Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells

ARCHIVES

OF

BIOCHEMISTRY

Selective

ADOLF0

AND

BIOPHYSICS

174,

273-290

(1976)

Synthesis and Modification of Nuclear Proteins Maturation of Avian Erythroid Cells1

RUIZ-CARRILLO,

LAWRENCE

The Rockefeller

J. WANGH,”

University, Received

New November

York,

New

AND

York

VINCENT

during

G. ALLFREY

10021

17, 1975

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions Hl, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in earlyand midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small. Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis. Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activitpof erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled

The experiments to be described deal with changes in the composition and metabolism of chromosomal proteins during the process of red cell maturation. Erythroid cells were separated according to their stage of development and compared with regard to their capacity to synthesize different classes of histones and nonhistone nuclear proteins. Parallel studies of postsynthetic modifications of histone structure (by phosphorylation and acetylation) suggest correlations with chromatin as-

sembly and with changes in the functional state of the nucleus. Although recent studies of chromatin structure have given new insights into the fundamental role of the histones in its organization (l-141, little is known about the contribution of histone-modifying reactions to the assembly and structural dynamics of chromatin. Postsynthetic modifications of histones, including phosphorylation, acetylation, and methylation, often involve the basic regions of the polypeptide chains that would be expected to interact with DNA. How such modifications of histone charge and structure are employed to regulate histone-DNA interactions in different regions or functional states of chromatin is not clear, but numerous studies of gene activity in a variety of eukaryotic cells have established tem-

’ This research was supported in part by grants from the National Foundation-March of Dimes (l289), the United States Public Health Service (GM173831, the American Cancer Society (VC-114E), and the Rockefeller Foundation Program on Reproductive Biology. ’ Present address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge, England. 273 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

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poral or spatial correlations between rates of RNA and/or DNA synthesis and rates of acetylation or phosphorylation of different histone classes. Cells responding to hormones, mitogenic agents, nutritional changes, drugs, and carcinogens have been found to alter coordinately their rates of transcription and histone acetylation (for a review, see (Xi)), while histone phosphorylation varies during the cell cycle (16-20) and is modified by cyclic AMP (2123). Despite the numerous kinetic and spatial correlations between histone-modifying reactions and chromatin function, no simple quantitative relationships have emerged to relate the synthetic activity of the nucleus to the extent and rate of histone modification. The maturation of the nucleated erythrocytes of birds involves a progressive inactivation of the nucleus and provides a good model system to investigate corresponding changes in chromosomal protein structure. In a previous communication (24), we described the use of density-gradient centrifugation for the separation of duck erythroid cells at different stages of maturation. This work, together with that of other investigators (25-42), has provided data on changes in cellular and nuclear morphology, on nucleic acid synthesis, and on nuclear protein composition of the maturing red cells. In addition, our studies of histone synthesis in avian erythroblasts have revealed new levels of complexity in histone modifications occurring at the time of H4 synthesis.:’ Nascent H4 molecules are modified in the cytoplasm by phosphorylation of the amino-terminal serine residue and by acetylation of a lysine e-amino group. These two modifying groups are removed quickly when the newly synthesized H4 molecule enters the cell nucleus, but intranuclear enzymes can further alter the level of acetylation of the polypeptide chain (43). a In the new International Nomenclature for Histones adopted at the CIBA Foundation Symposium in London, April 1974 (E. M. Bradbury, CUBA Found. Symp. 28, l-4 (1975)), H4 replaces the older designations F2a1, IV, GRK and GAR, H3 replaces F3, III and ARE; H2a replaces F2a2, IIb, and ALK; H2b replaces F2b, IIb, and KSA; Hl replaces Fl, I and KAP; H5 replaces F2c, V and KAR.

AND

ALLFREY

The present work deals with the question of whether the intranuclear acetylation and phosphorylation of histones reflects the synthetic activity of the nucleus during the process of erythroid cell maturation. We have also considered the timing of histone synthesis in relation to DNA synthesis, with particular emphasis on the programming of synthesis of histone H5, an erythrocyte-specific histone which accumulates in the nucleus of the maturing cell. As contrasted with the predominantly structural role of histones in the organization of chromatin structure, the nonhistone nuclear proteins (NHNP)4 are known to include components that regulate chromatin function, affecting both the rate of RNA synthesis (44-47) and the nature of the RNA transcript (48-54). In considering the complexity and multiple functions of nuclear nonhistone proteins, it seems likely that components which have a regulatory role in transcription (e.g., in the control of globin-messenger RNA synthesis) interact with only a small fraction of the total DNA. They would be expected to occur in small amounts relative to the proportions of other nuclear proteins involved in chromosome structure and mobility, in nucleic acid processing, in ribosome assembly, in histone-modifying reactions, and in other aspects of nuclear activity. To permit the detection of small amounts of NHNP which may be involved in the regulation of transcription in erythroid cells we have used radioisotopic labeling procedures. The experiments to be described show that the synthesis of NHNP is gradually phased down as the cells mature, except for the synthesis of a stage-specific group of nuclear proteins in the late-polychromatic erythrocytes. MATERIALS

AND

METHODS

Incubation and separation of duck erythroid cells. Hemolytic anemia was induced by phenylhydrazine 4 Abbreviations used: NHNP, nonhistone nuclear proteins; NKMA, 130 rnM NaCl, 5.2 mM KCl, 7.5 mM MgCl,, containing 100 units penicillin G and 50 pg of streptomycin per milliliter; PF, pooled fraction; SDS, sodium dodecyl sulfate; EPE, early-polychromatic erythrocytes; SE, small erythroblasts, LE, large erythroblasts.

NUCLEAR

PROTEINS

administration to adult ducks and erythroid cells were obtained as described previously (24). A mixed erythroid cell population was prepared by mixing 6 vol of peripheral blood cells from anemic birds with 1 vol of immature cells from anemic bone marrow and 2 vol of mature peripheral red blood cells from untreated ducks. The cells were suspended in phosphate-free and leucine-free Eagle’s minimal essential medium supplemented with 10% (v/v) dialyzed duck serum (24). The final concentration was 5-6 X lox cells/ml. The suspension was preincubated at 40.5”C for 30 min at which time carrier-free l”2P]orthophosphate (New England Nuclear, Inc., Boston, Mass.) was added at a concentration of 150 pCi/ml. After 40 min of incubation, 50 &i/m! of L[4,5-“HJleueine of specific activity 6 Ci/mmol (Schwarz/Mann, Orangeburg, N. Y.) and 25 $X/ml of sodium 1l-‘4Clacetate of specific activity 45 mCi/ mmol (Schwarz/Mann) were added. Twenty minutes later the cells were chilled by the rapid addition of 10 vol of ice-cold NKMA (130 rnM NaCl, 5.2 mM KC!, 7.5 rnM MgCl, containing 100 units of penicillin G and 50 fig of streptomycin per milliliter, and sterilized by filtration through 0.45-pm Millipore tilters). Separation of cells was achieved by zonal isopycnic centrifugation on gradients of isotonic bovine serum albumin as described previously (24). After centrifugation, 50 fractions were obtained from the gradient and the cells in each fraction were characterized cytologically (55) and their RNA synthetic activity was determined (24). Cells differing in buoyant density and stage of maturation were collected into five pooled fractions (PF-1 to PF-5) prior to isolation of the nuclei and extraction of the nuclear proteins. Tests for effects of inhibitors ofRNA synthesis on histone acetylation. In attempts to determine whether RNA synthesis and histone acetylation are tightly coupled, the two reactions were compared in the presence and absence of inhibitors of RNA synthesis. Two systems were employed: intact peripheral erythrocytes from anemic ducks, and “permeabilized” cells from the same source. The cells were obtained as described previously (24). RNA synthesis in intact cells was measured in cell suspensions containing 1-2 x 10” cells/ml in Joklik’s medium (Gibco) supplemented with 10% (v/v) dialyzed duck serum. Aliquots of the suspension were incubated at 40.5”C in the presence of 200 pg/ml of Rifamycin AF/ 013 (a gift of Dr. L. Silvestri, Gruppo Lepetit, Italy) or 75 pglml of Actinomycin D (Calbiochem). After 30 min, 15,6-“Hluridine of specific activity 49 Ciimmol (SchwarziMann) was added to control and inhibited cultures at a concentration of 50 pCi/ml. Incubation was continued for 60 min. Aliquots of 10 ~1 were taken in duplicate at the desired times and diluted with 1 ml of ice-cold water. One volume of 10% trichloroacetic acid (v/v) was then added and the precipitates were kept at 0°C for 30 min. After filtra-

