Chromatin-bound RNA Polymerase Research Articles

MAP Kinase-Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic Modification of Chromatin During Osteoblast Differentiation.

RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase-dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2-binding regions of Bglap2 and Ibsp. Growth of MC3T3-E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin-bound P-ERK, P-RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di-methylation, another gene activation-associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono-, di-, and tri-methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation-dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2-deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non-phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin-associated P-ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P-RUNX2-dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427-2435, 2017. © 2016 Wiley Periodicals, Inc.

Systems-level dynamic analyses of fate change in murine embryonic stem cells

Molecular regulation of embryonic stem cell (ESC) fate involves a coordinated interaction between epigenetic, transcriptional and translational mechanisms. It is unclear how these different molecular regulatory mechanisms interact to regulate changes in stem cell fate. Here we present a dynamic systems-level study of cell fate change in murine ESCs following a well-defined perturbation. Global changes in histone acetylation, chromatin-bound RNA polymerase II, messenger RNA (mRNA), and nuclear protein levels were measured over 5 days after downregulation of Nanog, a key pluripotency regulator. Our data demonstrate how a single genetic perturbation leads to progressive widespread changes in several molecular regulatory layers, and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. We observe that a large proportion of changes in nuclear protein levels are not accompanied by concordant changes in the expression of corresponding mRNAs, indicating important roles for translational and post-translational regulation of ESC fate. Gene-ontology analysis across different molecular layers indicates that although chromatin reconfiguration is important for altering cell fate, it is preceded by transcription-factor-mediated regulatory events. The temporal order of gene expression alterations shows the order of the regulatory network reconfiguration and offers further insight into the gene regulatory network. Our studies extend the conventional systems biology approach to include many molecular species, regulatory layers and temporal series, and underscore the complexity of the multilayer regulatory mechanisms responsible for changes in protein expression that determine stem cell fate.

Cytokinin signalling systems

A role of endogenous cytokinins as the hormonal signal, by which the roots regulate leaf metabolism and prevent leaf senescence in particular, is discussed. Cytokinin signal perception and transduction in leaf cells were studied on fully expanded first leaves of 10–13-day-old barley plants (Hordeum vulgare L. cv. Viner). Their high sensitivity to exogenous cytokinins depends on dramatic decrease in endogenous cytokinin content during leaf growth. Cytokinin-binding protein of 28–30 kD was isolated from barley leaf cytosol by its affinity to synthetic cytokinin benzyladenine (BA). The 30-kD protein was involved in cytokinin-dependent activation of RNA synthesis in vitro in the system containing chromatin-bound RNA polymerase I from barley leaves. Multistage purification of the protein included affinity chromatography on trans-zeatin-Sepharose or zeatin riboside-Sepharose resulted in isolation of barley leaf cytosol 67-kD protein up to electrophoretic homogeneity. The protein was revealed as a single band in Western blot analysis developed with anti-idiotype antibodies from antiserum to zeatin. In concert with trans-zeatin, the 67-kD protein activated transcription elongation directed by RNA polymerase I (in the system containing chromatin-bound RNA polymerase I from barley leaves) and by RNA polymerase II (in nuclei isolated from barley leaves). The protein effect strongly depended on cytokinin concentration. The maximum activation was observed at trans-zeatin concentration of 10−8M. cis-Zeatin had no effect. These results together with data on reversible [3H]zeatin-binding moiety of the 67-kD protein [8] provide a definitive proof to consider this protein as one of the cytokinin receptors in barley leaf cells which is responsible for cytokinin activation of transcription elongation directed by both RNA polymerase I and RNA polymerase II.

Isolation of a protein with cytokinin-receptor properties by means of anti-idiotype antibodies

A protein from cytosol of 10-day-old barley leaves with cytokinin-receptor properties was isolated and 12 000-fold purified by anti-idiotype antibodies from anti-BA serum. Cytokinin-binding properties of this protein were demonstrated by its competition with Ab ba for immobilized BA in competitive ELISA. In the presence of BA the protein activates in vitro rRNA synthesis in the transcription elongation system containing chromatinbound RNA-polymerase 1 from barley leaves. The protein with similar properties was isolated from barley leaf cytosol with BA-Sepharose. The control proteins isolated with adenine- and ethanolamine-Sepharose did not possess cytokinin-binding properties and had no effect on RNA synthesis in vitro.

