Volume 5 Number 5 May 1978
Nucleic Acids Research
Nucleosome-associated proteins and phosphoproteins of differentiating Friend erythroleukemia cells
Jeffrey Neumann, Robert Whittaker,* Barbara Blanchard and Vernon Ingram
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 8 March 1978 ABSTRACT
Mononucleosomes derived from brief digestion of uninduced Friend cell nuclei with micrococcal nuclease contain a set of non-histone chromosomal proteins which are partly or altogether missing in the oligomeric nucleosomes. On the other hand, the latter contain a protein of Mr 190,000 not seen in the mononucleosomes. Longer digestion removes most of these non-histone proteins, excepting the Mr 190,000 protein. Brief digestion of nuclei from Friend cells induced by DMSO or by n-butyrate removes most of the non-histone proteins from the nucleosomes, as did the prolonged digestion of uninduced nuclei. The Mr 190,000 protein remains, while a protein of Mr 27,000 is increased. The rate of phosphorylation of histone Hl associated with mononucleosomes was 3 to 4-fold greater in cells induced with DMSO. The major phosphoprotein and most of the other phosphorylated non-histones were modified at the same rate in control and induced cells. However, a Mr 95,000 protein was less phosphorylated in the induced cells. INTRODUCTION The repeating pairs of DNA wound histones H2A, H2B, region of "linker"
subunit of chromatin, the nucleosome, consists of 140 base about a central octamer consisting of two of each of the H3 and H4 (1-5). Monomeric nucleosomes are separated by a DNA containing 30-60 base pairs, depending on cell type and degree of transcriptional activity (6-7). Actively transcribed gene sequences remain associated with nucleosomes (8-10) after micrococcal nuclease (EC 3.1. 4.7.)digestion of nuclei. Incubation with pancreatic DNase I (EC 3.1.4.5), however, preferentially digests active or tissue-specific DNA sequences, both from intact chromatin and from chromatin previously digested with micrococcal nuclease (11-13). This differential sensitivity may be due to the presence or absence of certain non-histone chromosomal proteins or to the modification of Hl or the core histones in stretches of the genome where RNA synthesis is occurring. Non-histone proteins are enriched in "active" chromatin (15), as are phosphorylated forms of these proteins (16). It has been suggested that the
C Information Retrieval Umited 1 FalconbwgCourt London W1V 5FG England
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Nucleic Acids Research non-histones confer transcriptional specificity on reconstituted chromatin (17). Although the occurrence and distribution of non-histones and their phosphorylated derivatives in isolated mononucleosomes has recently been reported (18-20), their role is unknown. Friend erythroleukemia cells provide a useful system in which to investigate changes in nuclear proteins which presumably occur during the induction of genetic reprogramming brought about by agents which induce differentiation (21,22). We report here the distribution of histones, non-histone proteins and their phosphorylated forms in chromatin subunits of uninduced and induced Friend cells. METHODS Cell culture and Labelling. Friend erythroleukemia cells (clone 745-PC-4) were cultured as previously described (23). For induction experiments DMSO was used at 210 mM and sodium n-butyrate at 1.5 mM. Labelling with 9P04 was accomplished by incubating cells at 2 x 107/ml in conditioned medium for 1 hr in the presence of 100-200 uCi/ml32P-orthophosphate. (New England Nuclear, Boston, MA) Nucleosome Preparation. Purified nuclei were suspended at 5 x 107/ml in a digestion buffer (0.34M sucrose/0.06M KC1/0.015M NaCl/0.015M 2-mercaptoethanol/0.015M Tris-HCl (pH 7.4) containing 0.11 mM spermine and 0.38 mM spermidine (1). They were preincubated for 3 min at 370C with 2mM CaC12 and then digested with 96 units/ml of micrococcal nuclease (E.C. 3.1.4.7, Worthington Biochemicals). The reaction was stopped by placing the nuclei on ice and immediately bringing the suspension to 4 mM Na2EDTA. Centrifugation at SOOxg for 5 minutes yielded a supernatant fraction S1. The nuclei were then extracted for 30 to 45 minutes with 0.2 mM Na2EDTA, pH 7, and the nucleosome-containing supernatant (S2) was recovered after centrifuging the suspension at 12,000xg for 5 minutes. The S2 fraction was layered directly on a 5-28.8% linear sucrose gradient containing 1 mM Na2EDTA, pH 7, and centrifuged at 140,000 xg for 22 hours. Gradients were analyzed with a Gilford Model 240 spectrophotometer and fractions corresponding to monomers, dimers plus trimers, and higher order oligomers were combined. The pooled fractions were dialyzed overnight against 0.1 mM phenylmethylsulfonyl fluoride and then lyophilized. The dried samples were dissolved in 0.5M NaCl/0.05M MgC12/0.OlM Tris-HCl (pH 7.5) and digested for 15 min at 370C with 3000 units/ml each of pancreatic DNase and pancreatic RNase (E.C. 3.1.4.22.) to remove labelled nucleic acids. They were then dialyzed exhaustively against 0.05M Tris-HCl, pH 8.6, containing 0.1% SDS. 1676
Nucleic Acids Research Electrophoresis. Analysis on 5-15% acrylamide gradient slab gels in the presence of SDS and autoradiography of gels containing 9P-labelled proteins was done as previously described (23). Specific radioactivity of phosphoproteins was determined from densitometric scans of Coomassie Blue staining patterns and autoradiograms derived from the same gels. The calculated specific radioactivities referred to in RESULTS are the ratios of the area under the autoradiogram scan (9') to the area under the staining profile (protein). This method was used both for total protein in a fraction and for specific well-resolved proteins. Histones were extracted from sucrose gradient fractions with 2M NaCl0.4N H2S04. They were precipitated with 25% (w/v) trichloroacetic acid, dialyzed exhaustively against distilled water and lyophilized. They were dissolved in SM urea-0.9N acetic acid-0.38% (v/v) Triton DF-16 (Sigma Chemical Co., St. Louis, MO) and electrophoresed on 15% acrylamide gels as described by Alfageme et al. (24). Non-denaturing gels for the analysis of nucleosome fractions were run according to Varshavsky et al. (25). Aliquots of the gradient fraction corresponding to the monomer peak were applied directly to the gels. Following electrophoresis the gels were scanned at 260 nm. DNA was isolated from nucleosome fractions by the method of Maniatis et al. (26). Purified DNA was electrophoresed on 6% polyacrylamide slab gels containing 98% formamide. RESULTS Nucleosome-associated Proteins. We determined the kinetics of micrococcal nuclease digestion of Friend cell nuclei from uninduced cells. Sucrose gradient analysis was performed on the S2 nucleosome fraction derived from nuclei digested for times varying from 30 sec to 10 min (0.7% to 23% acidsoluble DNA). In addition, DNA was prepared from the S2 fraction and from monomers isolated after digestion for 2 or for 10 min. The purified DNA was fractionated on denaturing gels. Both the sucrose gradients and the denaturing gels of isolated DNA revealed the subunit profiles expected from the literature (2,27). Mononucleosomes and the corresponding monomer lengths of DNA accumulated after longer digestion times at the expense of larger fragments. After 2 min of digestion, DNA fragments isolated from mononucleosomes were on the average somewhat longer and more heterodisperse than those obtained after 10 min, presumably due to the presence of internucleosomal linker DNA. Non-denaturing gels (Figure 1A) showed that the monomer fraction from a 2 min digest contained a considerably higher proportion of the 170 base pair, Hl1677
Nucleic Acids Research
E
i
12mim
A260
0~~~~~~~A 5
10mi
()Electrophoresis
~ +()Elctrophoresis
W +
Figure 1.
