age of the donor. More specifically, under the influence of trichostatin A, PBL showed increasing H1 0 synthesis rates and increasing levels of histone H4 acetylation as a function of increasing age of the donor. Moreover, although trichostatin A induced an increasing expression of H1 0 with increasing age, it also concomitantly partially inhibited S phase total histone synthesis. This inhibition also increased as a function of increasing age of the donor.

Nuclear import of histone H2A and H2B is mediated by a network of karyopherins.

The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

Histone genes expression during the cell cycle in Trypanosoma cruzi.

Histones, the basic proteins which compact DNA into the nucleosomal and solenoidal fibers are synthesized in correlation with DNA replication during the S-phase of the cell cycle. This behavior is controlled both at transcriptional and postranscriptional levels in higher eukaryotes and yeasts. We have found that histone synthesis in synchronized trypanosomes is controlled by fluctuations on the levels of their mRNAs. Though we cannot preclude the existence of a transcriptional regulatory mechanism, our results point to the participation of changes in the stability of histone mRNAs as a regulatory mechanism of their levels during the cell cycle in Trypanosoma. We have also found a postranscriptional regulatory mechanism which could be acting at the translational level. These results show both similarities and differences between Trypanosoma and higher eukaryotes regarding the expression of their histone genes.

Chromatin assembly at kinetochores is uncoupled from DNA replication.

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.

Where the cell cycle and histones meet.

Since the discovery of mammalian cyclin E and the Dtype cyclins in 1991, much effort has been devoted towards elucidating their role in G1 progression and S phase entry. Key to this endeavor has been the identification and characterization of substrates for cyclin E–CDK2 and D-type cyclin–CDK4 and cyclin–CDK6 complexes. Whereas there appears to be only one critical substrate of CDK4 and CDK6, the list of CDK2 substrates continues to lengthen in line with the realization that the principal mediators of cyclin E–CDK2 action are yet to be identified. The papers by Ma et al. and Zhao et al. in this issue bring a new dimension to our understanding of cyclin E–CDK2, by showing that NPAT, a previously identified CDK2 substrate, links the cell cycle to histone gene transcription. This provides us with the beginnings of how the action of CDKs is linked to the periodic synthesis of histones in mammalian cells. These works illuminate our picture of how the cell cycle control elements may influence multiple biosynthetic processes as integral to S phase as duplication of the genetic material itself.

287. Histone H2B on lymphocyte membranes of patients with bipolar disorder

In a previous communication we reported that peripheral blood lymphocytes from patients with bipolar disorder show a histone synthetic profile indicative of an active cell state. Furthermore, histones have been detected on the plasma membranes of mitogenically activated peripheral blood lymphocytes. Based on the above, the aim of the present study is to determine whether histones are associated with the plasma membranes of lymphocytes of patients with bipolar disorder. Lymphocytes, isolated from the blood of 13 manic-depressive patients and 7 control subjects, were incubated with radioactive protein precursors (14C-lysine and 14C-arginine) for two hours at 37°C. Cells were gently homogenized using a Dounce homogenizer and nuclei and membranes were isolated by differential centrifugation. Histones were extracted from both nuclei and membranes with 0.1N HCI and the incorporated radioactivity per mg protein was measured. As expected, the lymphocytes from a significant number of patients (7 out of 13) showed an increased histone synthesis rate with respect to control subjects indicative of an activated state. The acid extracted membrane fraction was subjected to SDS PAGE electrophoresis. In continuation, proteins were transferred onto nitrocellulose membranes. Western analysis was accomplished for the detection of histone H2B using a polyclonal antibody raised against H2B. It was found that histone H2B can be detected in the acid extracted plasma membrane fraction obtained from the lymphocytes of patients with bipolar disorder (11 out of 13 cases). The localization of H2B on the plasma membranes of the lymphocytes of these patients is a finding which further characterizes the activated state of these cells which possibly, could be related to the elevated anti-histone antibodies found, by other investigators, in the serum of bipolar patients.