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tion through glass-fiber filter discs (GF/C, Whatman), the precipitates were washed with 15 ml of ice-cold 5% trichloroacetic acid, 70% ethanol, and absolute ethanol, in succession. Radioactivity was measured after digestion of the precipitates in Protosol (New England Nuclear)-4 M NH,OH (12/l, v/v) as described by Ruiz-Carrillo et ai. (56). The uptake and release of acety! groups of erythrocyte histones was measured in the presence and absence of 200 Fg/ml of Rifamycin AF/013 or 75 pg/ ml of Actinomycin D. After 30 min of incubation at 40,5”C, 1 mCi/ml of [methyl-‘Hlsodium acetate of specific activity 0.74 Ciimmol (SchwarziMann) was added and incubation was continued for 10 min. The incorporation of acetate was stopped by the addition of 10 vol of ice-cold Joklik’s medium containing 30 rnM sodium acetate (nonradioactive). The cells were harvested by centrifugation and washed three times in the same medium. An aliquot was withdrawn prior to measurements of radioactive acetate release under “cold-chase” conditions. The remainder of the cells were resuspended in prewarmed Joklik’s medium containing 10% duck serum in the presence or absence of 200 pgiml of Rifamycin AFI013 or 75 pg/ ml of Actinomycin D. Aliquots were withdrawn after 30 and 60 min of incubation at 40.5”C. All aliquots were quickly chilled, washed three times, and frozen at -80°C prior to isolation of the nuclei and extraction of the histones. RNA synthesis and histone acetylation were also compared in permeabilized red cells. Erythrocytes from anemic ducks were washed three times in 120 mM KC!, 5 rnM MgCl,, 7 mM 2-mercaptoethanol, 30 mM Tris-HC!, pH 7.5, and then lysed at 0°C in 2 vol of 10 mM KC!, 1.5 mM MgC12, 10 mM Tris-HC!, pH 7.5. After 3 min, the cells were dispersed, using a motor-driven Teflon-glass homogenizer with four strokes at 1700 rpm. One-tenth volume of 910 mM KC!, 50 mM MgCl,, 70 mM 2-mercaptoethanol, 230 mM Tris-HC!, pH 7.5, was then added. RNA polymerase assays were performed essentially as described by Wu and Zubay (57). Complete incubation mixtures of 250 /*-I contained: 225 ~1 of permeabilized cells in 88 mM KC!, 5.4 mM MgCl,, 6 mM 2-mercaptoethanol, 27 mM Tris-HC!, pH 7.5; 1 mM ATP; 0.275 rnM CTP; 0.275 mM GTP; 2.5 KCi of [5-“HlUTP of specific activity 15 Ciimmol (SchwarziMann); 10 mM phosphoenolpyruvate; 8 c(g of pyruvate kinase; and 1% (v/v) dimethyl sulfoxide. RNA polymerase inhibitors were tested at the following concentrations: Rifamycin AF/013, 100 /Iglml; a-amanitin (Calbiochem), 10 Kg/ml; Actinomycin D, 75 *g/ml. The inhibitors were added to the permeabilized cells at 0°C. After 10 min, the mixture of nucleoside triphosphates and energy-generating system was added and the suspensions were incubated for 1 h at 30°C. Aliquots of 10 ~1 were taken in duplicate for determination of RNA radioactivity as described above. Histone acetylation assays were carried out un-

276

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WANGH

der similar conditions, scaling up to a final volume of 3.0 ml and replacing the radioactive UTP with 0.275 mM nonradioactive UTP. The medium was supplemented with 0.045 mM Coenzyme A. Assays were started by exposing the cells to the inhibitors for 10 min at O’C, at which time the rest of the medium containing 100 &i/ml of lmethylSHlsodium acetate of specific activity 1 Ci/mmol was added. After 15 min of incubation at 3O”C, acetate uptake was stopped by the addition of 10 vol of icecold NKMA and the suspensions were centrifuged at 1lOOg for 5 min. The pellets were washed three times with NKMA and stored at - 80°C prior to isolation of the nuclei and extraction of the histones. Isolation of nuclei and extraction of nuclear proteins. Nuclei from cells separated by isopycnic centrifugation were purified by centrifugation in 2.5 M sucrose solutions as described by Ruiz-Carrillo et aE. (24). In studies of the effects of RNA polymerase inhibitors, the nuclei were purified by the Triton X100 method (43). Total histones were extracted in 0.25 N HCl. In some preparations, histones were selectively extracted, using 5% (v/v) perchloric acid to extract histones Hl and H5, and 1.25 N HClethanol (i/q, v/v) to extract histones H2a, H3, and H4 (58). Nonhistone nuclear proteins were extracted from the residues remaining after treatment with 0.25 N HCl by a modification of the sodium dodecylsulfate method of Elgin and Bonner (591, as previously described (24). Electrophoretic analysis of nuclear proteins and radioactivity measurements. Histone preparations were characterized by electrophoresis in 12 and 11% polyacrylamide gels containing 1.5 or 2.5 M urea, cross-linked with ethylene diacrylate (43). Gels 29 and 45 cm in length were routinely used to improve resolution of the modified histones (43). In some instances to further separate slowly-migrating histones, the leading bands of histone H4 were run off the gels. Histones were also separated as described by Panyim and Chalkley (601 in gels of 15% polyacrylamide containing 6.25 M urea except that a stock solution of 1% (w/v) TEMED was used (24, 43). All histone samples were reduced with dithiothreito1 in the presence of urea as described by RuizCarrillo and Allfrey (61). Densitometric tracings of the stained histone bands were obtained by scanning the gels at 615 nm in a Gilford 2000 spectrophotometer equipped with a Model 2410 linear transport device. The relative amounts of protein in particular bands were determined by electronic integration of the corresponding peaks in the denistometric tracing, using a Model 310 DuPont Curve Resolver (24, 62). Calculations of the specific activities of individual radioactive histone bands were carried out as follows: the gels were sliced at the interband minima and the radioactivity in each band was determined as described below. The amount of protein