(A) Electrophoresis in 5% non-denaturing polyacrylamide gels of S2 nucleosome fractions derived from 2 min and 10 min digests (7.5% and 18.596 acid-soluble DNA, respectively) . (B) Electrophoresis as in (A) of monosomes isolated from sucrose gradients after 10 min digestion (16.7% acid-soluble DNA). The gel was scanned at 260 nm to locate the DNA. After staining with Coomassie Blue, the gel was scanned again at 550 nm to detect the protein.
contaning mononucleosome species, corresponding to the MN 2 band described by Bakayev et al. (28), than monomers derived from a longer digest. The Coomassie Blue staining pattern superimposed on the A260 profile of 10 min mononucleosomes (Figure 1B) establishes that the proteins (presumably both histones and non-histones) are bound to the DNA and not merely tailing from the top of the gradient into the mononucleosome band during centrifugation in the sucrose gradient. The mononucleosomes from a brief digestion display a similar correspondence of ASS0 and A260 profiles. We show the 10 min pattern because the digestion mixture after the longer incubation would be expected to have a greater amount of protein from the top of the gradient which might artifactually co-sediment with the monomer peak. Note the absence of low molecular weight staining material. The proteins in fractions S1 and S2 from uninduced cells and portions of the sucrose gradient containing monomers, di- and trimer, and oligomers were analyzed by electrophoresis in SDS-containing gels (Figure 2). Samples of intact nuclei and fractions from the tops and bottoms of the gradients were also examined (data not shown). Sham-digested nuclei released negligible amounts of DNA or protein into either fraction S1 or S2. Quantitation of the area under densitometric tracings showed release of 1678
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Nucleic Acids Research
A_
AiO
I
9
ois g5-x4y
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H3, H41C_
|S1.
_
__
9
S2 mono di/tri oligol Si S2 mono di/tri oligo| l 2minute digest | Ominute digest
Figure 2. SDS-polyacrylamide gel electrophoresis of the protein in fractionated chromatin subunits. S1, S2, and pooled sucrose gradient fractions
corresponding to the indicated nucleosome fractions obtained from 2 min and 10 min digests (7.5 and 18.5% acid soluble DNA) were separated by electrophoresis as described in Methods. Each lane was loaded with one A260 unit of chromatin. The gel was stained with 0.1% Coomassie Blue and photographed using a green filter. Molecular weights of the indicated proteins were determined by comparison with a standard curve constructed with the following markers: soybean trypsin inhibitor (21,500), bovine serum albumin (68,000) and the subunits of RNA polymerase from E. coli (39,000, 155,000 and 165,000). Tne major bands, with their calculated molecular weights, are designated: A(180-190,000), B(140-150,000), C(125-130,000), D(ll0115,000), E(95-100,000), F(63-65,000), G(41-43,000), I(36-38,000), J(3335,000), K(32-33,000), L(26-28,000) and M(20-22,000). about one-third of the total extractable non-histone proteins (S1 plus S2) With further digestion into the S1 fraction after only 2 min of digestion. there was a slight increase in the amount of protein in this fraction, resulting in only small differences between the protein patterns of the S1 fraction after 2 or 10 min of digestion. The amount of histone associated with fraction S1 was variable. The non-histone proteins in the S1 fraction were probably released as free proteins or as very small deoxynucleoprotein fragments (subnucleosomes) (28). The S1 fraction, with an ionic strength of approxi1679
Nucleic Acids Research mately 0.1M, would also contain nucleoplasmic proteins and ribonucleoprotein particles. The patterns of non-histone proteins in the S2 fractions at the two time points were similar, but different from the Si patterns. The S2 fraction contains proteins that are complexed with nucleosomes and perhaps also free proteins released during the low salt extraction. Because of the likely presence of free proteins in the S2, we also examined gradient purified mono- and oligonucleosome fractions. In contrast to dimers and oligomers, the mononucleosomes from a 2 min digest were strikingly enriched in non-histone proteins. While all nucleosome fractions contained the expected complement of core histones, the mononucleosomes reproducibly had greater amounts of certain non-histone proteins, which we have for convenience designated as B, C, D, E, and I. Protein E (Mr 95-100,000) is interesting, because it was only a minor component of fraction S2. All of these non-histone proteins were greatly reduced in or absent from the multimer fractions. Conversely, protein A (Mr 190,000) was more prominent in the oligonucleosomes and in fact has never been detected in mononucleosomes. Further digestion with micrococcal nuclease for a total of 10 min produced a nucleosome population with a low content of non-histone proteins; only proteins I and M remained prominent in the mononucleosome fraction. The missing proteins were presumably released from the nucleosomes, since they were somewhat enriched in the top fraction of the sucrose gradient (data not shown). From quantitative measurements we deduce that they must also have precipitated in non-extractable form in the residual nuclei. Protein A, however, was not found in the top fraction, nor did it accumulate in the Sl fraction or with mononucleosomes. It was the most prominent non-histone protein associated with the di- and oligonucleosomes from 10 min digests. Since micrococcal nuclease generates early in digestion a population of mononucleosomes which is enriched in non-histone proteins, we thought it would be of interest to compare this chromatin-bound subset of nuclear proteins in uninduced Friend cells with those in DMSO-treated cells. Nuclei of control and 48 hr DMSO-treated cells were digested for 2 min. Approximately twice as much DNA in the nuclei from induced cells was rendered acid-soluble during this short digestion period. This increased susceptibility to digestion indicates an altered chromatin conformation in the induced cells, as suggested by the work of others (29-31). When the resulting sucrose gradient fractions were examined by electrophoresis (Fig. 3), mononucleosomes from 2 min diges1680
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Control
48 hr
DMSO SB
,165 155
K 0
-9
0
('I
-1
MH2A, H2B H3, H4
3 c-L N)I N. 0
t
3o3
9-
2 Z.I 1
