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• W2040722846 abstract "The dynamic character of core histone post-translational acetylation in the unicellular green algaChlamydomonas reinhardtii was studied by tritiated acetate incorporation. Histone H3 is the major target of acetylation, steady state, and in pulse and pulse-chase analyses. Acetylation turnover rates were measured by tracer labeling under steady-state conditions. Half-lives of 1.5–3 min were found for penta- to mono-acetylation of H3, dynamically acetylated to the 30% level. Twenty percent of H3 was multi-acetylated, on average with 3.2 acetyl-lysines, all with rapid turnover. Deacetylase inhibitor trichostatin A (TSA) caused doubling of average acetylation levels, primarily as penta-acetylated H3, but half of H3 was not acetylated at all. The level of histone H4 acetylation was only half that of H3 and a major fraction of mono- and di-acetylated forms appeared static. The dynamic fraction had an average half-life of 3.5 min with higher turnover rates for more highly acetylated H4 forms. TSA, inhibiting less effectively deacetylases active on H4, strongly increased multi-acetylated H4 levels and doubled average acetylation. As for H3, half of histone H4 remained unacetylated. Acetylation of histone H2B was low and of H2A was barely measurable. Despite turnover with half-lives of approximately 2 min, no increase beyond di-acetylation was seen upon TSA treatment. The dynamic character of core histone post-translational acetylation in the unicellular green algaChlamydomonas reinhardtii was studied by tritiated acetate incorporation. Histone H3 is the major target of acetylation, steady state, and in pulse and pulse-chase analyses. Acetylation turnover rates were measured by tracer labeling under steady-state conditions. Half-lives of 1.5–3 min were found for penta- to mono-acetylation of H3, dynamically acetylated to the 30% level. Twenty percent of H3 was multi-acetylated, on average with 3.2 acetyl-lysines, all with rapid turnover. Deacetylase inhibitor trichostatin A (TSA) caused doubling of average acetylation levels, primarily as penta-acetylated H3, but half of H3 was not acetylated at all. The level of histone H4 acetylation was only half that of H3 and a major fraction of mono- and di-acetylated forms appeared static. The dynamic fraction had an average half-life of 3.5 min with higher turnover rates for more highly acetylated H4 forms. TSA, inhibiting less effectively deacetylases active on H4, strongly increased multi-acetylated H4 levels and doubled average acetylation. As for H3, half of histone H4 remained unacetylated. Acetylation of histone H2B was low and of H2A was barely measurable. Despite turnover with half-lives of approximately 2 min, no increase beyond di-acetylation was seen upon TSA treatment. trichostatin A acid/urea/Triton X-100 high pressure liquid chromatography high salt medium. The study of histone acetylation has its origin more than 30 years ago when Allfrey (1Allfrey V.G. Li H.J. Eckhardt R.A. Chromatin and Chromosome Structure. Academic Press, New York1977: 167-191Crossref Google Scholar) established for the first time the correlation between high levels of histone acetylation and gene transcription. This was followed by the realization that histone acetylation is dynamic (2Matthews H.R. Waterborg J.H. Freedman R.B. Hawkins H.C. The Enzymology of Post-translational Modification of Proteins. 2. Academic Press, London1985: 125-285Google Scholar). In animal cells, histone acetylation has turnover half-lives of 3–30 min (3Sealy L. Chalkley R. Cell. 1978; 14: 115-121Abstract Full Text PDF PubMed Scopus (554) Google Scholar, 4Jackson V. Shires A. Chalkley R. Granner D.K. J. Biol. Chem. 1975; 250: 4856-4863Abstract Full Text PDF PubMed Google Scholar, 5Covault J. Chalkley R. J. Biol. 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Chem. 1982; 257: 1565-1568Abstract Full Text PDF PubMed Google Scholar). In recent years, histone H4 and H3, highly acetylated at some or all amino-terminal lysines, has been studied intensively by specific antibodies (11Turner B.M. Birley A.J. Lavender J. Cell. 1992; 69: 375-384Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 12Lin R. Leone J.W. Cook R.G. Allis C.D. J. Cell Biol. 1989; 108: 1577-1588Crossref PubMed Scopus (121) Google Scholar, 13Braunstein M. Sobel R.E. Allis C.D. Turner B.M. Broach J.R. Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Scopus (328) Google Scholar, 14Hebbes T.R. Thorne A.W. Crane-Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (707) Google Scholar). This has confirmed the general relationship between high acetylation and gene transcription, although high acetylation at some sites is more clearly correlated with deposition of newly synthesized histone or even with gene or chromosome silencing (15Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2388) Google Scholar). The recognition that n-butyrate inhibited histone deacetylase in vivo and caused general and extensive hyperacetylation of chromatin led to experiments that in general supported correlation between high acetylation and gene transcription. It allowed measurements of acetylation turnover rates upon release of inhibition (5Covault J. Chalkley R. J. Biol. Chem. 1980; 255: 9110-9116Abstract Full Text PDF PubMed Google Scholar, 16Zhang D.E. Nelson D.A. Biochem. J. 1988; 250: 241-246Crossref PubMed Scopus (40) Google Scholar). In recent years, many and more specific inhibitors of histone deacetylase have been discovered, including the reversible inhibitor trichostatin A (TSA)1 and irreversible inhibitor trapoxin (17Yoshida M. Horinouchi S. Beppu T. BioEssays. 1995; 17: 423-430Crossref PubMed Scopus (666) Google Scholar, 18Kwon H.J. Owa T. Hassig C.A. Shimada J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. 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Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1282) Google Scholar), and the recognition that many transcription factors, co-activators, and basal transcription initiation complex proteins are, localize, or contain acetyltransferases in species from yeast to man, it has become apparent that multiple acetylating enzymes, each with potentially unique enzymatic substrate specificities and/or with unique localization mechanisms, act on chromatin at and away from the transcription initiation complex (15Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2388) Google Scholar, 21Kadonaga J.T. Cell. 1998; 92: 307-314Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 22Mizzen C.A. Allis C.D. Cell. Mol. Life Sci. 1998; 54: 6-20Crossref PubMed Scopus (189) Google Scholar). Also, more and diverse histone deacetylase activities, and their localization adapters (23Hassig C.A. Tong J.K. Fleischer T.C. Owa T. Grable P.G. Ayer D.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524Crossref PubMed Scopus (331) Google Scholar, 24Wolffe A.P. Nature. 1997; 387: 16-17Crossref PubMed Scopus (254) Google Scholar), have been identified, from yeast to man and higher plants (22Mizzen C.A. Allis C.D. Cell. Mol. Life Sci. 1998; 54: 6-20Crossref PubMed Scopus (189) Google Scholar, 25Lopez-Rodas G. Georgieva E.I. Sendra R. Loidl P. J. Biol. Chem. 1991; 266: 18745-18750Abstract Full Text PDF PubMed Google Scholar, 26Lusser A. Brosch G. Loidl A. Haas H. Loidl P. Science. 1997; 277: 88-91Crossref PubMed Scopus (192) Google Scholar, 27Lechner T. Lusser A. Brosch G. Eberharter A. Goralik-Schramel M. Loidl P. Biochim. Biophys. Acta. 1996; 1296: 181-188Crossref PubMed Scopus (71) Google Scholar, 28Sendra R. Rodrigo I. Salvador M.L. Franco L. Plant Mol. Biol. 1988; 11: 857-868Crossref PubMed Scopus (49) Google Scholar). To date, all deacetylase activities described and tested can be inhibitedin vitro by TSA, but with different efficiencies (29Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar). In general, the correlation between acetylation and gene expression has been confirmed (22Mizzen C.A. Allis C.D. Cell. Mol. Life Sci. 1998; 54: 6-20Crossref PubMed Scopus (189) Google Scholar, 30Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (506) Google Scholar, 31Wade P.A. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-131Abstract Full Text PDF PubMed Scopus (409) Google Scholar, 32Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1546) Google Scholar, 33Almouzni G. Khochbin S. Dimitrov S. Wolffe A.P. Dev. Biol. 1994; 165: 654-669Crossref PubMed Scopus (124) Google Scholar) even while repression of some genes and functions has also become more clearly described (23Hassig C.A. Tong J.K. Fleischer T.C. Owa T. Grable P.G. Ayer D.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524Crossref PubMed Scopus (331) Google Scholar, 29Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 34Rundlett S.E. Carmen A.A. Suka N. Turner B.M. Grunstein M. Nature. 