Histone synthesis in Leishmania infantum is tightly linked to DNA replication by a translational control

We have analysed the regulation of histone synthesis in Leishmania infantum following inhibition of DNA replication. Run-on experiments indicated that transcription rates of the genes coding for the four core histones (H2A, H2B, H3 and H4) were not affected by the inhibition with hydroxyurea of DNA synthesis. However, a dramatic decrease was observed in the newly synthesized histones after inhibition of DNA synthesis. Furthermore, the synthesis of both the histones and DNA resumed in promastigotes after removal of hydroxyurea, indicating that inhibition was reversible. Unlike most eukaryotes, in which the replication-dependent histone transcripts decrease upon a replication blockade, the levels of L. infantum histone mRNAs do not change under similar conditions. Thus the present data indicate that histone synthesis in Leishmania is tightly coupled to DNA replication by a mechanism operating at the translational level.

Histone synthesis in Leishmania infantum is tightly linked to DNA replication by a translational control

We have analysed the regulation of histone synthesis in Leishmania infantum following inhibition of DNA replication. Run-on experiments indicated that transcription rates of the genes coding for the four core histones (H2A, H2B, H3 and H4) were not affected by the inhibition with hydroxyurea of DNA synthesis. However, a dramatic decrease was observed in the newly synthesized histones after inhibition of DNA synthesis. Furthermore, the synthesis of both the histones and DNA resumed in promastigotes after removal of hydroxyurea, indicating that inhibition was reversible. Unlike most eukaryotes, in which the replication-dependent histone transcripts decrease upon a replication blockade, the levels of L. infantum histone mRNAs do not change under similar conditions. Thus the present data indicate that histone synthesis in Leishmania is tightly coupled to DNA replication by a mechanism operating at the translational level.

Two Xenopus proteins that bind the 3' end of histone mRNA: implications for translational control of histone synthesis during oogenesis.

Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3' end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3' end of histone mRNA.

Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression.

Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription.

Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I.

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacDelta mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacDelta mutants, although hirDelta mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacDelta mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.

TATA element-binding protein is important to epidermal growth factor-dependent induction of H2B histone gene expression in primary hepatocytes from rat.

Epidermal growth factor (EGF) is a potent mitogen for rat hepatocytes and mammalian histone synthesis is functionally and temporally coupled to DNA replication. To gain an insight on the role of EGF in the regulation of H2B histone gene expression in primary hepatocyte cultures, the binding patterns of nuclear proteins to various elements in the H2B histone gene upstream region have been investigated. EGF induced H2B histone mRNA with maximal stimulation reached at 36 hours. The induction of H2B histone mRNA was dependent on the concentration of EGF and almost reduced by actinomycin-D pretreatment. In DNase I footprinting analysis, one nuclear factor (TATA element-binding protein, TBP) bound at -20 bp (TATA element) in either the absence or presence of EGF. One DNA-protein complex was formed by DNA mobility shift assay when TATA element was incubated with nuclear extract prepared from EGF-free hepatocytes, and the amount of TBP was increased after EGF treatment. These results suggest that TBP may be correlated with transcriptional regulation of H2B histone gene by EGF in primary hepatocytes.

Preliminary analysis of histone and DNA synthesis of mitogenically activated peripheral blood lymphocytes of patients with bipolar disorder : T.G. Sourlingas a, A. Karpouza a, M.R. Issidorides b,c, G. Trikkas c, D. Tsapali a, K.E. Sekeri-Pataryas a. aNational Center for Scientific Research “DEMOKRITOS” Institute of Biology, Athens Greece. bNeurobiology Research Institute, Theodor Theohari Cozzika Foundation, Athens, GreececDepartment of Psychiatry, University

Preliminary analysis of histone and DNA synthesis of mitogenically activated peripheral blood lymphocytes of patients with bipolar disorder : T.G. Sourlingas a, A. Karpouza a, M.R. Issidorides b,c, G. Trikkas c, D. Tsapali a, K.E. Sekeri-Pataryas a. aNational Center for Scientific Research “DEMOKRITOS” Institute of Biology, Athens Greece. bNeurobiology Research Institute, Theodor Theohari Cozzika Foundation, Athens, GreececDepartment of Psychiatry, University