-
0
o-ol~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure 3. Comparison of the proteins associated with the nucleosomes of Friend cells before and after 48 hours of stimulation with DMSO. Each batch of nuclei was digested for 2 min, leading to 7 and 13% acid-soluble DNA for control and induced nuclei, respectively. Samples were prepared as described in Methods and each lane was loaded with 1.1 A260 units of chromatin. The bands indicated on the left correspond to those in Figure 2. tion of the induced cells were found to have a reduced content of non-histones, reminiscent of the control mononucleosomes after a 10 min digestion. The relative amount of proteins, E, F, G, I, and J was greatly decreased in mononucleosomes from DMSO-treated cells compared to control cells. Protein L, however, was increased. The Mr 190,000 protein (band A) was confined to di- and oligonucleosomes, as it was in control cell chromatin. The effect of another potent inducer of Friend cell differentiation, 1.5 mM sodium n-butyrate (32), on the nucleosomal proteins was also examined (data not shown). Protein L was increased to a greater extent than with DMSO treatment and proteins F and J were depressed, as with DMSO. In addition, protein A followed the pattern described for control and DMSO chromatin. 1681
Nucleic Acids Research Phosphorylation of nucleosomal proteins. The phosphoproteins associated with chromatin subunits in the S2 fraction from control and DMS0-treated cells were qualitatively similar (Figure 4). However, 73% of the 32P-labelled protein was lost from the S2 nucleosomes of induced cells, based on an analysis of the scans of the autoradiogram shown in Figure 4. As mentioned above, the non-histone content of nucleosomes also decreased with induction. Quantitation based on scans of the gels shown in Figure 3 shows a 63% loss of nonhistone protein, roughly equivalent to the NtP-protein loss. Therefore, the specific radioactivity of the S2 proteins remains constant during induction, as was found for total nuclear phosphoproteins (23). Both S2 fractions contained a major phosphoprotein (pD, Figure 4) of molecular weight 110-115,000 which co-electrophoreses with protein D of Figures 2 and 3. Control and DMS0-treated cells labelled with 3P04 gave rise A
B
Con1roi
W
48 hour DMSO
E~T oE
~0
.
_____
E.lectroohoresls
Electrophoresis
Figure 4. Electrophoretic separation of labelled phosphoproteins associated with nucleosomes in the S2 fraction derived from a 2 min digest of (A) uninduced Friend cell iluclei (7% acid-soluble) and (B) nuclei of cells after 48 hours of stimulation by DMS0 (13% acid-soluble). Cells were labelled for 1 hr and fractions were prepared for electrophoresis as described in Methods. The sample load was 0.30 A260 units of control S2 and 0.60 A260 units of DMSO S2. Because the DMS0 S2 was depleted of protein (see text), this 2-fold greater load of A26n resulted in approximately half the intensity of stain in bands D and E for the DMS0 sample. After staining, the gel slab was dried and exposed to X-ray film. The identity of the radioactive phosphoproteins pD and pE was determined by comparison with the proteins D and E in the gel stained with Coomassie Blue. At the bottom of panels A and B are photographs of each channel as it appeared in the autoradiogram. The insets show scans of the same gels at expanded scale for better visualization of band pE. 1682
Nucleic Acids Research to pD with identical specific radioactivity.
This was true both in the S2 fraction and in isolated mononucleosomes. However, a second major phosphoprotein in the S2 fraction (pE, molecular weight 95-100,000) was prominent in control cells but was almost undetectable in cells treated with DMSO for 48 or 96 hr (Figure 4). All size classes of sucrose gradient-fractionated nucleosomes from control cells contained pE. It was greatly reduced in each of the corresponding size classes from induced cells. The amount of proteins D and E in the S2 fraction decreased 3- to 5-fold during induction, similar to the loss of total S2 non-histones mentioned above. In addition, the specific radioactivity pE/E decreased sharply compared to pD/D, which did not change during induction. Resolution on the gels, however, was not sufficient to allow quantitation of this decrease. We also observed a phosphorylated protein (pA) that comigrated with the Mr 190,000 protein in oligonucleosomes. Like the corresponding protein band, it appeared as a minor component in the unfractionated S2 and was never observed in gradient-purified mononucleosomes. Amido Block Stoin
A H2
48 hr DMSO
Control
H 5t
H4
Autorodiogron 48 hr DMISO-, |
2 (+)
3
4
5
6
7
Electrophoresis (cm)
8
9 (-)
Figure S. Electrophoretic separation of the phosphohistones associated with mononucleosomes of control and 48 hr DMSO-treated cells after a 2 min digestion (7 and 13% acid-soluble DNA). The histones were extracted from isolated mononucleosomes and electrophoresed as described in Methods. (A) Amido Black staining pattern; (B) scans of the autoradiograms derived from the same gels shown in (A). 1683
Nucleic Acids Research Phosphorylated histones were only minor components of the nucleosomal phosphoprotein pattern (Figure 4) and had to be characterized after acid extraction. Figure 5 shows clearly that mononucleosome-associated Hl and, to a lesser extent, H2A (but see ref. 23) are more highly phosphorylated in DMSO-treated than in untreated cells. After DMSO treatment a 3- to 4-fold greater specific activity was calculated for phospho-Hl. DISCUSSION Mononucleosomes derived from brief micrococcal nuclease digestion of uninduced Friend cell nuclei contain a specific set of non-histone proteins not found in the oligonucleosomes. This finding is of interest, because it may indicate that the early mononucleosomes are derived from a specific region of chromatin with a particular protein composition. Many of these proteins may be specifically associated with the linker regions of DNA which are removed after prolonged treatment with the nuclease. We cannot discount the possibility of non-histone protein rearrangement during our digestions, which could lead to an artifactual distribution pattern. There is, however, no reason to suppose that displaced proteins would preferentially reassociate with mononucleosomes. The distribution of the 190,000 dalton protein A, found only in oligonucleosomes, is of great interest. This protein persists after prolonged digestion and after stimulation of the cells with DMSO