1998; 392: 831-835Crossref PubMed Scopus (363) Google Scholar). Most studies of histone acetylation have been performed in animal cells, in fungi like S. cerevisiae or protists likeTetrahymena. In all these, histone H4 is the predominant target of histone acetylation, followed by histone H3, whereas acetylation of H2B and H2A core histones is lower. In higher plants, histone H3 is the predominant target of histone acetylation with rather high steady-state levels, especially of replacement variant forms which are preferentially localized in transcriptionally active chromatin domains (35Waterborg J.H. Plant Physiol. (Bethesda). 1991; 96: 453-458Crossref PubMed Scopus (36) Google Scholar, 36Waterborg J.H. Plant Mol. Biol. 1992; 18: 181-187Crossref PubMed Scopus (20) Google Scholar, 37Waterborg J.H. J. Biol. Chem. 1993; 268: 4912-4917Abstract Full Text PDF PubMed Google Scholar). Acetylation levels of histone H4 are lower, and levels of H2B are lower still, whereas acetylation of histone H2A is barely detectable (38Waterborg J.H. Harrington R.E. Winicov I. Biochim. Biophys. Acta. 1990; 1049: 324-330Crossref PubMed Scopus (15) Google Scholar). In the unicellular green algaeChlamydomonas reinhardtii, this difference is even more pronounced, and steady-state levels of multi-acetylated H3 are remarkably high (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). Also, histone acetylation in Chlamydomonas appeared rather fast, as detected during analysis of histone synthesis throughout the cell cycle (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). This prompted study of the dynamics of histone acetylation. Described below is a unique quantitative analysis of the rate of turnover of acetylation with the highest rates of turnover for histone acetylation reported to date. It describes how fast and to which extent TSA induces hyperacetylation of histones, limited by histone methylation, and reports that a remarkably high fraction of the algal chromatin cannot be acetylated. Cell wall-deficient strain CC-400cw-15 mt+ clone DG-1 (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar) was maintained on Sueoka's high salt medium (HSM) on 0.8% agarose at 25 °C and continuous light. Phototrophic culture at 28 °C, exposed to continuous white light at 75 microeinsteins photosynthetic active radiation (PAR) m−2 and 3% CO2-enriched air bubbling, was inoculated directly from plates at an initial density of 105 cells/ml into HSM minimal medium in 800-ml glass cylinders (4 cm in diameter) (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). Typically, growing cultures at a density of 2–6 × 106 cells/ml were collected by centrifugation for 10 min at 7000 × g in 1-liter buckets at room temperature and were resuspended at a density of 5 × 107 cells/ml. Translation inhibitor cycloheximide was added to 10 μg/ml from fresh, non-sterile stock (2 mg/ml in 50% ethanol) 10 min prior to the addition of acetate. For labeling to steady state, 0.1 m NH4Ac was added to 0.2 mm final concentration 5 min prior to the addition of tritiated acetate. High specific activity [3H]NaAc (9 × 1013 Bq/mol, NEN Life Science Products) was added at 3.7 × 107 Bq (1 mCi) per 40 ml of concentrated culture (to 10 μm acetate), and cells were incubated at room temperature for various lengths of time. Acetate incorporation was stopped by transfer to melting ice and collection of cells by centrifugation for 5 min at 650 × g and 0 °C. The chase condition in pulse-chase experiments was created by the addition of 1 volume of ammonium acetate-containing HSM (9.34 mm acetate) 2 min after tritiated acetate was added to 20 μm. Acetate labeling at reduced specific radioactivity was performed by addition of ammonium acetate to 200 μmfollowed after 5 min by the addition of tritiated acetate to 10 μm. Trichostatin A (Wako Bioproducts) was desolved in dimethyl sulfoxide at 0.5 mg/ml (1.67 mm) and was used at a final concentration of 100 ng/ml unless otherwise indicated. Isolation of nuclei and preparation of histones was essentially done as described previously (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). Briefly, histones from 2 × 109 cells were solubilized by sonication of the nuclear pellet in 40% guanidine HCl at pH 6.8, acidified, centrifuged, neutralized, bound to and eluted from 0.25 ml of Bio-Rex 70 resin (Bio-Rad) by 40% guanidine HCl, dialyzed extensively against 2.5% (v/v) acetic acid, and lyophilized. At all steps, 2-mercaptoethanol in excess of 1 mm was present. Histones were solubilized and subjected to reversed-phase HPLC on a Zorbax Protein-Plus column (4.6 × 25 mm) as described (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar), developed by a 90-min gradient at 1 ml/min