Histone Synthesis Research Articles

Zinc: An Essential Trace Element for Parenteral Nutrition

Zinc is an essential trace element for human nutrition that is an integral part of many enzyme systems, including DNA polymerase complex. Zinc deficiency has been associated with stunting of growth and sexual immaturity. In children, deficiency causes a fatal condition called acrodermatitis enteropathica. The same syndrome has been observed in patients on total parenteral nutrition (TPN) who do not receive zinc. In TPN the requirements have been estimated by balance studies to be 3 mg/d in patients without gastrointestinal losses and a mean of 12 mg/d in patients with diarrhea and fistula losses.

Establishment of Histone Modifications after Chromatin Assembly

Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin ‘matures’ and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine (15N4) that is 4 Da heavier than the naturally occurring 14N4 isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.

Kinetics of histone and protamine synthesis during meiosis and spermiogenesis in the mouse.

The separation of mouse spermatogenic cell nuclei by sedimentation velocity at unit gravity has been used to determine the timing of histone and "mouse protamine" synthesis, and the turnover of basic nuclear proteins throughout spermatogenesis. Animals were injected with 3H-arginine or 3H-lysine and at various time intervals (2 hours post-label or from 1 to 30 days post-label) germinal cell nuclei preparations were separated on the staput. Labelled histones and mouse protamine were extracted from staput separated nuclei with hydrocholoric acid and fractionated by polyacrylamide gel electrophoresis. Results indicate that histones are synthesized in association with DNA replication in spermatogonia and preleptotene spermatocytes, in pachytene primary spermatocytes and in spermatids stages 11-16, simultaneously with "mouse protamine". Experiments are reported showing that histones synthesized in pachytene primary spermatocytes and in spermatids stages 11-16 are retained in epididymal spermatozoa, while histones synthesized before meiosis are no longer detectable onto chromatin after meiosis.

DNA-activated protein kinase functions in a newly observed S phase checkpoint that links histone mRNA abundance with DNA replication

DNA and histone synthesis are coupled and ongoing replication is required to maintain histone gene expression. Here, we expose S phase–arrested cells to the kinase inhibitors caffeine and LY294002. This uncouples DNA replication from histone messenger RNA (mRNA) abundance, altering the efficiency of replication stress–induced histone mRNA down-regulation. Interference with caffeine-sensitive checkpoint kinases ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) does not affect histone mRNA down- regulation, which indicates that ATR/ATM alone cannot account for such coupling. LY294002 potentiates caffeine's ability to uncouple histone mRNA stabilization from replication only in cells containing functional DNA-activated protein kinase (DNA-PK), which indicates that DNA-PK is the target of LY294002. DNA-PK is activated during replication stress and DNA-PK signaling is enhanced when ATR/ATM signaling is abrogated. Histone mRNA decay does not require Chk1/Chk2. Replication stress induces phosphorylation of UPF1 but not hairpin-binding protein/stem-loop binding protein at S/TQ sites, which are preferred substrate recognition motifs of phosphatidylinositol 3-kinase–like kinases, which indicates that histone mRNA stability may be directly controlled by ATR/ATM- and DNA-PK–mediated phosphorylation of UPF1.

The structure of chromatin at meiotic prophase I

The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I.

Stem–loop binding protein expressed in growing oocytes is required for accumulation of mRNAs encoding histones H3 and H4 and for early embryonic development in the mouse

Growing oocytes accumulate mRNAs and proteins that support early embryogenesis. Among the most abundant of these maternal factors are the histones. Histone mRNA accumulation and translation are mainly restricted to S-phase in somatic cells, and the mechanism by which oocytes produce histones is unknown. In somatic cells, replication-dependent histone synthesis requires the stem–loop binding protein (SLBP). SLBP is expressed during S-phase, binds to the 3′-untranslated region of non-polyadenylated transcripts encoding the histones, and is required for their stabilization and translation. SLBP is expressed in oocytes of several species, suggesting a role in histone synthesis. To test this, we generated transgenic mice whose oocytes lack SLBP. mRNAs encoding histones H3 and H4 failed to accumulate in these oocytes. Unexpectedly, mRNAs encoding H2A and H2B were little affected. Embryos derived from SLBP-depleted oocytes reached the 2-cell stage, but most then became arrested. Histones H3 and H4, but not H2A or H2B, were substantially reduced in these embryos. The embryos also expressed high levels of γH2A.X. Injection of histones into SLBP-depleted embryos rescued them from developmental arrest. Thus, SLBP is an essential component of the mechanism by which growing oocytes of the mouse accumulate the histones that support early embryonic development.