or n-butyrate, circumstances which lead to the disappearance of most of the other non-histones from nucleosomes. The distribution pattern of protein A suggests that it requires at least a dinucleosome structure for binding; it might even stabilize chromatin against further digestion. The chromatin-associated enzyme poly (ADP-ribose) synthase reportedly has a similar distribution among nucleosome size classes (33). Alternative explanations for the unequal distribution of protein A are that it exists in the S2 as an aggregate or that due to its size any mononucleosome to which it is bound sediments in the dinucleosome (or larger) region of sucrose gradients. Nucleosomes prepared from DMSO-treated Friend cells have a much lower content of non-histone proteins than those prepared from uninduced cells. The gel patterns resemble those obtained from a longer digestion of control cell nuclei. Non-histone proteins might be less tightly bound to the DNA of induced cells, causing a basic structural alteration of the chromatin structure. Darzynkiewitz et al. (29) and Terada et al. (30) have found evidence of such an alteration, since they see changes in the dye-binding capacity of Friend cell nuclei after DMSO treatment. These changes in chromatin struc1684
Nucleic Acids Research ture which we and others have seen in induced cells might be related to the onset of production of differentiated products
might relate to the accompanying withdrawal of Friend cells from the cell cycle. We find that the disappearance of some non-histone proteins from mononucleosomes is similar following treatment with the two inducers we have used; other proteins respond differently to DMS0 and n-butyrate. Ross and Sautner (34) found that DMS0 and hemin induce Friend cell differentiation with different time courses and probably work via different biochemical mechanisms. The non-histone protein L (Mr 27,000), which is increased in all the nucleosome fractions after induction by DMS0 or by n-butyrate is similar in molecular weight to an inducible protein described by others (35,36). The rate of phosphorylation of nucleosomal proteins is the same for control and 48 hr DM50-treated cells. This result is not unexpected, since we previously reported little or no decrease in total nuclear protein phosphorylation at this time point. The decreased phosphate content of the DMS0 S2 merely parallels the decreased amount of protein in this fraction. However, the phosphoprotein pE (Mr 95-100,000) appears to be selectively decreased in or
its specific activity and, therefore, in its rate of phosphorylation relative This selective decrease in pE phosphorylation during induction may result from a change in protein kinase and/or phosphatase activities during the differentiation process. Certain compounds, including n-butyrate, are known to increase the level of phosphatase activity in some cells (37). It is also possible that pE is already fully and stably phosphorylated in the induced cells, and hence cannot incorporate more P04 during a 1 hr pulse. Alternatively, pE could be precipitated selectively in the residue fraction during the digestion or the subsequent low ionic strength extraction of the nuclei. Whether the change we observe in pE phosphorylation is related to the process of differentiation or merely to the cessation of cell cycle activity in induced cells is not yet clear. It is interesting, however, that Kletzien et al. (38) recently reported the decreased phosphorylation of a Mr 96,000 protein in cultured hamster kidney cells when they were in stationary phase. Lough and Ingram (39) have also seen significantly decreased incorporation of phosphate into a Mr 100,000 protein during in vitro myogenesis. The rate of phosphorylation of histone Hl associated with mononucleosomes was 3- to 4-fold greater in cells treated with DMS0. Perhaps the increased phosphorylation of Hl contributes to the greater digestibility of the chromatin of stimulated cells. to the other nucleosome-associated phosphoproteins.
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Nucleic Acids Research The striking changes which we report here are in nucleosome-associated histones and non-histone proteins. We previously described (23) similar experiments in which we studied the composition of total nuclear proteins of Friend cells, without seeing most of these changes. Apparently, the changes occur only in the proteins which are associated with DNA in nucleosome structures.
ACKNOWLEDGEMENTS The authors wish to thank Dr. A. Varshavsky for reviewing this manuscript. This work was supported by a grant from the U.S. Public Health Service
(AM13945). *Present address: Genetics Research Laboratories, North Ryde, P. 0. Box 184, North Ryde, N.S.W., 2113, N.S.W. Australia 2121.
REFERENCES 1. Hewish, D.R. and Burgoyne, L.A. (1973) Biochem. Biophys. Res. Comm. 52, 504-510. 2. Noll, M. (1974) Nature 251, 249-251. 3. Olins, A.L. and Olms, D.E. (1974) Science 183, 330-32. 4. Kornberg, R.D. and Thomas, J.O. (19745 Science 184, 865-68. 5. Kornberg, R.D. (1974) Science 184, 868-71. 6. Compton, J.L., Bellard, H. and Chambon, P. (1976) Proc. Natl. Acad. Sci. U.S. 73, 4382-86. Lohr, D., Corden, J., Tatchell, K., Kovacic, R.T. and Van Holde, K.E. 7. (1977) Proc. Natl. Acad. Sci. U.S. 74, 79-83. 8. Axel, R., Cedar, H. and Felsenfeld, G. (1975) Biochemistry 14, 2489-95. 9. Lacy, E. and Axel, R. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3978-82. 10. Kuo, M.T., Sahasrabuddhe, C.G. and Saunders, G.F. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1572-75. 11. Weintraub, H. and Groudine, M. (1976) Science 193, 848-856. 12. Garel, A. and Axel, R. (1976) Proc. Natl. Acad. Sci. U.S 73, 3966-70. 13. Garel, A., Zolan, M. and Axel, R. (1977) Proc. Natl. Acad. Sci. U.S. 74, 4867-71. 14. Flint, S.J. and Weintraub, H.M. (1977) Cell 12, 783-94. 15. Montagna, R.A., Rodriguez, L.V. and Becker, F.F. (1977) Arch. Biochem. Biophys. 179, 617-24. 16. Allfrey, V.G., Johnson, E.M., Karn, J. and Vidali, G. (1973) in Protein Phosphorylation in Control Mechanisms (Huijing, F. and Lee, E.Y.C., ed.) pp. 217-49, Academic Press, New York. 17. Park, W.D., Stein, J.L. and Stein, G.S. (1976) Biochemistry 15, 3296-3300. 18. Liew, C.C. and Chan, P.K. (1976) Proc. Natl. Acad.-Sci. U.S. 73, 3458-62. 19. Chan, P.K. and Liew, C.C. (1977) Can. J.Bioce;m. 53847-55. 20. Bohm, J., Keil, G. and Knippers, R. J. Biochem. 78, 251-66. 21. Friend, C., Scher, W., Holland, J.G. and Sato, T. (1971) Proc. Natl. Acad. Sci. U.S. 68, 378-82.