RNA Synthesis by Nuclei and Chromatin Isolated from Adult and Senescent Wistar Rat Liver

Nuclei isolated from senescent (22-26 months) female Wistar rat liver show a decreased RNA synthesis compared to nuclei from adult (12 months) liver. Kinetic analyses demonstrate a decreased rate (25-42%) and maximal incorporation (41-54%) of labeled uridine triphosphate into both rRNA and mRNA in senescent nuclei. Chromatin-bound RNA polymerase activity is decreased by 36%, whereas free RNA polymerase activity, i.e., not bound to chromatin, is increased by 41% in senescent nuclei, but the total bound plus free activity is the same in senescent and adult nuclei. Isolated senescent chromatin shows reduced transcriptional capacity and requires a higher temperature to initiate melting. A decreased ability of chromatin to bind RNA polymerases appears to underlie the observed decreased RNA synthesis in senescent liver nuclei.

Poliovirus-induced modification of host cell RNA polymerase IIO is prevented by cycloheximide and zinc.

Infection of HeLa cells with poliovirus results in a decrease in the level of RNA polymerase IIO, the transcriptionally active form of the enzyme, and a shutdown of host transcription (Rangel, L. M., Fernández-Tomas, C., Dahmus, M. E., and Gariglio, P. (1987) J. Virol. 61, 1002-1006). The effect of cycloheximide on poliovirus-induced modification of host RNA polymerase IIO was investigated. The inhibition of protein synthesis, at sequential stages during viral replication, prevents the modification of both total and chromatin-bound RNA polymerase IIO. Furthermore, the inclusion of zinc at a concentration that inhibits the proteolytic post-translational processing of viral polyprotein also prevents the modification of RNA polymerase IIO. These results suggest that host cell enzyme modification depends on the synthesis and processing of protein(s) encoded by the viral genome.

Decreased Transcription of Neuronal Polyadenylated RNA During Senescence in Nuclei from Rat Brain Cortex

Neuronal and glial cell-enriched nuclei were prepared from the brain cortex of rats of different ages for study of alterations in the synthesis of cellular RNA with age. RNA synthesis by isolated neuronal nuclei was substantially reduced in senescent rats, whereas that of glial nuclei remained constant throughout development. The nuclear content of polyadenylic acid-containing RNA in neurons was particularly decreased in old rats. The results show that the activity of the chromatin-bound neuronal RNA polymerase declines during senescence, an observation indicating an age-related reduction in template activity in neuronal nuclei. The activity of nuclear poly(adenylate) polymerase also progressively decreases in aging neurons. The decrease in the transcription and polyadenylation of nuclear RNA may contribute to the decline in neuronal protein synthesis observed in old animals.

Intranuclear compartmentalization of DNA-dependent RNA polymerases: association of RNA polymerase I with nucleolar organizing chromosomes.

The compartmentalization of chromatin-bound RNA polymerases was examined with HeLa chromosomes fractionated according to their size in sucrose/glycerol gradients. There was a good correlation between the enrichment of nucleolus-organizing chromosomes, i.e., D and G group chromosomes, and the level of chromosome-associated RNA polymerase form I activity. A profile of DEAE-Sephadex chromatography of enzymes solubilized from chromosome fractions also supported the view that form I was associated with D and G group chromosomes. The amount of form I associated with unfractionated chromosomes was nearly identical to that of nuclei, indicating that this enzyme is probably quantitatively conserved even when chromatin undergoes extensive condensation. Since the chromatin-bound form I enzyme can be reactivated with heparin, it seems that this enzyme is in the initiated state, probably being bound to rDNA throughout the mitotic cycle. Thus, the absence of rRNA synthesis in mitosis is due to neither unavailability of enzyme to rDNA nor to the release of some factors necessary for transcriptional processes. Form II enzyme was associated uniformly with all chromosome fractions. Taken together, the present findings suggest that the intranuclear compartmentalization of RNA polymerases persists not only in interphase stages, but also in mitosis, during which most other nuclear proteins are released into the cytoplasm.

RNA polymerase activities and chromatin protein composition of rat liver during methionine deprivation and refeeding

1. 1. The reversible effect of dietary methionine deficiency was studied in young adult rats. 2. 2. The sensitivity of nuclear chromatin to micrococcal nuclease (EC 3.1.4.7) digestion and the composition of the chromatin proteins were unaffected by the dietary regimens. 3. 3. The specific chromatin-bound RNA polymerase II activity decreased during methionine deficiency. Refeeding of methionine for 2 days restored the activity in the nuclease-released chromatin. RNA polymerase I plus III activity remained unchanged. 4. 4. Total RNA polymerase activity changed with the liver wet weight which was reduced during methionine deficiency and was not restored to control level after 2 days of methionine refeeding. 5. 5. RNA polymerase activity was altered by methionine deficiency. The recovery was independent of major modifications of the chromatin structure and protein composition.

Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats.

When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei. We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.