AND

ALLFREY

present in each band was determined by integration of the peak areas in the densitometric tracing, using a Model 9864 Hewlett-Packard Digitizer programmed to sum the ordinate values at each i/lo-in. along the abscissa. In the case of band 4 (the triacetylated subfraction of H4, see Fig. 1) the area was determined in the best scans to be about 0.6 times that of band 3 (the diacetylated form of H4, see Fig. 1). This value was used to calculate the area of band 4 in other gels, assuming a coupled variation of the di- and triacetylated forms of H4 in cells at different stages of maturation. The integrated area corresponding to each histone band was converted to moles of protein by multiplying by the appropriate dye-binding coefficient as determined by Johns (63) and dividing by the molecular weight of the corresponding histone fraction as determined for calf and rabbit thymus histones (64-69). The modified histones were assumed to have the same dye-binding coefficients as the nonmodified forms. Dye-binding by histone H5 was taken as the average of the dyeaffinities of Hl and H2b, and the molecular weight of H5 was taken as 18,500 (70). The amount of radioactivity incorporated into each of the histone bands for each isotopic precursor was determined as follows: for measurement of [“‘PIphosphate incorporation, each band was sliced at the interband minima and placed in a vial containing 1 ml of concentrated NH,OH. The gel was hydrolyzed at 6570°C and the contents of the vial was evaporated to dryness. The residue was dissolved in 0.3 ml of Soluene (Packard, Inc., Downers Grove, 111.) and radioactivity was measured in a toluene-based scintillator (561. The incorporation of lgH]leucine and of [‘4C]acetate into individual histone bands was measured in slices from appropriate replicate gels, after oxidation of the slices as described previously (71). The counts per minute obtained for each of the isotopes was corrected appropriately for efficiency of counting, channel crossover, and decay. Histone synthesis is expressed as moles x 1Om’z of “H-labeled protein, as calculated from the amount and radioactivity of the protein in each band, the number of leucine residues in each histone, and the specific activity of the isotopic precursor. The specific activities of histones labeled with [14C]acetate and lY’P]phosphate are expressed as disintegrations per minute and counts per minute per lo-” mole of protein, respectively. The relative amount of acetate incorporated into the mono-,di-,tri-, and tetraacetylated subfractions of histone H4 was determined by electronic integration of the areas beneath the peaks of the [“HIacetate radioactivity profile after separation of the acetylated forms of H4 by electrophoresis in 45cm long polyacrylamide gels (431. The nuclear nonhistone proteins were analyzed in 10% polyacrylamide gels containing 0.1% sodium dodecylsulfate (SDS) as described previously (24).

NUCLEAR

PROTEINS

OF

Migration distances for individual bands were normalized to permit comparisons of different gels. Densitometric tracings of the stained bands were prepared, and ordinate values along scans 17 in. in length were read at O.l-in. intervals along the abscissa using a Model 9864 Hewlett-Packard Digitizer. Normalized scans were plotted with a Model 9862 Hewlett-Packard Plotter using internal absorbancy markers. Gels were sectioned in l-mm slices and the amount of radioactivity in each slice was measured (24, 561. The distribution of [“Hlleucine activity along the gel was plotted similarly, using internal radioactivity markers to permit normalization of the radioactivity profiles. RESULTS

Separation Different

of Avian Erythroid Stages of Maturation

Cells

at

Maturation of the avian erythrocyte is accompanied by diminishing size and increasing limitations of nuclear function. As the red cells mature, their buoyant density increases and this permits their separation according to the stage of development. Isopycnic zonal centrifugation (72) was employed as described previously (24) to separate duck erythroid cells into five fraction: (PF-1 to PF-5). Each of the fractions contained the following cell types as major components: PF-1, mature erythrocytes (ME); PF-2, late-polychromatic erythrocytes (LPE); PF-3, midpolychromatic erythrocytes (MPE); PF-4, early-polychromatic erythrocytes (EPE); PF-5, EPE plus small erythroblasts (SE) and large erythroblasts (LE). Histone Cells

Comp1exit.y

in, Avian

Erythroid

Histones were extracted in 0.25 N HCl from the purified nuclei of each of the cell types separated by isopycnic zonal centrifugation. The heterogeneity of the nuclear basic proteins was examined by electrophoresis in 12% polyacrylamide gels containing 2.5 M urea. The resulting banding patterns are shown in Fig. 1. The bands are identified as follows: bands 1 to 4 correspond to H4 molecules differing in their degree of acetylation; band 1 has no E-Nacetyllysine; bands 2, 3, and 4 contain 1, 2, and 3 l -N-acetyllysine residues, respectively. Tetraacetylated forms of histone H4 also occur, but in very small proportions

ERYTHROID

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(less than 1% of the total H4 molecules); they can be readily detected in histone samples labeled with radioactive acetate (see below, and also (43) and (71)). Other minor forms of H4 are also detectable in erythroblasts but not in more mature cell types. These appear as a result of cytoplasmic modifications and early nuclear processing of newly synthesized H4 chains. The modifications include phosphorylation of the amino-terminal serine residue and acetylation of a lysine residue, both of which lower the mobility of the parent polypeptide chain (see below and (43)). Band 5 on the gels is a basic protein which does not belong to the H4 group. Although it is frequently observed in a variety of whole histone preparations, its origin is uncertain, because partial degradation of histone H3 from calf thymus has been shown to yield a band of comparable mobility (71). Because the identity of band 5 is not clear, its proportions and labeling properties have been omitted from subsequent discussions of histone biosynthesis and postsynthetic modifications. Band 6 corresponds to the parental form of histone H2a; molecules in this band lack l -N-acetyllysine residues or phosphoserine residues. The main component in band 7 is the unmodified form of histone H2b. However, acetylated and phosphorylated forms of histone H2a also migrate in the region of band 7. They can be identified and distinguished from H2b by selective extraction procedures after labeling with the appropriate precursors (see below and (73)). Bands 8, 9, and 10 correspond to H3 molecules differing in their degree of acetylation; band 8 has no C-N-acetyllysine; bands 9 and 10 contain 1 and 2 l -N-acetyllysine residues, respectively. Minor modified forms of histone H2b also migrate in the region of band 8. The erythrocyte-specific histone, H5, occurs in two bands (11A and 11B in Fig. 1B) with the relative proportions 35 and 65%, respectively. The identification of these bands has been confirmed by selective extraction of histone H5 (and Hl) in 5% perchloric acid; electrophoretic analysis of the residual histones shows these two bands to be missing, together with 4-5 missing