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Ross, J., Gielen, J., Packman, S., Ikawa, T. and Leder, P. (1974) J. Mol. Biol. 87, 687-714. Neumann, J.R., Housman, D., and Ingram, V.M. (1978) Exptl. Cell Res., in press. Alfageme, C.R., Zweidler, A., Mahowald, A. and Cohen, L.H. (1974) J. Biol. Chem. 249. 3729-36. Varshavsky, A.J., Bakayev, V.V. and Georgiev, G.P. (1976) Nucl. Acids Res. 3, 477-92. Maniatis, T., Jeffrey, A., and van de Sande, H. (1975) Biochemistry 14, 3787-94. Sollner-Webb, B. and Felsenfeld, G. (1975) Biochemistry 14, 2915-20. Bakayev, V.V., Bakayeva, T.G. and Varshavsky, A.J. (1977) Cell 11, 619-29. Darzynkiewicz, Z., Traganos, F., Sharpless, T., Friend, C. and Melamed, M.R. (1976) Exptl. Cell Res. 99, 301-309. Terada, M., Fried, J., Nudel, U., Rifkind, R.A. and Marks, P.A. (1977) Proc. Natl. Acad. Sci. U.S. 74, 248-52. Gottesfeld, J.M. anWPartington, G.A. (1977) Cell 12, 953-62. Leder, A. and Leder, P. (1975) Cell 5, 319-22. Mullins, D.W., Jr., Giri, C.P. and Samulson, M. (1977) Biochemistry 16, 506-13. Ross, J. and Sautner, D. (1976) Cell 8, 513-20. Keppel, F., Allet, B. and Eisen, H. (1977) Proc. Natl. Acad. Sci. U.S. 74, 653-56. Peterson, J.L. and McConkey, E.H. (1976) J. Biol. Chem. 251, 555-58. Griffin, M.J., Price, G.H. and Bazzell, K.L.T(174) rch. Biochem. Biophys. 164, 619-23. Kletzien, R.F., Miller, M.R. and Pardee, A.B. (1977) Nature 270, 57-59. Lough, J.W. and Ingram, V.M. (1978) Exptl. Cell Res., in press.
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Nucleosome-associated proteins and phosphoproteins of differentiating Friend erythroleukemia cells
Jeffrey Neumann, Robert Whittaker,* Barbara Blanchard and Vernon Ingram
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 8 March 1978 ABSTRACT
Mononucleosomes derived from brief digestion of uninduced Friend cell nuclei with micrococcal nuclease contain a set of non-histone chromosomal proteins which are partly or altogether missing in the oligomeric nucleosomes. On the other hand, the latter contain a protein of Mr 190,000 not seen in the mononucleosomes. Longer digestion removes most of these non-histone proteins, excepting the Mr 190,000 protein. Brief digestion of nuclei from Friend cells induced by DMSO or by n-butyrate removes most of the non-histone proteins from the nucleosomes, as did the prolonged digestion of uninduced nuclei. The Mr 190,000 protein remains, while a protein of Mr 27,000 is increased. The rate of phosphorylation of histone Hl associated with mononucleosomes was 3 to 4-fold greater in cells induced with DMSO. The major phosphoprotein and most of the other phosphorylated non-histones were modified at the same rate in control and induced cells. However, a Mr 95,000 protein was less phosphorylated in the induced cells. INTRODUCTION The repeating pairs of DNA wound histones H2A, H2B, region of "linker"
subunit of chromatin, the nucleosome, consists of 140 base about a central octamer consisting of two of each of the H3 and H4 (1-5). Monomeric nucleosomes are separated by a DNA containing 30-60 base pairs, depending on cell type and degree of transcriptional activity (6-7). Actively transcribed gene sequences remain associated with nucleosomes (8-10) after micrococcal nuclease (EC 3.1. 4.7.)digestion of nuclei. Incubation with pancreatic DNase I (EC 3.1.4.5), however, preferentially digests active or tissue-specific DNA sequences, both from intact chromatin and from chromatin previously digested with micrococcal nuclease (11-13). This differential sensitivity may be due to the presence or absence of certain non-histone chromosomal proteins or to the modification of Hl or the core histones in stretches of the genome where RNA synthesis is occurring. Non-histone proteins are enriched in "active" chromatin (15), as are phosphorylated forms of these proteins (16). It has been suggested that the
C Information Retrieval Umited 1 FalconbwgCourt London W1V 5FG England
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Nucleic Acids Research non-histones confer transcriptional specificity on reconstituted chromatin (17). Although the occurrence and distribution of non-histones and their phosphorylated derivatives in isolated mononucleosomes has recently been reported (18-20), their role is unknown. Friend erythroleukemia cells provide a useful system in which to investigate changes in nuclear proteins which presumably occur during the induction of genetic reprogramming brought about by agents which induce differentiation (21,22). We report here the distribution of histones, non-histone proteins and their phosphorylated forms in chromatin subunits of uninduced and induced Friend cells. METHODS Cell culture and Labelling. Friend erythroleukemia cells (clone 745-PC-4) were cultured as previously described (23). For induction experiments DMSO was used at 210 mM and sodium n-butyrate at 1.5 mM. Labelling with 9P04 was accomplished by incubating cells at 2 x 107/ml in conditioned medium for 1 hr in the presence of 100-200 uCi/ml32P-orthophosphate. (New England Nuclear, Boston, MA) Nucleosome Preparation. Purified nuclei were suspended at 5 x 107/ml in a digestion buffer (0.34M sucrose/0.06M KC1/0.015M NaCl/0.015M 2-mercaptoethanol/0.015M Tris-HCl (pH 7.4) containing 0.11 mM spermine and 0.38 mM spermidine (1). They were preincubated for 3 min at 370C with 2mM CaC12 and then digested with 96 units/ml of micrococcal nuclease (E.C. 3.1.4.7, Worthington Biochemicals). The reaction was stopped by placing the nuclei on ice and immediately bringing the suspension to 4 mM Na2EDTA. Centrifugation at SOOxg for 5 minutes yielded a supernatant fraction S1. The nuclei were then extracted for 30 to 45 minutes with 0.2 mM Na2EDTA, pH 7, and the nucleosome-containing supernatant (S2) was recovered after centrifuging the suspension at 12,000xg for 5 minutes. The S2 fraction was layered directly on a 5-28.8% linear sucrose gradient containing 1 mM Na2EDTA, pH 7, and centrifuged at 140,000 xg for 22 hours. Gradients were analyzed with a Gilford Model 240 spectrophotometer and fractions corresponding to monomers, dimers plus trimers, and higher order oligomers were combined. The pooled fractions were dialyzed overnight against 0.1 mM phenylmethylsulfonyl fluoride and then lyophilized. The dried samples were dissolved in 0.5M NaCl/0.05M MgC12/0.OlM Tris-HCl (pH 7.5) and digested for 15 min at 370C with 3000 units/ml each of pancreatic DNase and pancreatic RNase (E.C. 3.1.4.22.) to remove labelled nucleic acids. They were then dialyzed exhaustively against 0.05M Tris-HCl, pH 8.6, containing 0.1% SDS. 1676