Chromatin-bound and free RNA polymerase A activities in rat thymus cells following glucocorticoid treatment

Treatment of rat thymus cell suspensions with dexamethasone resulted in inhibition of engaged RNA polymerase A, without significant change in free pool activity. Studies with the re-initiation inhibitor, rifamycin AF/0–13, and measurements of numbers of RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis resulted from a decrease in elongation rate. This effect was selectively abolished by mild proteolysis of nuclei. It is concluded that glucocorticoid treatment of rat thymus cells suppresses 45 S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate, rather than by effecting a change in enzyme redistribution or concentration.

RNA synthesis in regenerating mouse liver evaluated by incorporation of [methyl-14C]methionine and by determination of RNA polymerase activity.

The synthesis of RNA during mouse liver regeneration was studied by two different methods at 24 and 48 h after partial hepatectomy. Total chromatin-bound RNA polymerase activity showed an increase of 32% at 24 h after partial hepatectomy. At 48 h a slight increase in total activity was also observed in regenerating liver, but the difference was not significant. The increase in total RNA polymerase activity was due to a rise in RNA polymerase I plus III activity. This enzyme activity was increased at both 24 and 48 h. The increase was 57% at 24 h and 51% at 48 h. When [methyl-14C]methionine was used for labelling of methyl groups in rRNA, there was an increased specific radioactivity of this class of RNA at both 24 h and 48 h. The increases were 263 and 103% at 24 and 48 h respectively. Thus both methods revealed an increased synthesis of rRNA during mouse liver regeneration. The results are discussed in relation to previous results from this laboratory [Yngner, Carlberg, Lewan & Engelbrecht (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1069-1074; Yngner, Engelbrecht, Lewan & Annerfeldt (1979) Biochem. J. 178, 1-8; Yngner, Bengtsson, Carlberg, Engelbrecht & Wieslander (1980) Exp. Cell. Biol. 48, 393-403], which have shown that the incorporation of orotic acid or uridine into RNA is not increased in mouse liver regenerating after partial hepatectomy.

Differential changes of tightly chromatin-bound RNA polymerase II in starved and cycloheximide-treated rat liver nuclei.

In order to investigate the physiological function of loosely and tightly chromatin-bound RNA polymerase II in vivo (1), the changes of these enzyme activities in cycloheximide-treated or starved rat liver nuclei were studied. Total nuclear mRNA synthesis activity in starved rats was considerably decreased, but that of cycloheximide-treated rats was not affected significantly. In starved rats, tightly bound enzyme activity was much more repressed as compared with that of loosely bound enzyme. On the other hand, cycloheximide-treated rats showed the reverse relationship. Thus, mRNA synthesis activity in hepatic nuclei seems to be dependent on the tightly bound RNA polymerase II activity. However, the difference of nuclear mRNA synthesis in both cases can not be explained by the change of chromatin-bound enzyme activity of Yu (2).

Dietary sugar promotes gene activation in intestinal cell chromatin of adult rats.

The effect of dietary sucrose on transcription activity of chromatin from intestinal cells of adult rats was investigated. Chromatin-bound RNA polymerase II activity was significantly enhanced after feeding sucrose to rats. RNA polymerase II transcribes the genes coding for pre-messenger RNA. The results indicate that feeding of sucrose increased the synthesis of pre-messenger RNA molecules coding for specific proteins which are involved in the adaptive processes of sucrose hydrolysis in intestinal cells.

Effects of partial hepatectomy on rna polymerase activities in mouse liver

1. 1. Chromatin-bound and poly[d(A-T)]dependent RNA polymerase 1 plus III and II activities of mouse liver were analysed 24 and 48 hr after partial hepatectomy. 2. 2. Chromatin-bound RNA polymerase I plus III activity showed an increase of 57%, at 24 hr and 51%, at 48 hr after partial hepatectomy. 3. 3. There was a decrease in chromatin-bound RNA polymerase II activity of 15% at 24 hr and 34% at 48 hr after partial hepatectomy. 4. 4. There was no significant changes in poly[d(A-T)] dependent RNA polymerase activities. 5. 5. Heparin caused an approximately 10-fold increase in chromatin-bound RNA polymerase II activity. The stimulation by heparin was significantly increased 48 h after partial hepatectomy. 6. 6. Anaesthesia and/or surgery had great influence on RNA polymerase activities. At 24 hr after operation, chromatin-bound RNA polymerase I plus III and II activities were depressed, and the liver cell chromatin was more susceptible to stimulation by heparin.

Non-random effect on RNA synthesis in liver chromatin by administration of dimethylnitrosamine to mice.