278

RUIZ-CARRILLO,

Pooled-fraction A

WANGH

AND

ALLFREY

number

12345

Pooled-fraction

B

I

2

3

number 4

5

.J --I36 7 -b3A

2 t :! ,” 2

--/I4 I5

12 JB IA

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0 9 0 7 6

H3 H2b

tizo

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FIG. 1. Electrophoretic separations of histones and their modified forms from duck erythroid cells at different stages in maturation. The histones were extracted in acid from nuclei isolated from cells separated according to buoyant density. About 150 c(g of reduced whole histone from each cell population was appled to 0.6 x 29 cm cylindrical gels containing 12% polyacrylamide and 2.5 M urea. (A) The histone banding patterns are compared for mature erythrocytes (Pooled Fraction-l), late-polychromatic erythrocytes (PF-2), midpolychromatic erythrocytes (PF-31, early-polychromatic erythrocytes (PF-4), and a mixture of large and small erythroblasts plus some EPE (PF-5). Bands l-4 correspond to histone H4 molecules containing 0, 1, 2, or 3 r-l\r-acetyllysine residues, in that order. Band 5 contains an unidentified basic protein as well as the tetraacetylated form of H4. Band 6 corresponds to unmodified H2a. Band 7 is a mixture of unmodified H2b and small amounts of H2a molecules of diminished mobility containing acetyl or phosphate groups. Bands 8-10 correspond to H3 molecules with 0, 1, or 2 l N-acetyllysine residues, respectively. Band 11 corresponds to histone H5, and bands 12-15 correspond to different forms of histone Hl. (B) Resolution of the slower-moving histone bands was improved by extending the time of electrophoresis. Histone H4 molecules move out of the gel, but there is improved resolution of the H3 subfractions (bands 8-10); a doublet of H5 appears in bands 11A and llB, and band 13 is resolved into two components, 13A and 13B. The latter band disappears as the cells mature and it is not detectable in the mature erythrocytes of PF-1. The direction of migration is from top to bottom.

bands of lower mobility corresponding to different components of the Hl fraction. An electrophoretic doublet for histone H5 has recently been observed by others in embryonic chick histones (74). This heterogeneity is likely to be the result of allelic polymorphism which is known to result in a substitution of arginine for glutamine in H5 molecules of the chicken erythrocyte (75).

The proteins in bands 12 to 15 are different electrophoretic variants of histone fraction Hl. Interestingly, Hl from immature erythroid cells shows an additional complexity (bands 13A and 13B in Fig. IB) that is not observed in Hl from more mature red cells. Measurements of the relative concentrations of these two Hl subfractions in the different cell populations reveals that one of the changes occur-

ring in the Hl fraction at the later stages of cell maturation is the conversion of band 13B into 13A, presumably as a result of dephosphorylation. Histone Synthesis Cd Maturation

at Different

Stages

of

When mixed erythroid cell populations are incubated in the presence of \:‘H]leucine and the cells are subsequently fractionated according to their buoyant densities, the labeling of the histones is found to vary in the different cell fractions. Figure 2 shows the specific activities of each histone class (as separated by polyacrylamide gel electrophoresis) from cells at different stages of development. The results show that amino acid incorporation into all histones occurs mainly in the erythroblasts of cell fraction PF-5 and to a

NUCLEAR

PROTEINS

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ERYTHROID

279

CELLS

The exception is histone H5, which continues to be synthesized after the synthesis of the other histones has virtually ceased. This is evident in the histograms of Fig. 3, which show that l:‘H]leucine uptake into H5 persists in early- and midpolychromatic erythrocytes at rates higher than are observed for other histone fractions. It also appears that H5 can be synthesized in the absence of DNA synthesis, which does not occur at the MPE stage. The conclusion that there is no obligatory coupling of DNA synthesis and H5 synthesis was confirmed directly, by comparing the synthesis of different histone classes in cells in which DNA synthesis was blocked by hydroxyurea. Under these conditions the synthesis of all other his-

t

PF

no

FIG. 2. Comparative rates of synthesis of four major histone classes in duck erythroid cells at different stages of maturation. A mixed population of erythroid cells was incubated in the presence of 13H]leucine. The &ells were then separated in a density gradient and the nuclei were prepared from each cell type. Histones were extracted and separated electrophoretically. The specific activity of each histone class is plotted as a function of maturation-stage, beginning with the erythroblasts of PF-5 and progressing to the mature erythrocytes of PF-1. The [“HIactivities of histones at each stage of development are expressed relative to those of the erythroblast histones (taken as 100%). Note that the synthesis of histones H2a, H2b, H3, and H4 is largely restricted to the erythroblast stage, while H5 synthesis continues through the LPE stage. HI synthetic rates are not included because of globin contamination in the corresponding region of the gel.

lesser extent in PF-4, a fraction comprised largely of early polychromatic erythrocytes. The same cell fractions showed high levels of DNA synthesis (24). This result is in agreement with the findings of other investigators that there is a tight coupling between histone and DNA synthesis in a variety of eukaryotic cells (for a review, see (76)) and it confirms earlier reports that histone synthesis is largely restricted to the erythroblast stage of red cell differentiation (39, 74, 77).

nnn 13al3b SI ice

14

number

FIG. 3. Histogram indicating the electrophoretic distribution of newly synthesized histone molecules from avian red cells at successive stages in maturation. A mixed population of erythroid cells was incubated with lSH]leucine for 20 min and the cells were then separated on a density gradient. The nuclear histones were extracted and separated electrophoretically, using the conditions shown in Fig. IA for bands 1-7, and in Fig. IB for bands 8-15. Each histone band was analyzed for protein content and radioactivity. Vertical comparisons of the histograms indicate the differences in rate of synthesis of each histone band during red cell differentiation. Horizontal comparisons indicate the distribution of newly synthesized histone molecules in the modified and unmodified forms of each histone class.

280

RUIZ-CARRILLO,

WANGH

tones was suppressed but that of H5 remained unaffected (Ruiz-Carrillo, unpublished experiments). Appels and Wells (39) have reported that H5 is the only histone made in early polychromatic erythrocytes and our results agree with that finding except that we also observed continuing low levels of [“Hlleucine incorporation into Hj even in late-polychromatic erythrocytes. Our result is in accord with the continuing increase in the absolute amount of this histone fraction in the nuclei of maturing erythroid cells (24, 38, 40, 78). Newly synthesized H4 molecules are known to be phosphorylated and acetylated in the cytoplasm; the new molecules have a lower electrophoretic mobility until the modifying groups are removed in the nucleus (43). For this reason the electrophoretic analysis of erythroblast histones after a short “pulse” with l”H]leucine shows that an appreciable proportion of the newly synthesized H4 molecules have retarded electrophoretic mobilities (Fig. 3 and (43)). In contrast, nascent chains of histone H3 do not undergo corresponding modifications in the cytoplasm (43). The electrophoretie analysis of erythroblast histone H3 after a short “pulse” with [“Hlleucine shows that the newly synthesized molecules are distributed in proportion to the relative amounts of the three -major H3 subfractions (bands 8-10, Fig. 3). The most direct explanation of these divergent results is that H4 molecules, but not H3 molecules, are obligatorily modified during or shortly after synthesis (43). A disproportionate distribution of newly synthesized H4 molecules in low mobility forms also has been observed in developing trout spermatocytes by Louie and Dixon (79). The synthesis of histone Hl is indicated by [“Hlleucine uptake into bands 12-15 of the analytical gels (Figs. 1 and 3). This activity is higher at the early stages of cell maturation (PF-5) than in more mature erythrocyte populations, but precise quantitation has not been attempted because small amounts of highly radioactive hemoglobin also migrate in the Hl region of the gel.