Continuous Histone H2B and Transcription-Dependent Histone H3 Exchange in Yeast Cells outside of Replication

We investigated the dynamics of histone-DNA interactions in yeast by using inducible forms of epitope-tagged histones H2B and H3. Chromatin assembly of newly synthesized histones was assessed by chromatin immunoprecipitation in G1-arrested cells to prevent replication-coupled histone incorporation. We find that while histone deposition within a subtelomeric region is strictly linked to DNA replication, histone H2B is continuously incorporated at the promoter and coding regions of both transcriptionally active and inactive loci. In contrast, incorporation of histone H3 occurs only at active genes, being predominant at the promoter and showing a dynamics along the gene that inversely correlates with the average nucleosomal density. Similar results were obtained with N-terminally truncated H2B and H3 variants. We infer that replication-independent incorporation of H2B and H3 are distinct events, each occurring independently of the histone tail, and that nucleosome loss at active promoters reflects a dynamic equilibrium between histone deposition and dissociation.

Review of "The Cell Cycle: Principles of Control" by David O. Morgan

Review of "The Cell Cycle: Principles of Control" by David O. Morgan

Anti-HER2 Antibody Trastuzumab Inhibits CDK2-Mediated NPAT and Histone H4 Expression via PI3K Pathway

Anti-HER2 antibody trastuzumab (Herceptin®) has been used effectively to treat patients with breast cancer that overexpresses HER2. We have demonstrated that p27Kip1 upregulation is one of the key events that cause G1 arrest upon trastuzumab treatment. Here, we have examined the effect of trastuzumab on CDK2, Rb, E2F1, NPAT, histone H4, and their associated signaling pathway. Results showed that trastuzumab was able to dramatically inhibit the kinase activity and expression of CDK2, whereas the kinase activity and expression of CDK4 were not affected by trastuzumab treatment. Unlike the p27Kip1 upregulation that occurs primarily through post-translational mechanisms, CDK2 was mainly downregulated at a transcriptional level as shown by Northern blotting and real-time RT-PCR analyses. Trastuzumab decreased the kinase activity of cyclin E (because of CDK2 inhibition) although it had little effect on cyclin E protein level. Overexpression of wild-type cyclin E or its lower molecular weight forms did not influence the response to trastuzumab. Levels and activities of CDK6, cyclin A, and cyclin D1 were all suppressed by trastuzumab. As a result, Rb phosphorylations that associates with CDK2, cyclin E, CDK6, cyclin A, or cyclin D1 were substantially inhibited by trastuzumab. Trastuzumab decreased the DNA-binding activity of E2F1. Trastuzumab decreased the level of NPAT protein. Trastuzumab decreased the level of histone H4 mRNA. Blockade of PI3K pathway with LY294002 produced similar effects to trastuzumab treatment. Taken together, these results demonstrated that anti-HER2antibody trastuzumab inhibits CDK2, Rb phosphorylation, E2F1 activity, NPAT, and histone H4 via PI3K signaling pathway, which are essential to both DNA and histone synthesis during the progression of G1 phase to S phase of the cell cycle.