Nucleic Acids Research Electrophoresis. Analysis on 5-15% acrylamide gradient slab gels in the presence of SDS and autoradiography of gels containing 9P-labelled proteins was done as previously described (23). Specific radioactivity of phosphoproteins was determined from densitometric scans of Coomassie Blue staining patterns and autoradiograms derived from the same gels. The calculated specific radioactivities referred to in RESULTS are the ratios of the area under the autoradiogram scan (9') to the area under the staining profile (protein). This method was used both for total protein in a fraction and for specific well-resolved proteins. Histones were extracted from sucrose gradient fractions with 2M NaCl0.4N H2S04. They were precipitated with 25% (w/v) trichloroacetic acid, dialyzed exhaustively against distilled water and lyophilized. They were dissolved in SM urea-0.9N acetic acid-0.38% (v/v) Triton DF-16 (Sigma Chemical Co., St. Louis, MO) and electrophoresed on 15% acrylamide gels as described by Alfageme et al. (24). Non-denaturing gels for the analysis of nucleosome fractions were run according to Varshavsky et al. (25). Aliquots of the gradient fraction corresponding to the monomer peak were applied directly to the gels. Following electrophoresis the gels were scanned at 260 nm. DNA was isolated from nucleosome fractions by the method of Maniatis et al. (26). Purified DNA was electrophoresed on 6% polyacrylamide slab gels containing 98% formamide. RESULTS Nucleosome-associated Proteins. We determined the kinetics of micrococcal nuclease digestion of Friend cell nuclei from uninduced cells. Sucrose gradient analysis was performed on the S2 nucleosome fraction derived from nuclei digested for times varying from 30 sec to 10 min (0.7% to 23% acidsoluble DNA). In addition, DNA was prepared from the S2 fraction and from monomers isolated after digestion for 2 or for 10 min. The purified DNA was fractionated on denaturing gels. Both the sucrose gradients and the denaturing gels of isolated DNA revealed the subunit profiles expected from the literature (2,27). Mononucleosomes and the corresponding monomer lengths of DNA accumulated after longer digestion times at the expense of larger fragments. After 2 min of digestion, DNA fragments isolated from mononucleosomes were on the average somewhat longer and more heterodisperse than those obtained after 10 min, presumably due to the presence of internucleosomal linker DNA. Non-denaturing gels (Figure 1A) showed that the monomer fraction from a 2 min digest contained a considerably higher proportion of the 170 base pair, Hl1677
Nucleic Acids Research
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i
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~ +()Elctrophoresis
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Figure 1.
(A) Electrophoresis in 5% non-denaturing polyacrylamide gels of S2 nucleosome fractions derived from 2 min and 10 min digests (7.5% and 18.596 acid-soluble DNA, respectively) . (B) Electrophoresis as in (A) of monosomes isolated from sucrose gradients after 10 min digestion (16.7% acid-soluble DNA). The gel was scanned at 260 nm to locate the DNA. After staining with Coomassie Blue, the gel was scanned again at 550 nm to detect the protein.
contaning mononucleosome species, corresponding to the MN 2 band described by Bakayev et al. (28), than monomers derived from a longer digest. The Coomassie Blue staining pattern superimposed on the A260 profile of 10 min mononucleosomes (Figure 1B) establishes that the proteins (presumably both histones and non-histones) are bound to the DNA and not merely tailing from the top of the gradient into the mononucleosome band during centrifugation in the sucrose gradient. The mononucleosomes from a brief digestion display a similar correspondence of ASS0 and A260 profiles. We show the 10 min pattern because the digestion mixture after the longer incubation would be expected to have a greater amount of protein from the top of the gradient which might artifactually co-sediment with the monomer peak. Note the absence of low molecular weight staining material. The proteins in fractions S1 and S2 from uninduced cells and portions of the sucrose gradient containing monomers, di- and trimer, and oligomers were analyzed by electrophoresis in SDS-containing gels (Figure 2). Samples of intact nuclei and fractions from the tops and bottoms of the gradients were also examined (data not shown). Sham-digested nuclei released negligible amounts of DNA or protein into either fraction S1 or S2. Quantitation of the area under densitometric tracings showed release of 1678
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Nucleic Acids Research
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S2 mono di/tri oligol Si S2 mono di/tri oligo| l 2minute digest | Ominute digest
Figure 2. SDS-polyacrylamide gel electrophoresis of the protein in fractionated chromatin subunits. S1, S2, and pooled sucrose gradient fractions
corresponding to the indicated nucleosome fractions obtained from 2 min and 10 min digests (7.5 and 18.5% acid soluble DNA) were separated by electrophoresis as described in Methods. Each lane was loaded with one A260 unit of chromatin. The gel was stained with 0.1% Coomassie Blue and photographed using a green filter. Molecular weights of the indicated proteins were determined by comparison with a standard curve constructed with the following markers: soybean trypsin inhibitor (21,500), bovine serum albumin (68,000) and the subunits of RNA polymerase from E. coli (39,000, 155,000 and 165,000). Tne major bands, with their calculated molecular weights, are designated: A(180-190,000), B(140-150,000), C(125-130,000), D(ll0115,000), E(95-100,000), F(63-65,000), G(41-43,000), I(36-38,000), J(3335,000), K(32-33,000), L(26-28,000) and M(20-22,000). about one-third of the total extractable non-histone proteins (S1 plus S2) With further digestion into the S1 fraction after only 2 min of digestion. there was a slight increase in the amount of protein in this fraction, resulting in only small differences between the protein patterns of the S1 fraction after 2 or 10 min of digestion. The amount of histone associated with fraction S1 was variable. The non-histone proteins in the S1 fraction were probably released as free proteins or as very small deoxynucleoprotein fragments (subnucleosomes) (28). The S1 fraction, with an ionic strength of approxi1679