AND

ALLFREY

Histone Phosphorylation Erythroid Cells

in

Maturing

Duck erythroid cells were separated after a period of [“zP]phosphate incorporation, and the histones of each cell fraction were analyzed by polyacrylamide gel electrophoresis. Figures 4 and 5 compare the specific 32P activities of each of the major histone classes at different stages of cell maturation. In many, but not all respects, the patterns of phosphorylation resemble those observed for [“Hlleucine incorporation in Fig. 2. All of the histone bands, with the exception of the unmodified forms of H4 and H2a (bands 1 and 6, respectively;

5

4

3 PF

2

I

no

FIG. 4. Comparative rates ofhistone phosphorylation and acetylation in erythroid cells at different stages of maturation. A mixed population of erythroid cells was incubated in the presence of 1”‘Plphosphate or lL4C1acetate, for 20 min and the cells were then separated on a density gradient. The nuclei were isolated from each cell type. The histones were extracted and separated electrophoretitally. The nrP or ‘“C activities of each histone class are plotted as a function of maturation-stage (expressing the data relative to that of the erythroblast histones of fraction PF-5, taken as 100%). Note the rapid decline in phosphorylation of all histone classes (except HZa) as cells progress beyond the erythroblast stage. Acetyiation persists in the more mature erythrocytes but at a greatly diminished rate.

NUCLEAR “PO,

lobeled

HISTONE

PROTEINS

FRACT!ONS

FIG. 5. Histogram indicating the patterns of histone phosphorylation at successive stages of red cell maturation. A mixed population of erythroid ceils was pulse-labeled with 13’Plphosphate for 20 min and the cells were then separated according to buoyant density. Nuclei were isolated from each cell layer and the histtmes were extracted and separated electrophoretically. Each histone band was analyzed for protein content and radioactivity. Vertical comparisons of the histograms indicate that histone phosphorylation (except for H2a) is largely restricted to the dividing cells engaged in histone synthesis (compare with Fig. 3). Horizontal comparisons show that histone phosphorylation diminishes the electrophoretic mobility of the corresponding polypeptide chain.

Figs. 1A and 5) show incorporation of [ “‘PIphosphate at early stages of maturation (i.e., in the erythroblasts and early polychromatic erythrocytes of fractions PF-5 and PF-4). Most histone fractions show little or no :‘“P-uptake in the more mature erythroid cells. This result is in accord with the observations of Seligy and Neelin (80) that IZi”P]phosphate incorporation into the histones of immature circulating erythrocytes of anemic geese exceeds that observed in the mature erythrocytes from normal birds. Studies of histone phosphorylation during the cell cycle of mammalian cells have indicated great complexity in the timing

OF

ERYTHROID

281

CELLS

and sites of phosphorylation of different histone classes (16-20, 81). For example, certain serine and threonine residues in histone Hl are phosphorylated at precise times in the cycle (20, 82) while other histones, such as H2a, seem to be phosphorylated irrespective of the DNA-replication state or histone-synthetic activity of the cell (18, 19, 81). We have found that the phosphorylation of H2a differs from that of other histones of avian erythrocytes in its persistence throughout maturation. (The electrophoretic detection of the modified H2a is complicated by the fact that phosphorylation reduces the net positive charge and electrophoretic mobility of the parent polypeptide chain; the phosphorylated H2a molecules comigrate with histone H2b (band 7; Fig. 1A and 5). However, H2a can be separated from H2b by selective extraction of the nuclei with acidethanol (58); subsequent electrophoretic analysis of the extracted H2a shows the unmodified main band (band 6) and the :i2P-labeled band of retarded mobility (band 7). Unlike most of the other histone bands, band 7 continues to show phosphate incorporation in more mature erythroid cells that have ceased DNA and H2a synthesis (Figs. 4 and 5). The phosphorylation of H2b has also been measured in red cells at different stages of maturation. The phosphorylated H2b molecules have a diminished electrophoretic mobility and comigrate with the unmodified form of histone H3 (band 8) (73). The uptake of [‘j’P]phosphate into H2b decreases rapidly after the erythroblast stage and it is not detectable in late-polychromatic or mature erythrocytes (Fig. 5). Similar limitations of histone phosphorylation to the early stages of red cell maturation are also evident for the erythrocyte-specific histone H5 and for the various forms of histone Hl (bands 11 to 15; Fig. 5). Histone Acetylation roid Cells

in

Maturing

Eryth-

Duck erythroid cells were incubated with radioactive acetate and separated by isopycnic centrifugation. The histones of each cell fraction were analyzed by polyacrylamide gel electrophoresis (Figs. 4 and

282

RUIZ-CARRILLO.

WANGH

6). The [14C]acetate-labeling patterns of the histone bands are strikingly different from those observed after labeling with [“Hlleucine or ]“‘P]phosphate. First, the vast majority of [‘“Clacetate counts incorporated into histones occurs in two fractions, H3 and H4. The predominance of acetylation of these fractions resembles that observed in adult rat liver (83) and in other cell types, and it is largely due to the formation of e-N-acetyllysine residues within the polypeptide chains (for a review, see (15, 84)). There are multiple sites of lysine acetylation in each of these histone classes. Mono-, di-, and triacetylated forms of H4 occur in bands 2, 3, and 4, respectively 14C acelote

labeled

HlSTOhE

FRKTIONS

lld Sllc?

;8

i3e

138

rbnlber

FIG. 6. Histogram indicating the patterns of histone acetylation at successive stages of red cell maturation. A mixed population of erythroid cells was incubated with l’%]acetate for 20 min and the cells were then separated according to buoyant density. Nuclei were prepared from each cell layer and the histones were extracted and separated electrophoretically. Each histone band was analyzed for protein content and radioactivity. Vertical comparisons of the histograms indicate the persistence of histone acetylation during red cell maturation. Horizontal comparisons show that acetylation decreases the electrophoretic mobility of the modified histone molecules.

AND

ALLFREY

(Figs. 1A and 6, and (43)). The uptake of radioactive acetate into these H4 bands occurs at all stages of cell maturation but diminishes as the cells grow older (Figs. 4 and 6). The situation is complicated in the erythroblasts of cell fraction PF-5, because some of the acetylation represents a cytoplasmic modification of nascent H4 chains (43). These acetyl groups are rapidly lost, and most of the uptake of [‘“Clacetate represents the postsynthetic modification of histones within the erythroid cell nucleus (43). The important point is that the acetylation of H4 persists in developing erythroid cells after its synthesis and phosphorylation have ceased. Similarly, the acetylation of histone H3 gives rise to subfractions of diminished electrophoretic mobility containing 1 or 2 l -N-acetyllysine residues, respectively (bands 9 and 10, Fig. 1A). Incorporation of [14C]acetate into these bands continues during red cell maturation and after H3 synthesis has ceased (compare Fig. 2 with Figs. 4 and 6). Hence, it can be concluded that the acetylation of histones H3 and H4 in mature erythrocytes is not coupled to histone synthesis nor to chromatin replication. However, the polypeptide chains of several histone classes (H4, H2a, and HI) are known to begin with N-acetyl serine residues (67, 85, 86). The acetylation of the serine residues occurs in the cytoplasm at the time of histone synthesis (87). Thus, the low levels of radioactivity found in bands 1, 6, and 12-15 probably reflect acetylation of the amino-terminal serine residues of the corresponding histone fractions. As expected, this labeling is most prominent in the immature erythroid cells that are capable of histone synthesis (Fig. 6). The erythrocyte-specific histone H5 also incorporates [‘%Jacetate (bands 11A and llB, Fig. 6). If the amino-terminal threonine residue of this histone is acetylated (88), rather than free (75)), then the uptake of ] “Clacetate represents terminal acetylation associated with H5 synthesis. The persistence of H5 acetylation during the more mature stages of erythroid cell development would be expected in view of the