Photoradiation could influence the cytoskeleton organization and inhibit the survival of human hepatoma cells in vitro

Low-power laser therapy has become popular in clinical applications including promoting wound healing and pain relief. However, effects of this photoradiation on human hepatoma cells are rarely studied. Previously, we found 808 nm gallium aluminum arsenide (GaAlAs) continuous wave laser had an inhibitory effect on the proliferation of human hepatoma cell lines HepG2 and J-5 at the energy density of 5.85 and 11.7 J/cm(2), respectively. The aim of the present study was to evaluate the possible mechanism of action of this photoradiation on HepG2 and J-5 cells. HepG2 and J-5 cells were cultured in 24-well plates for 24 h. After photoradiation by 130 mW 808 nm GaAlAs continuous wave laser for different time intervals (0, 30, 60, 90, 120, 150, and 180 s), Western blot and immunofluorescent staining were used to examine the expression and distribution of histone and cytoskeletal proteins. The cell counts as well as histone and synemin expression of HepG2 and J-5 cells were reduced by photoradiation at the energy density of 5.85 and 11.7 J/cm(2), respectively. Furthermore, the architecture of cytoskeletons and the distribution of intermediate filament-associated proteins (plectin and synemin) were disorganized by photoradiation. Photoradiation by 808 nm GaAlAs continuous wave laser at the energy density of 5.85 and 7.8 J/cm(2) inhibited the survival of human hepatoma cell lines. The mechanism might reduce synthesis of histone and synemin. Reduced histone synthesis might further reduce the proliferation rate of these cells. Reduced synemin synthesis might result in the destruction of the cytoskeleton. Therefore, the net effects by this photoradiation were reduced cell survival.

The stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis

Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3′-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3′-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP.

Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upf1

Eukaryotic cells coordinately regulate histone and DNA synthesis. In mammalian cells, most of the regulation of histone synthesis occurs post-transcriptionally by regulating the concentrations of histone mRNA. As cells enter S phase, histone mRNA levels increase, and at the end of S phase they are rapidly degraded. Moreover, inhibition of DNA synthesis causes rapid degradation of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is the only cis-acting element required for coupling regulation of histone mRNA half-life with DNA synthesis. Here we show that regulated degradation of histone mRNAs requires Upf1, a key regulator of the nonsense-mediated decay pathway, and ATR, a key regulator of the DNA damage checkpoint pathway activated during replication stress.

Obligate Heterodimerization of the Archaeal Alba2 Protein with Alba1 Provides a Mechanism for Control of DNA Packaging

Organisms growing at elevated temperatures face a particular challenge to maintain the integrity of their genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of the Alba (Sac10b) gene. Alba is an abundant, dimeric, highly basic protein that binds cooperatively and at high density to DNA. Sulfolobus solfataricus encodes a second copy of the Alba gene, and the Alba2 protein is expressed at approximately 5% of the level of Alba1. We demonstrate by NMR, ITC, and crystallography that Alba2 exists exclusively as a heterodimer with Alba1 at physiological concentrations and that heterodimerization exerts a clear effect upon the DNA packaging, as observed by EM, potentially by changing the interface between adjacent Alba dimers in DNA complexes. A functional role for Alba2 in modulation of higher order chromatin structure and DNA condensation is suggested.

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks. In contrast, DNA polymerase alpha (Pol alpha) accumulates to higher than normal levels at stalled forks in asf1Delta cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.

Regulation of histone synthesis and nucleosome assembly

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.

The human histone gene expression regulator HBP/SLBP is required for histone and DNA synthesis, cell cycle progression and cell proliferation in mitotic cells

Histone proteins are essential for chromatin formation, and histone gene expression is coupled to DNA synthesis. In metazoans, the histone RNA binding protein HBP/SLBP is involved in post-transcriptional control of histone gene expression. In vitro assays have demonstrated that human HBP/SLBP is involved in histone mRNA 3' end formation and translation. We have inhibited human HBP/SLBP expression by RNA interference to determine its function during the mitotic cell cycle. Inhibition of HBP/SLBP expression resulted in the inhibition of histone gene expression and DNA synthesis, the inhibition of cell cycle progression in S phase and the inhibition of cell proliferation. These findings indicate that human HBP/SLBP is essential for the coordinate synthesis of DNA and histone proteins and is required for progression through the cell division cycle.