The effect of dimethylnitrosamine on functional activities of liver chromatin was studied in mice. After a single dose of dimethylnitrosamine injected i.v. (25 mg/kg body wt, 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcal nuclease (EC 3.1.4.7) to an acid-solubility of 2.5% of total DNA. Chromatin was fractionated into a 1,200 g pellet P1, 102,000 g pellet P2 and supernatant fraction S2. Chromatin-bound RNA polymerase I plus III activity decreased 15% in the P1 and 25% in the P2 fraction. No changes in activity were observed in the S2 fraction. Chromatin-bound RNA polymerase II activity decreased 19% in the P1, 49% in the P2 and 32% in the S2 fraction. Heparin stimulated RNA polymerase II activity decreased 10% in the P1 and 44% in the P2 fraction. Formation of initiation in nuclear lysates with RNA polymerase from Escherichia coli increased after administration of dimethylnitrosamine suggesting an increase in the number of sites available for the start of new RNA chains. The results show that limited digestion of nuclei with endonuclease cleaves chromatin regions which are more affected by dimethylnitrosamine than the total chromatin suggesting a non-random effect of the hepatotoxin on chromatin. Modifications of the DNA template by dimethylnitrosamine is indicated by the change in number of initiation complexes.

Modulation of RNA Polymerase Activities in the Intestine of Adult Rats by Dietary Sucrose

The effect of sucrose feeding on endogenous intestinal RNA polymerase activities and on chromatin structure was studied in rats. Adult rats were given a 70% sucrose solution for 15 hours following a 48-hour starvation period. Comparison was made with rats starved for 63 hours and with ad libitum nourished animals. Chromatin-bound RNA polymerase I activity was significantly reduced by starvation. Sucrose feeding provoked a significant rise in the activity, but the level found in the nourished rats was not reached. The free poly[d(A-T)]-dependent RNA polymerase I activity of the sucrose-fed rats exceeded that of the starved and the nourished animals. Chromatin-bound RNA polymerase II activity was enhanced most markedly by sucrose feeding. The balance between the chromatin-bound and free enzymes was shifted towards the chromatin-bound state when compared to the starved and nourished rats. Starvation caused a reduction in the size of oligonucleosomes but sucrose feeding restored almost entirely the original pattern obtained in the nourished animals. These results reflect modifications in the structure of chromatin after sucrose feeding. The present report demonstrates that the adaptive processes triggered in the intestine by dietary sucrose are associated with changes in gene expression.

Lysine deficiency reduces transcription activity and concentration of chromatin proteins reversibly in rat liver.

The reversible effect of dietary lysine deficiency was studied in young adult rats. During 6 days on a lysine diet the rats maintained the same body weight. During 2 days of recovery body weight gain was that of the controls. Liver nuclei were isolated, incubated with micrococcus nuclease and chromatin fractionated in to a 2 000 X g pellet, 102 000 X g pellet and supernatant fraction. Chromatin-bound RNA polymerase 1 plus III activity decreased by 15% per mg of fractional and nuclear DNA and by 30% per total liver. The corresponding decrease of RNA polymerase II activity was 30% and 40%. Recovery from lysine deficiency was complete after 2 days of refeeding the amino acid. Chromatin proteins of the 102 000 X g pellet were characterized by polyacrylamide gel electrophoresis in sodium dodecylsulfate and by 2-dimensional gel electrophoresis. Quantitative but no qualitative differences between the proteins of the dietary groups were observed. Relative to DNA the non-histone proteins decreased in the lysine deficient group by 43% and histones by 10%. It is concluded that RNA synthesis is restored to its original level within 2 days of refeeding lysine after 6 days of lysine deficiency.

Cortisone acetate does not change free and chromatin-bound RNA polymerase II activities early after injection

FEBS LettersVolume 149, Issue 1 p. 41-43 Full-length articleFree Access Cortisone acetate does not change free and chromatin-bound RNA polymerase II activities early after injection Yasuji Okai, Yasuji Okai Department of Immunology, OEH University Medical School, Yawatanishi-ku, Kitakyushu, 807, JapanSearch for more papers by this author Yasuji Okai, Yasuji Okai Department of Immunology, OEH University Medical School, Yawatanishi-ku, Kitakyushu, 807, JapanSearch for more papers by this author First published: November 22, 1982 https://doi.org/10.1016/0014-5793(82)81066-1Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume149, Issue1November 22, 1982Pages 41-43 ReferencesRelatedInformation

DNA-binding property of free and chromatin-bound RNA polymerase II: Analysis by DNA—Sephadex G-25 chromatography

At present, the biological determinants that localize RNA polymerase molecules in the cell nucleus are not fully elucidated. Since it seemed possible that the enzyme’s DNA-binding property might be one of the determinants, we examined DNA-binding activity of the extracted RNA polymerase II by affinity chromatography of native DNA that was covalently coupled to Sephadex G-25 [ 1,2]. We found that: (1)