NUCLEAR

PROTEINS

OF

ERYTHROID

283

CELLS

incorporated into the mono-, di-, tri-, and corresponding prolongation of H5 synthesis (compare Figs. 4 and 6 with Figs. 2 and tetracetylated forms of H4 was measured 3). We have not determined whether any after their separation on 45-cm long polyof the radioactive acetate in H5 occurs as E- acrylamide gels (24, 43). Table I summarizes the results of these analyses. ComN-acetyllysine. Some acetylation of histone H2b also oc- parisons of the percentage of the total curs in avian erythroid cells. It diminishes I:‘H]acetate present in each H4 subfraction as the red cells mature (Fig. 6). This is not show little or no change in distribution of due to terminal acetylation (histone H2b the label between 5 and 20 min, suggesting has an amino-terminal proline residue) some coordination in acetylation of the but represents the acetylation of lysine various subfractions. The specific activity of each band increases as its degree of residues at specific sites in the polypeptide chain (84, 89). As a result of the decrease in acetylation increases (Table I), but the rapositive charge, acetylated H2b has a di- tio of specific activities is not simply prominished electrophoretic mobility and it portional to the number of acetyl groups comigrates with unmodified molecules of present; i.e., the diacetylated form has histone H3 (band 8, Fig. 6). Acetylation of more than twice the [:$H]acetate activity of H2b also has been shown to occur in calf the monoacetylated form, and the triacetylated H4 band has more than three times thymus (go), trout testis (89, 91), avian the specific activity of the monoacetylated erythrocytes (92), and HTC cells (93). form (Table I). Interconversion of Acetylated Forms of We have considered a number of models Histone H4 to explain this disproportionate increase in As noted above, the acetylation of his- specific activity of H4 subfractions with tone H4 continues in erythroid cells that increasing acetyl content. The situation is are not engaged in H4 synthesis. It repre- complicated by the fact that the proporsents a postsynthetic modification of previtions of the various acetylated and nonaceously existing histone molecules. To study tylated forms of H4 differ in erythrocytes the parameters of acetylation of various at different stages of maturation (24). H4 subfraction, erythrocytes were pulse- Therefore, acetylation kinetics in a mixed labeled with [:‘H]acetate for periods rang- population of red cells reflect cell-to-cell ing from 5 to 20 min. The radioactivity differences as well as different rates of aceTABLE EVIDENCE