Nucleic Acids Research mately 0.1M, would also contain nucleoplasmic proteins and ribonucleoprotein particles. The patterns of non-histone proteins in the S2 fractions at the two time points were similar, but different from the Si patterns. The S2 fraction contains proteins that are complexed with nucleosomes and perhaps also free proteins released during the low salt extraction. Because of the likely presence of free proteins in the S2, we also examined gradient purified mono- and oligonucleosome fractions. In contrast to dimers and oligomers, the mononucleosomes from a 2 min digest were strikingly enriched in non-histone proteins. While all nucleosome fractions contained the expected complement of core histones, the mononucleosomes reproducibly had greater amounts of certain non-histone proteins, which we have for convenience designated as B, C, D, E, and I. Protein E (Mr 95-100,000) is interesting, because it was only a minor component of fraction S2. All of these non-histone proteins were greatly reduced in or absent from the multimer fractions. Conversely, protein A (Mr 190,000) was more prominent in the oligonucleosomes and in fact has never been detected in mononucleosomes. Further digestion with micrococcal nuclease for a total of 10 min produced a nucleosome population with a low content of non-histone proteins; only proteins I and M remained prominent in the mononucleosome fraction. The missing proteins were presumably released from the nucleosomes, since they were somewhat enriched in the top fraction of the sucrose gradient (data not shown). From quantitative measurements we deduce that they must also have precipitated in non-extractable form in the residual nuclei. Protein A, however, was not found in the top fraction, nor did it accumulate in the Sl fraction or with mononucleosomes. It was the most prominent non-histone protein associated with the di- and oligonucleosomes from 10 min digests. Since micrococcal nuclease generates early in digestion a population of mononucleosomes which is enriched in non-histone proteins, we thought it would be of interest to compare this chromatin-bound subset of nuclear proteins in uninduced Friend cells with those in DMSO-treated cells. Nuclei of control and 48 hr DMSO-treated cells were digested for 2 min. Approximately twice as much DNA in the nuclei from induced cells was rendered acid-soluble during this short digestion period. This increased susceptibility to digestion indicates an altered chromatin conformation in the induced cells, as suggested by the work of others (29-31). When the resulting sucrose gradient fractions were examined by electrophoresis (Fig. 3), mononucleosomes from 2 min diges1680
Nucleic Acids Research
Control
48 hr
DMSO SB
,165 155
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-9
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MH2A, H2B H3, H4
3 c-L N)I N. 0
t
3o3
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2 Z.I 1
-
0
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Figure 3. Comparison of the proteins associated with the nucleosomes of Friend cells before and after 48 hours of stimulation with DMSO. Each batch of nuclei was digested for 2 min, leading to 7 and 13% acid-soluble DNA for control and induced nuclei, respectively. Samples were prepared as described in Methods and each lane was loaded with 1.1 A260 units of chromatin. The bands indicated on the left correspond to those in Figure 2. tion of the induced cells were found to have a reduced content of non-histones, reminiscent of the control mononucleosomes after a 10 min digestion. The relative amount of proteins, E, F, G, I, and J was greatly decreased in mononucleosomes from DMSO-treated cells compared to control cells. Protein L, however, was increased. The Mr 190,000 protein (band A) was confined to di- and oligonucleosomes, as it was in control cell chromatin. The effect of another potent inducer of Friend cell differentiation, 1.5 mM sodium n-butyrate (32), on the nucleosomal proteins was also examined (data not shown). Protein L was increased to a greater extent than with DMSO treatment and proteins F and J were depressed, as with DMSO. In addition, protein A followed the pattern described for control and DMSO chromatin. 1681
Nucleic Acids Research Phosphorylation of nucleosomal proteins. The phosphoproteins associated with chromatin subunits in the S2 fraction from control and DMS0-treated cells were qualitatively similar (Figure 4). However, 73% of the 32P-labelled protein was lost from the S2 nucleosomes of induced cells, based on an analysis of the scans of the autoradiogram shown in Figure 4. As mentioned above, the non-histone content of nucleosomes also decreased with induction. Quantitation based on scans of the gels shown in Figure 3 shows a 63% loss of nonhistone protein, roughly equivalent to the NtP-protein loss. Therefore, the specific radioactivity of the S2 proteins remains constant during induction, as was found for total nuclear phosphoproteins (23). Both S2 fractions contained a major phosphoprotein (pD, Figure 4) of molecular weight 110-115,000 which co-electrophoreses with protein D of Figures 2 and 3. Control and DMS0-treated cells labelled with 3P04 gave rise A
B
Con1roi
W
48 hour DMSO
E~T oE
~0
.
_____
E.lectroohoresls
Electrophoresis
Figure 4. Electrophoretic separation of labelled phosphoproteins associated with nucleosomes in the S2 fraction derived from a 2 min digest of (A) uninduced Friend cell iluclei (7% acid-soluble) and (B) nuclei of cells after 48 hours of stimulation by DMS0 (13% acid-soluble). Cells were labelled for 1 hr and fractions were prepared for electrophoresis as described in Methods. The sample load was 0.30 A260 units of control S2 and 0.60 A260 units of DMSO S2. Because the DMS0 S2 was depleted of protein (see text), this 2-fold greater load of A26n resulted in approximately half the intensity of stain in bands D and E for the DMS0 sample. After staining, the gel slab was dried and exposed to X-ray film. The identity of the radioactive phosphoproteins pD and pE was determined by comparison with the proteins D and E in the gel stained with Coomassie Blue. At the bottom of panels A and B are photographs of each channel as it appeared in the autoradiogram. The insets show scans of the same gels at expanded scale for better visualization of band pE. 1682
Nucleic Acids Research to pD with identical specific radioactivity.