with acetonitrile between 30 and 60% (v/v) and 0.1% trifluoroacetic acid, and monitored by absorbance at 214 nm. Radioactivity in fractions was determined by liquid scintillation counting. Fractions were made 1 mm in 2-mercaptoethanol, pooled based on absorbance and/or radioactivity, and lyophilized. Histones were electrophoresed in 30-cm long discontinuous acid-urea-Triton (AUT) gels with 8 m urea and 9 mm Triton X-100, essentially as described (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar, 40Waterborg J.H. J. Biol. Chem. 1990; 265: 17157-17161Abstract Full Text PDF PubMed Google Scholar), until the methylene blue dye front had traversed one gel length (approximately 20 h at 300 V), except that histone H3 was run until methylene blue had traversed two gel lengths (approximately 44 h at 300 V). Procedures of AUT Coomassie gel staining, densitometry, and fluorography have been described previously (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). SigmaPlot version 4.0 (SSPS, Chicago) was used for nonlinear regression data analysis. Post-translational modification of the histones of unicellular green alga C. reinhardtii has been studied by incorporation of tritiated acetate into growing cells, synchronized by a light-dark regimen in acetate-free minimal medium during phototrophic growth (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). Labeled acetate is rapidly metabolized and incorporated into newly synthesized protein, which is observed as label incorporation into all histone species, separated by reversed-phase HPLC, after labeling for 60 min. Reducing the time of labeling to 5 min abolished most but not all label incorporation into non-acetylated histone forms (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar) (results not shown). Preincubation of cells with translation inhibitor cycloheximide (10 μg/ml) completely abolished acetate incorporation into the co-translationally, amino-terminally acetylated linker histone H1 species and into the many low abundance proteins observed during HPLC fractionation of the crude histone preparation. Acid-urea-Triton (AUT) gel analysis and fluorography (Fig. 1) confirmed these observations and the absence of any acetate label incorporation into non-acetylated core histones, including histone H4 and H2A, histones that are typically co-translationally acetylated at the amino termini of newly synthesized polypeptides. Provided cycloheximide was added at least 5 min prior to the addition of tritiated acetate, (co)translational labeling of histones by acetate was prevented for more than 2 h. The relative levels of post-translational incorporation of acetate into core histones following a short incubation with tritiated acetate differed markedly between histone species (Table I) and correlated well with steady-state acetylation levels of each histone, determined by Coomassie staining and densitometry of AUT gels (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar) (compare lanes G and H in Fig. 1). Whereas in animal cells histone acetylation is highest in histone H4 and significant for all other core histones, in Chlamydomonas, even more than in higher plants (35Waterborg J.H. Plant Physiol. (Bethesda). 1991; 96: 453-458Crossref PubMed Scopus (36) Google Scholar, 36Waterborg J.H. Plant Mol. Biol. 1992; 18: 181-187Crossref PubMed Scopus (20) Google Scholar, 37Waterborg J.H. J. Biol. Chem. 1993; 268: 4912-4917Abstract Full Text PDF PubMed Google Scholar, 38Waterborg J.H. Harrington R.E. Winicov I. Biochim. Biophys. Acta. 1990; 1049: 324-330Crossref PubMed Scopus (15) Google Scholar), histone acetylation of H3 is predominant, acetylation of H4 is lower but significant, and acetylation of H2B and H2A is low, especially when judged by the relative post-translational incorporation of radioactive acetate (Table I).Table IHistone comparison by steady-state acetylation levels and post-translational acetate labelingHistoneHPLC labeling1-aCells were incubated with tritiated acetate as shown in Fig. 2. The specific radioactivity “labeling” of histones was measured as cpm per absorbance at 214 nm during HPLC fractionation (39) and by densitometry of Coomassie-stained and fluorographed AUT gels (Fig. 1). Values were standardized on histone H3 which incorporated approximately 50,000 cpm per nmol of histone H3 when 1.8 × 109 cells were incubated for 2 min with 1 mCi of tritiated acetate.AUT gel: labeling1-aCells were incubated with tritiated acetate as shown in Fig. 2. The specific radioactivity “labeling” of