FOR RAPID ___.___

H4 subfraction

SEQUENTIAL

Total -

H4 (AC), H4 - (AC), H4 - (AC):; H4 - (AC),

ACETYLATION

l:‘HJacetate incorporated into each H4 ban@ at ~~ ~ 5 min 20 min” 34 28 22 16

I

OF HISTONE

35 26 23 16

-~

H4 MOLECULES

IN

Fraction of total H4 present in each band

Specific

~26.0 5.5 3.5 ND

~~~~~

~

AVIAN

CELLS

~~. -. Specific activity ratio”

.____

~~.

ity

1.3 4.9 6.4 -

ERYTHROID

activ-

~~1.0

3.8 4.9

~ -~ ~~-~~ -. U Duck erythrocytes were incubated in the presence of 1 mCi/ml of I”Hlacetate (Na) as described in Materials and Methods, and the nuclei were isolated. The histones were extracted and separated by electrophoresis in 45cm gels containing 12% polyacrylamide and 2.5 M urea. The /“HIacetate content ofeach H4 band was obtained by electronic integration of the areas beneath the respective peaks in the radioactivity profile of the gel. ’ Average of two determinations. ’ The protein content of each H4 band was determined by electronic integration of the appropriate peaks in the densitometric tracings of the electrophoretic banding patterns (24). ” The specific activity ratios are expressed relative to that of the monoacetylated (H4 (AC),) form. ’ The relative amount of subfraction H4 - (AC), in avian erythrocytes is too small to permit accurate determinations.

284

RUIZ-CARRILLO,

WANGH

tate turnover in each H4 subfraction. Moreover, chromatin fractionation and autoradiographic studies of [“HIacetate uptake have shown that acetylation involves only a fraction of the total chromatin in a given nucleus (94-96). Therefore, calculations based on the average specific activity of all the histone H4 in the nucleus may be misleading. A further complication is that the activity of histone deacetylating enzymes increases during red cell maturation (92). Although the present results suggest that histone acetylation is sequential and that acetate uptake is more likely to occur on a form which is already acetylated, more definitive studies of the kinetics of H4 acetylation and deacetylation in homogeneous cell populations are required before the interconversion of H4 subfractions can be quantitatively analyzed. Tests for Coupling lation and RNA

between Histone Synthesis

Acety-

Rates of acetylation of histones H3 and H4 decrease during red cell maturation (Figs. 4 and 6) and the proportion of the acetylated molecules also diminishes (24). These changes do not coincide with rates of chromatin replication (as judged by the synthesis of DNA or the corresponding histones) because replication is limited to the erythroblast stages of development. However, the fivefold decline in acetylation of histones H3 and H4 during maturation TABLE EFFECTS

OF INHIBITORS ___--

AND

(Fig. 4) appears to correlate with a progressive decline in RNA synthesis (24-27). Rates of transcription in mature duck erythrocytes are only one-eighth as high as those measured in erythroblasts and early-polychromatic erythrocytes (24). In order to test whether the acetylation of histones is directly coupled to transcription we have compared the effects of inhibitors of RNA synthesis on the uptake of [“Hluridine into RNA and on the uptake and turnover of I”H]acetyl groups in histones H3 and H4. Intact peripheral erythrocytes from anemic ducks were labeled as described in Materials and Methods in the presence and absence of rifamycin AF/013 or actinomycin D. The results (Table II) show that both antibiotics effectively inhibit RNA synthesis without appreciable effects on the acetylation or deacetylation of histones H3 and H4. Other experiments comparing RNA synthesis and histone acetylation in permeabilized erythrocytes are summarized in Table III. Actinomycin D, rifamycin AF/013, and a-amanitin all inhibit the utilization of [“H]UTP for RNA synthesis without effect on ]:‘H]acetate uptake into the histones. Thus, it is clear that the acetylation of histones H3 and H4 can occur independently of RNA synthesis, and that the modification of lysine residues in these histones is not tightly coupled to the movement of the RNA polymerases along the chromatin II

OF RNA SYNTHESIS ON [:‘HIURIDINE ACETYLATION IN INTACT AVIAN

Inhibitor

Control (no inhibitor) Rifamycin AFiOl3 (200 pglml) Actinomycin D (75 pg/ml)

]:‘H]uridine corporation” (e)

in-

ALLFREY

INCORPORATION INTO ERYTHRO~D CELLS

[“H]acetate incorporation into histones” __ H3 (%) H4 (%l ___~____~ 100 100 100 20 100 109 5 104 95

RNA

AND

ON HISTONE

[:‘H]acetate release from histones’ H3 t%r)

H4 f7r)

50 47 59

15 18 13 -__

li L”H]uridine incorporation into the RNA of intact avian erythroid cells was measured in the presence and absence of the indicated inhibitors as described in Materials and Methods. Results are expressed relative to the uptake in control cells. The levels of inhibition are constant for periods of incubation from 10 to 60 min. h [:‘H]acetate incorporation into the histones was determined after labeling intact cells for 10 min. The results are expressed relative to the activity of the histones in control cells. The figures represent the average of two determinations. ’ The cells were pulse-labeled with L:‘H]acetate for 10 min and then incubated under cold-chase conditions for an additional 60 min. The release of [“HIacetate from H3 and H4 is expressed relative to the activity of each histone at the lo-min time point.

NUCLEAR TABLE EFFECTS

OF INHIBITORS

1JHJUTP ACETYLATION

OF

III OF

INCORPORATION IN

PROTEINS

RNA

SYNTHESIS

AND

PERMEABILIZED

ON

ON

HISTONE

AVIAN

ERYTHROID

-1 “H]UMP incorporated into RNA” (%‘r)

1“Hlacetate incorporation into histones” (%)

ERYTHROID

285

CELLS

ods. The proteins were then analyzed by electrophoresis in SDS-polyacrylamide gels. Figure 7 shows the molecular weight distributions and [“Hlleucine activities of

CELLS

_~~....

Inhibitor

--_.Control (no inhibitor) Actinomycin D (75 pgiml) Rifamycin AFiOlY (200 ~gimll a-Amanitin (10 kg/ml) _-.

100 100 104

100 7 42 23

~.

100

~-

‘I Permeabilized duck erythrocytes were incubated with I:‘H]UTP in the presence or absence of the indicated inhibitors of RNA synthesis as described in Materials and Methods. The uptake of the isotopic precursor into RNA is expressed relative to that observed in the cells without added inhibitors. ” The incorporation of I”Hlacetate into the total histones of the permeabilized cells was measured after a 15min incubation. The results are expressed relative to the specific activity (dpm/mg) of the histones in control cells.

template. This is not surprising if the template-active reiions of the chromatin are devoid of histones. It remains to be seen whether acetylation of the histones is a prerequisite to the “opening-up” of DNA in transcriptionally active regions of the chromatin ias suggested by numerous experiments on the timing of histone acetylation and RNA synthesis during gene activation (15, 95)) and whether histone deacetylation correlates with the assembly of template-inactive histone-DNA complexes. Stage-Specific Synthesis of Nuclear Nonhistone Proteins during Erythrocyte Maturation

Both the amount and the relative proportions of nuclear nonhistone proteins are known to change during the maturation of avian red cells (24, 97-99). The present experiments were designed to compare the rates of nonhistone protein synthesis in erythrocytes at different stages. After labeling with [:‘Hlleucine, the cells were separated by isopycnic centrifugation and the NHNP were extracted from each cell fraction as described in Materials and Meth-

i )0

0

./jG 0

.-

h

..i i-

2-=i

20

40

Running

60 Dtstance

so

100

J

o

120

(mm)

FIG. 7. Patterns of synthesis of nuclear nonhistone proteins 1NHNP) in avian red cells at successive stages in maturation. A mixed population of erythroid cells was pulse-labeled with [“Hlleucine for 20 min and the cells were then separated in a density gradient. Nuclei were isolated from each cell layer in the gradient, The nonhistone proteins were extracted from the dehistonized nuclei in 1% SDS at pH 8.0. About 200 pg of protein was applied to 0.6 x 10 cm cylindrical gels containing 10% polyacrylamide and 0.1% SDS at pH 7.4. The solid lines indicate the densitometric tracings of the stained protein bands. The dashed lines show the distribution of [“Hlleucine activity in the NHNP. Molecular weights are indicated in the scale above each panel. Note the progressive decrease in the content of NHNP of molecular weights greater than 20,000 during red cell maturation. The synthesis of these proteins is also curtailed except for a selective labeling of particular peaks in late polychromatic erythrocytes (PF-2).

286

RLJIZ-CARRILLO.

WANGH