This was true both in the S2 fraction and in isolated mononucleosomes. However, a second major phosphoprotein in the S2 fraction (pE, molecular weight 95-100,000) was prominent in control cells but was almost undetectable in cells treated with DMSO for 48 or 96 hr (Figure 4). All size classes of sucrose gradient-fractionated nucleosomes from control cells contained pE. It was greatly reduced in each of the corresponding size classes from induced cells. The amount of proteins D and E in the S2 fraction decreased 3- to 5-fold during induction, similar to the loss of total S2 non-histones mentioned above. In addition, the specific radioactivity pE/E decreased sharply compared to pD/D, which did not change during induction. Resolution on the gels, however, was not sufficient to allow quantitation of this decrease. We also observed a phosphorylated protein (pA) that comigrated with the Mr 190,000 protein in oligonucleosomes. Like the corresponding protein band, it appeared as a minor component in the unfractionated S2 and was never observed in gradient-purified mononucleosomes. Amido Block Stoin
A H2
48 hr DMSO
Control
H 5t
H4
Autorodiogron 48 hr DMISO-, |
2 (+)
3
4
5
6
7
Electrophoresis (cm)
8
9 (-)
Figure S. Electrophoretic separation of the phosphohistones associated with mononucleosomes of control and 48 hr DMSO-treated cells after a 2 min digestion (7 and 13% acid-soluble DNA). The histones were extracted from isolated mononucleosomes and electrophoresed as described in Methods. (A) Amido Black staining pattern; (B) scans of the autoradiograms derived from the same gels shown in (A). 1683
Nucleic Acids Research Phosphorylated histones were only minor components of the nucleosomal phosphoprotein pattern (Figure 4) and had to be characterized after acid extraction. Figure 5 shows clearly that mononucleosome-associated Hl and, to a lesser extent, H2A (but see ref. 23) are more highly phosphorylated in DMSO-treated than in untreated cells. After DMSO treatment a 3- to 4-fold greater specific activity was calculated for phospho-Hl. DISCUSSION Mononucleosomes derived from brief micrococcal nuclease digestion of uninduced Friend cell nuclei contain a specific set of non-histone proteins not found in the oligonucleosomes. This finding is of interest, because it may indicate that the early mononucleosomes are derived from a specific region of chromatin with a particular protein composition. Many of these proteins may be specifically associated with the linker regions of DNA which are removed after prolonged treatment with the nuclease. We cannot discount the possibility of non-histone protein rearrangement during our digestions, which could lead to an artifactual distribution pattern. There is, however, no reason to suppose that displaced proteins would preferentially reassociate with mononucleosomes. The distribution of the 190,000 dalton protein A, found only in oligonucleosomes, is of great interest. This protein persists after prolonged digestion and after stimulation of the cells with DMSO or n-butyrate, circumstances which lead to the disappearance of most of the other non-histones from nucleosomes. The distribution pattern of protein A suggests that it requires at least a dinucleosome structure for binding; it might even stabilize chromatin against further digestion. The chromatin-associated enzyme poly (ADP-ribose) synthase reportedly has a similar distribution among nucleosome size classes (33). Alternative explanations for the unequal distribution of protein A are that it exists in the S2 as an aggregate or that due to its size any mononucleosome to which it is bound sediments in the dinucleosome (or larger) region of sucrose gradients. Nucleosomes prepared from DMSO-treated Friend cells have a much lower content of non-histone proteins than those prepared from uninduced cells. The gel patterns resemble those obtained from a longer digestion of control cell nuclei. Non-histone proteins might be less tightly bound to the DNA of induced cells, causing a basic structural alteration of the chromatin structure. Darzynkiewitz et al. (29) and Terada et al. (30) have found evidence of such an alteration, since they see changes in the dye-binding capacity of Friend cell nuclei after DMSO treatment. These changes in chromatin struc1684
Nucleic Acids Research ture which we and others have seen in induced cells might be related to the onset of production of differentiated products
might relate to the accompanying withdrawal of Friend cells from the cell cycle. We find that the disappearance of some non-histone proteins from mononucleosomes is similar following treatment with the two inducers we have used; other proteins respond differently to DMS0 and n-butyrate. Ross and Sautner (34) found that DMS0 and hemin induce Friend cell differentiation with different time courses and probably work via different biochemical mechanisms. The non-histone protein L (Mr 27,000), which is increased in all the nucleosome fractions after induction by DMS0 or by n-butyrate is similar in molecular weight to an inducible protein described by others (35,36). The rate of phosphorylation of nucleosomal proteins is the same for control and 48 hr DM50-treated cells. This result is not unexpected, since we previously reported little or no decrease in total nuclear protein phosphorylation at this time point. The decreased phosphate content of the DMS0 S2 merely parallels the decreased amount of protein in this fraction. However, the phosphoprotein pE (Mr 95-100,000) appears to be selectively decreased in or
its specific activity and, therefore, in its rate of phosphorylation relative This selective decrease in pE phosphorylation during induction may result from a change in protein kinase and/or phosphatase activities during the differentiation process. Certain compounds, including n-butyrate, are known to increase the level of phosphatase activity in some cells (37). It is also possible that pE is already fully and stably phosphorylated in the induced cells, and hence cannot incorporate more P04 during a 1 hr pulse. Alternatively, pE could be precipitated selectively in the residue fraction during the digestion or the subsequent low ionic strength extraction of the nuclei. Whether the change we observe in pE phosphorylation is related to the process of differentiation or merely to the cessation of cell cycle activity in induced cells is not yet clear. It is interesting, however, that Kletzien et al. (38) recently reported the decreased phosphorylation of a Mr 96,000 protein in cultured hamster kidney cells when they were in stationary phase. Lough and Ingram (39) have also seen significantly decreased incorporation of phosphate into a Mr 100,000 protein during in vitro myogenesis. The rate of phosphorylation of histone Hl associated with mononucleosomes was 3- to 4-fold greater in cells treated with DMS0. Perhaps the increased phosphorylation of Hl contributes to the greater digestibility of the chromatin of stimulated cells. to the other nucleosome-associated phosphoproteins.
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Nucleic Acids Research The striking changes which we report here are in nucleosome-associated histones and non-histone proteins. We previously described (23) similar experiments in which we studied the composition of total nuclear proteins of Friend cells, without seeing most of these changes. Apparently, the changes occur only in the proteins which are associated with DNA in nucleosome structures.
ACKNOWLEDGEMENTS The authors wish to thank Dr. A. Varshavsky for reviewing this manuscript. This work was supported by a grant from the U.S. Public Health Service
(AM13945). *Present address: Genetics Research Laboratories, North Ryde, P. 0. Box 184, North Ryde, N.S.W., 2113, N.S.W. Australia 2121.
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