histones was measured as cpm per absorbance at 214 nm during HPLC fractionation (39) and by densitometry of Coomassie-stained and fluorographed AUT gels (Fig. 1). Values were standardized on histone H3 which incorporated approximately 50,000 cpm per nmol of histone H3 when 1.8 × 109 cells were incubated for 2 min with 1 mCi of tritiated acetate.Coomassie densitometryFluorograph densitometryMultiAc1-bDensitometry of Coomassie-stained AUT gels yields the percentage of histone existing in each acetylated form and the fraction of histone with more than 1 acetylated lysine per molecule (multiAc). Constant values are given as average with standard deviation and number of independent samples (n). Densitometry of fluorographs yields the same data, for those histones labeled by tritiated acetate (*multiAc). The values obtained after 2 min and, within parentheses, after 60 min are given when levels decreased.AcLys/H1-cThe level of lysine acetylation of each histone species can be expressed as the average number of acetylated lysines per unlabeled (AcLys/H) and per labeled histone molecule (*AcLys/H), taking into account the number of acetylated lysines in each form (39). It facilitates comparison of acetylation levels among histone forms that differ in distribution patterns (Fig. 1).*MultiAc1-bDensitometry of Coomassie-stained AUT gels yields the percentage of histone existing in each acetylated form and the fraction of histone with more than 1 acetylated lysine per molecule (multiAc). Constant values are given as average with standard deviation and number of independent samples (n). Densitometry of fluorographs yields the same data, for those histones labeled by tritiated acetate (*multiAc). The values obtained after 2 min and, within parentheses, after 60 min are given when levels decreased.*AcLys/H1-cThe level of lysine acetylation of each histone species can be expressed as the average number of acetylated lysines per unlabeled (AcLys/H) and per labeled histone molecule (*AcLys/H), taking into account the number of acetylated lysines in each form (39). It facilitates comparison of acetylation levels among histone forms that differ in distribution patterns (Fig. 1).H3≡1.0≡1.024 ± 1% (n = 6)0.92 ± 0.05 (n = 6)83 ± 1% (n = 5)2.94 ± 0.17 (n = 8)H40.390.5210.0 ± 0.4% (n = 7)0.47 ± 0.01 (n = 6)66%2.25(29%)(1.6 ± 0.1 (n = 5))H2B0.150.083.3 ± 0.3% (n = 7)0.18 ± 0.01 (n = 7)36%1.38 ± 0.03 (n = 3)(14%)(1.15 ± 0.08 (n = 3))H2A0.140.03—1-dAUT gel overlap of variant H2A forms prevents quantitation.—1-dAUT gel overlap of variant H2A forms prevents quantitation.1-a Cells were incubated with tritiated acetate as shown in Fig. 2. The specific radioactivity “labeling” of histones was measured as cpm per absorbance at 214 nm during HPLC fractionation (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar) and by densitometry of Coomassie-stained and fluorographed AUT gels (Fig. 1). Values were standardized on histone H3 which incorporated approximately 50,000 cpm per nmol of histone H3 when 1.8 × 109 cells were incubated for 2 min with 1 mCi of tritiated acetate.1-b Densitometry of Coomassie-stained AUT gels yields the percentage of histone existing in each acetylated form and the fraction of histone with more than 1 acetylated lysine per molecule (multiAc). Constant values are given as average with standard deviation and number of independent samples (n). Densitometry of fluorographs yields the same data, for those histones labeled by tritiated acetate (*multiAc). The values obtained after 2 min and, within parentheses, after 60 min are given when levels decreased.1-c The level of lysine acetylation of each histone species can be expressed as the average number of acetylated lysines per unlabeled (AcLys/H) and per labeled histone molecule (*AcLys/H), taking into account the number of acetylated lysines in each form (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar). It facilitates comparison of acetylation levels among histone forms that differ in distribution patterns (Fig. 1).1-d AUT gel overlap of variant H2A forms prevents quantitation. Open table in a new tab Turnover rates of dynamic, post-translational acetylation of histones are high with reported half-lives measured by pulse-chase experiments ranging from 3 min in mammalian cells in culture for the fastest dynamic component (4Jackson V. Shires A. Chalkley R. Granner D.K. J. Biol. Chem. 1975; 250: 4856-4863Abstract Full Text PDF PubMed Google Scholar) to 2 h for the rather stable acetylation of H4 in S. cerevisiae (10Nelson D.A. J. Biol. Chem. 1982; 257: 1565-1568Abstract Full Text PDF PubMed Google Scholar). Less quantitative analyses provide rough estimates of half-lives of 10 or 20 min for higher plants (38Waterborg J.H. Harrington R.E. Winicov I. Biochim. Biophys. Acta. 1990; 1049: 324-330Crossref PubMed Scopus (15) Google Scholar). Chlamydomonas grown phototrophically, free of a carbon source other than CO2, appeared to be well suited to perform pulse-chase experiments. Very rapid incorporation of radioactive acetate, applied at a concentration of 10 μm, was followed by apparent rapid exhaustion of the small labeled acetyl-CoA pool, as judged by the pattern of acetate incorporation into individual histones (Fig. 2) and into acetylated forms of these histones (Fig. 1). Incorporation of acetate into cells upon addition of tritiated acetate, immediately followed by collection of cells and lysis as the start of preparing nuclei, typically gave label incorporation between 50 and 80% of the maximum value reached after incubation for 2 or 3 min. Between 2 and 20 min, apparent exponential decay of the specific radioactivity in each core histone species was observed with a half-life of 6 ± 1 min (Fig. 2). This value should be considered an upper limit for the turnover rate because no real chase, i.e. dilution of the acetate pool, was applied. The relative rate of acetyl-lysine turnover of a histone species at each level of modification can be determined from the change in specific radioactivity of each modification level by densitometry of Coomassie-stained and -fluorographed AUT gels. The pattern observed for histone H3, acetylated at 5 sites (Fig. 1 A), is clearly different from that of histone H4 (Fig. 1 B), despite the fact that H4 is also acetylated at 5 lysines. The rate of acetyl-lysine turnover is essentially identical for each level of modification of H3 (Fig. 3) yielding a pattern that is fading over time as radioactively labeled acetyl-lysines are turned into non-radioactive ones, without a change in distribution (Fig. 1 A). In contrast, it is clear from the much more rapid fading of the multi-acetylated bands in the histone H4 pattern (Fig. 1 B) that turnover is faster for penta- and tetra-acetylated forms than for di- or mono-acetylated species (Fig. 3 B). This does not imply that highly acetylated forms of H4 lose acetyl groups and are converted into less and less modified forms because the steady-state pattern of H4 acetylation, measured from the Coomassie Blue distribution in AUT gels, does not change over time. The labeled acetylation pattern of histone H2B, with most label in mono- and di-acetylated forms, showed a slight shift over time (Fig. 1 C) and a slight tendency for a higher turnover rate for higher modified forms (Fig. 3 C). A similar result was deduced for histone H2A, but the overlapping pattern of two variant protein forms (39Waterborg J.H. Robertson A.J. Tatar D.L. Borza C.M. Davie J.R. Plant Physiol. (Bethesda). 1995; 109: 393-407Crossref PubMed Scopus (37) Google Scholar) prevents quantitative analysis. A pulse-chase protocol was used to measure turnover rates of histone acetylation. A 450-fold excess of unlabeled acetate was added 2 min after addition of tritiated acetate to cultures, pre-treated with cycloheximide. Histones were prepared, and their specific radioactivity was determined during HPLC fractionation (results not shown) and by densitometric analysis of stained and fluorographed AUT gels (Fig. 2). The instantaneous sharp drop in labeling of all histone species clearly demonstrated that the acetyl-CoA pool in Chlamydomonas, growing phototrophically, is very small. It also showed that acetylation turnover is very fast, faster than can be measured with reasonable accuracy by the experimental method used. Although cooled quickly at the end of each incubation, cells must be collected by centrifugation before they can be lysed in the procedure to prepare nuclei and histones. During this time, acetylation incorporation and turnover will occur, as judged by loss of acetate incorporation after “0”-min chase (Fig. 2). One can measure rates of turnover from the pattern of specific radioactivity if conditions a" @default.
• W2040722846 created "2016-06-24" @default.
• W2040722846 creator A5026387452 @default.
• W2040722846 date "1998-10-01" @default.
• W2040722846 modified "2023-10-13" @default.
• W2040722846 title "Dynamics of Histone Acetylation in Chlamydomonas reinhardtii" @default.
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