Transcriptional frequency and cell determination

The relative base composition of DNA regulatory sequences of certain genes of undetermined multipotent progenitor cells may account for the frequency of transcription of these genes in cell determination. The sequences of these regulatory regions of cell determination genes that are more AT-rich would create the potential for transcription at a higher frequency due to their lower melting temperature, as well as propensity to bend. An increase of one or more of the high mobility group (HMG) chromatin proteins would preferentially bind the more AT-rich regulatory sequences, thereby increasing the rate of transcription. The amount of unphosphorylated H1 histone reacting with these same regulatory sites may decrease transcription frequency. The level of cell growth, i.e. total protein synthesis of a cell, is correlated positively with the synthesis of HMG proteins. H1 histone synthesis is linked to DNA replication. Unbalanced growth would alter the amounts of HMG proteins and H1 histone, thus changing transcriptional frequency. The greater the enrichment of AT sequences in the regulatory regions of the cell determination genes, the greater may be the extent of evolutionary conservation. Higher frequency of transcription of the cell determination genes with the more AT-rich regulatory sequences could account for the earlier expression of the more conserved cell determination genes during embryonic development. Preferential binding of H1 histone to the more AT-rich regulatory sequences would subsequently restrict their transcription before that of less conserved cell determination genes.

Histone metabolic pathways and chromatin assembly factors as proliferation markers

The structural organization of DNA into chromatin is of key importance to regulate genome function and stability. Maintenance of such an organization is thus crucial to preserve cellular identity. At each cell cycle, during S phase, this is achieved by duplication of chromatin structure in tight coordination with DNA replication. Such a coordinate process requires histone synthesis and their deposition onto DNA by chromatin assembly factors to be efficiently coupled to DNA synthesis. In this review, we highlight the intimate relationship between these chromatin-related events and DNA replication and we show how it is possible to take advantage of this coupling in order to identify cells with high replicative potential such as tumor cells. On the basis of recent data, we discuss the potential use of chromatin-associated factors as new proliferation markers of interest for cancer diagnosis and prognosis.

Functional characterization of a human histone gene cluster duplication

Histones are the major protein component of nucleosomes, and de novo histone synthesis is essential for packaging newly replicated DNA into chromatin. As a result, histone gene expression is exquisitely and functionally coupled with DNA replication. Vastly divergent organisms such as yeast, fly and human all demonstrate the phylogenetically conserved propensity to maintain clustering of histone genes at one or more genomic loci. Although specific mechanisms are unclear, clustering is presumed to be important for common stringent transcriptional control of these genes at the G1/S phase transition. In this study, we describe a genomic duplication of the human histone gene cluster located at chromosome 1q21, which effectively doubles the previously known size and gene number of that cluster. The duplication persists in all examined tissues and cell lines, and the duplicated genes are transcriptionally active. Levels of messenger RNAs for duplicated histone H4 genes are high relative to those for non-duplicated H4 genes. Our data suggest that transcriptionally robust histone H4 genes may have been preferentially duplicated during evolution.

Cell-cycle-dependent translation of histone mRNAs is the key control point for regulation of histone biosynthesis in Leishmania infantum.

The cell-cycle-dependent expression of the four core histones (H2A, H2B, H3 and H4) has been studied in the protozoan parasite Leishmania infantum. For that purpose, the cell cycle was arrested by incubation of promastigotes with the DNA synthesis inhibitor hydroxyurea, which induced an accumulation of cells stalled in G1 phase. Hydroxyurea release resulted in a semi-synchronous entry into the cell cycle, as determined by flow cytometry. The steady-state levels of histone mRNAs in the G1, S and G2/M phases were found to be constant along the cell cycle. However, the levels of histone synthesis increased when parasites enter the S phase, in agreement with previous results showing that histone synthesis in Leishmania is tightly coupled with DNA replication. In addition, we analysed the distribution of histone mRNAs on polyribosomes at different stages of the cell cycle by separation of cytoplasmic RNAs in sucrose gradients. Remarkably, a drastic change in the polysome profiles of histone mRNAs was observed during the progression from G1 to S phase. Thus, in the S phase, histone mRNAs are present in ribosome-bound fractions, but in the G1 phase, the histone transcripts are exclusively found in the ribosome-free fractions. These results support a regulatory model in which the cell-cycle-regulated synthesis of histones in Leishmania is controlled through a reversible interaction between translational repressors and histone mRNAs.