the protein bands at each stage of erythroid cell maturation. Under these conditions, proteins with apparent molecular weights ranging from 9500 to 140,000 can be separated. (The complexity of the NHNP complement of the erythrocyte nucleus is underestimated by these methods, because proteins of similar molecular weight or varying degrees of phosphorylation would not be resolved.) In accord with previous findings (24, 97-99) the electrophoretic analyses indicate that the nonhistone protein complement of avian red cell nuclei is altered and diminished during cell maturation. The synthesis of these proteins is also stage-dependent. Significantly, incorporation of [:‘H]leucine into NHNP is observed at all stages of maturation, even in the mature erythrocytes of cell fraction PF-1. Therefore, unlike the synthesis of most histone fractions, the synthesis of the nonhistone nuclear proteins is not coupled to DNA synthesis. Studies of other cell types, including HeLa cells (100-103) and WI-38 fibroblasts (104) have also shown that the synthesis of NHNP proceeds in phases other than the S-phase of the cell cycle. Several quantitative and qualitative conclusions can be drawn from the radioactivity profiles shown in Fig. 7. The erythroblasts of cell fraction PF-5 have a higher overall level of NHNP synthesis than do more mature erythroid cells, but there is a selective synthesis of nuclear nonhistone proteins in the late-polychromatic erythrocytes (LPE) of cell fraction PF-2. A transient synthesis of proteins in the molecular weight range 54,000 to 130,000 is observed at the LPE stage but not in the mature erythrocyte (Fig. 7). (Nuclei of the latter fraction show a highly radioactive peak in the region where globin chains migrate; this probably represents the transit of newly synthesized hemoglobin across the nuclear envelope (29, 105).) The synthesis of NHNP at successive stages of maturation was further analyzed by constructing longitudinal plots of the ratios of \“H]leucine specific activity of individual protein bands from cells at consecutive stages of maturation. This type of

AND

ALLFREY

analysis visualizes changes in the relative rates of synthesis of individual protein bands as fluctuations in the ratio value, while constant ratios indicate the synthe: sis of the corresponding proteins is coordinately regulated. The ratios in Fig. 8 reveal that extent for the dramatic svnthesis of a set of high-molecular-weight nonhistone nuclear proteins in LPE cells (and the globin peak in mature erythrocytes), changes in the synthesis of all other NHNP occur in concert. In the progression from the erythroblasts of fraction PF-5 to the midpolychromatic erythrocytes of fraction PF-3 there are no abrupt changes in the synthesis of any nuclearprotein>long the entire length of the gel. However, in every case, the ratio of [CsH]leucine activities is less than 1, indicating that the rate of synthesis of the NHNP declines progressively with age. The decreasing rates of NHNP synthesis during maturation, coupled with a sixfold decrease in their rela-

FIG. 8. Evidence for selective synthesis of nuclear proteins in late polychromatic erythrocytes. The distribution of newly synthesized NHNP molecules in erythroid cells at different stages of maturation (Fig. 7) was further analyzed by computer plotting of the ratios of LSHlleucine specific activities of corresponding protein bands from cells at consecutive stages of development. Note that the ratios are essentially invariant for most components of the NHNP, indicating a coordinate regulation of their rates of synthesis in erythroblasts, early- and midpolychromatic erythrocytes. However, there is a burst of NHNP synthesis in the LPE cells which selectively elevates the ratios of [3H]leucine uptake at particular regions of the gel.

NUCLEAR

PROTEINS

OF

ERYTHROID

CELLS

287

For most histones, phosphorylation appears to be limited to the erythroblast stage. In the case of histone H4 phosphorylation of the terminal serine residue is an early event in its biosynthesis (43). Phosphorylation of histones in cells that are capable of histone and DNA synthesis also has been observed in spermatogenesis of the trout (84, 106). Histone H2a is exceptional; it continues to be phosphorylated at later stages of red cell maturation. Similar differences between H2a phosphorylation DISCUSSION and that of other histone classes have been The development of erythroid cells, like noted in cultured mammalian cells (18, 19, other specialized cell types, involves at 81). Acetylation of the histones of avian red least two distinct processes. The first recells persists after histone and DNA synquires the commitment of a multipotenthesis have ceased. There is a fivefold drop tial stem cell to differentiate as an erythroid cell. The second process is the proin rates of histone acetylation as the cells and EPE grammed differentiation of the now com- progress from the erythroblast mitted state. The results presented in this stages to mature erythrocytes. An eightpaper and in preceding studies of avian red fold decrease in RNA synthetic capacity cells (24, 39, 77, 97-99) constitute a partial occurs over the same period of maturation. description of changes occurring in nuclear Although the results suggest some form of proteins during successive stages of erythcoupling between the two processes, experrocyte development. Since the early studiments using inhibitors of RNA synthesis ies of Swift and his co-workers (26) and of show that acetylation of the histones does Cameron and Prescott (25) it has been not require simultaneous RNA synthesis, clear that maturation of avian erythroid and it follows that acetylation and trancells involves a progressive inactivation of scription are not tightly coupled. This is nuclear function. DNA synthesis ceases not unexpected in view of numerous indiafter the erythroblast stage (24, 36) and cations that acetylation of the histones RNA synthesis declines rapidly as the cells precedes increases in RNA synthesis durgrow older (24-27). ing gene activation in a variety of cell Our work shows that the erythroblasts types (15, 95). Further studies are necesare the only red cells that synthesize all sary to determine whether acetylation and the major histone classes, and that the deacetylation of erythrocyte histones are synthesis of histones Hl, H2a, H2b, H3, part of the mechanism for controlling the and H4 is coupled to DNA synthesis. Syn“availability” of DNA for transcription thesis of the erythrocyte-specific histone during red cell development. H5 continues after synthesis of the other Acetylation of histone H3 and H4 inhistone classes has stopped and it can be volves multiple sites at which lysine residetected in early (39) and late-polychrodues are modified, and molecules which matic erythrocytes (Fig. 2). This result is differ in their content of e-N-acetyllysine in keeping with the accumulation of H5 in can be separated. Studies of the kinetics of the nucleus of maturing red cells (24, 40, 1:‘Hlacetate uptake into each histone 78) and with the increase in condensation subfraction strongly suggest that acetylaof the chromatin (35, 41). The fact that tion is a sequential process (Table I). The inhibitors of DNA synthesis (such as hy- fact that multiacetylated forms of histones droxyurea) block the synthesis of all hisH3 and H4 do not accumulate reflects the tones except H5, suggests that different activity of the histone deacetylating encontrol mechanisms regulate the tranzymes. The activity of these enzymes (92) scription of the H5 message. and the balance between acetylated and

tive amounts (24, 97), indicates that these proteins are subject to continuous turnover. The burst of synthesis of specific nonhistone nuclear proteins at the LPE stage is transient and it does not result in a detectable accumulation of these proteins in the mature erythrocyte. Whether these particular stage-specific proteins play a role in the suppression of nuclear activity in the mature erythrocyte remains to be determined.

288

RUIZ-CARRILLO.

WANGH

nonacetylated forms of the histones change during red cell maturation (24). Numerous studies have pointed to the nonhistone nuclear proteins as involved in genetic control mechanisms (44-54). Nuclear nonhistone proteins appear to control the transcription of the globin gene (54, 107, 108) and are also likely to be involved in its suppression. As erythroid cells mature, the nonhistone protein content of the nucleus diminishes (24,97) and the rates of synthesis of the NHNP generally decrease. However, there is a burst of synthesis of selected nuclear proteins at the LPE stage. This phase-specific event, involving the synthesis of small amounts of protein, suggests that some of the proteins produced at that time participate in the ensuing shut-down of nuclear function.

AND

histones Plenum, 16. BALHORN,

The authors wish to thank L. Morioka and J. Feinsod for their assistance. We are indebted to Dr. F. R. Kramer for the use of equipment and for his enthusiastic help in computer programming. REFERENCES 1. VAN HOLDE, K. E., AND ISENBERG, I. (1975) AC&. Chem. Res. 8, 327-335. 2. VARSHAVSKY, A., AND GEORGIEV, G. P. (1972) Biochim. Biaphys. Aeta 281, 669-674. 3. OLIN& A. L., AND OLINS, D. E. (1974) Science 183, 330-332. 4. D’ANNA, J. A., AND ISENBERG, I. (1974) Biochemistry 13, 4992-4997. 5. KORNBERG, R. D., AND THOMAS, J. 0. (1974) Science 184, 865-868. R. D. (19’74)Science 184,868-871. 6. KORNBERG, 7. WEINTRAUB, H., PALTER, K., AND VAN LENTE, F. (1975) Cell 6, 85-110. H. G., AND MCCARTHY, B. J. (1975) 8. MARTINSON, Biochemistry 14, 1073-1078. 9. CHALKLEY, R. (1975) Biochem. Biophys. Res. Commun. 64, 587-594. 10. SOLLNER-WEBB, B., AND FELSENFELD, G. (1975) Biochemistry 14, 2915-2920. 11. BALDWIN, J. P., BOSELEY, P. G., BRADBURY, E. M., AND IBEL, K. (1975) Neture ilondon) 253, 245-249. 12. OLINS, A. L., CARLSON, D., AND OLINS, D. E. (1975) J. Cell Biol. 64, 528-537. 13. OUDET, P., GROSS-BELLARD, M., AND CHAMBON, P. (1975) Cell 4, 281-300. 14. LANGMORE, J. P., Wooley, J. C. (1975) Proc. Nat. Acad. Sci. USA 72, 2691-2695. 15. ALLFREY, V. G. (1971) in Histones and Nucleo-

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