Studies on Highly Metabolically Active Acetylation and Phosphorylation of Histones*

SUMMARY The capacity to effectively label tumor cell histones using very short pulses of [3H]acetate and [32P]phosphate (1 to 10 min) has been developed. Four histone fractions Fa, Fzal, F2a2r and FPb are extensively acetylated in short time periods. About 70% of the acetate accumulated on the histone during a short pulse is removed with a half-life of -3 min. The rest of the metabolically active acetate is removed with a half-life of 30 to 40 min. Histones Ffalr Fza2, and FL are acetylated at the NH, terminus and this modification is metabolically stable. In short pulses, histones are labeled with 32P in the order Fzaz > F1 > Fa > Fzal > F2b. All fractions have a fairly rapid turnover time (f,,, = 20 to 40 min) except F1 phosphate which turns over some 5 times more slowly. Histones are an unusual group of proteins in that they are modified extensively by acetylation, phosphorylation, and methylation 2). Histones FZaz are phosphorylated extensively is phosphorylated slightly interphase

The capacity to effectively label tumor cell histones using very short pulses of [3H]acetate and [32P]phosphate (1 to 10 min) has been developed.
Four histone fractions Fa, Fzal, F2a2r and FPb are extensively acetylated in short time periods. About 70% of the acetate accumulated on the histone during a short pulse is removed with a half-life of -3 min. The rest of the metabolically active acetate is removed with a half-life of 30 to 40 min. Histones Ffalr Fza2, and FL are acetylated at the NH, terminus and this modification is metabolically stable. In short pulses, histones are labeled with 32P in the order Fzaz > F1 > Fa > Fzal > F2b. All fractions have a fairly rapid turnover time (f,,, = 20 to 40 min) except F1 phosphate which turns over some 5 times more slowly.
Histones are an unusual group of proteins in that they are modified extensively by acetylation, phosphorylation, and methylation (1,2). Histones F1 and FZaz are phosphorylated extensively in dividing cells, F, is phosphorylated slightly in interphase cells, but extensively in metaphase (3,4). F2b is apparently not phosphorylated in mammalian somatic cells, although it, along with the other histone molecules, is extensively phosphorylated during spermatogenesis in trout (5,6). Histones F1 and FZaz are phosphorylated quite rapidly after synthesis in trout testes, but Fsal is phosphorylated much more slowly in a process which does not begin until 24 hours after synthesis.
In mammalian cells, Allfrey and colleagues have observed acet,ylation in the arginine-rich histones FZal and F, (7,8) and the sites of acetylation have been established by the sequence analyses of DeLange et al. (9,10). More recently McCarty and co-workers (11) indicated that histones Fzb and F2a2 from duck erythrocytes were also capable of being acetylated, in agreement with the work of Candid0 and Dixon who made similar findings in trout (12)(13)(14). Dixon and his co-workers (15) and Shepherd et al. (16,17) have argued that a substantial degree of acetylation occurs on newly synthesized histone, although Sanders et al. (11) have shown that acetylation can occur in avian erythrocytes at a time when histone synthesis is essentially nonexistent. Three histone molecules are acetylated at the NHz-terminus and this is thought to be a highly stable modification (1). Sites of internal acetylation have been documented in the work cited above, and this acetate appears to turn over. Analysis of the preceding work indicates that the turnover of acetate at internal positions is quite rapid, and it is possible to conclude from the work of Allfrey (7, 8) and of Sanders et al. (11) that acetate turnover has a half-life of 20 min or less, though Shepherd has reported that histone from CHO cells contains a stable internal acetylation site (17), and Uyvoet has described turnover times ranging from 1 hour to 24 hours (18).
Many of the preceding studies utilized rather long pulses of [3H]acetate incorporation, which would tend to decrease the final yield of any modified forms that were turning over very rapidly. We have reinvestigated the incorporation and turnover of both acetylated and phosphorylated histones after very short pulses of radiolabel.
We show that -70%) of the acetate incorporated in a 2-min pulse into histones FSalr Fa, FZit2, turns over with a half-life of -3 min. It is possible to estimate that at any given time, approximately 307, of all of these his&one molecules in the nucleus are modified in this way. We have also assayed for the dependence of acetylation and phosphorylation on active DNA synthesis.

RESULTS
Biosynthesis and Turnover of Histone Acetate-The incorporation of [3H]acetate during a lo-min pulse and its subsequent turnover is shown in Fig. 1. It is apparent that substantial incorporation of acetate is observed even following so short a pulse. Major incorporation is found in the several levels of acetylation of Fzal and F3. Both Fta2 and FZb show the presence of a lower but nonetheless significant degree of modification due to acetylation. not reported from sequence studies, though McCarty and his co-workers (11) had indicated previously that this histone fraction could undergo acetylation.
It is known that the parental forms of FZal, Fzaz, and Fi contain a NHz-terminal acetate group (1). Since this acetate group becomes rapidly associated with the molecule, and does not turnover (see below), it provides a measure of the extent of incorporation of acetate onto newly synthesized histone. The level of incorporation into the multiacetylated forms of the histone molecules is greatly in excess of that on the NHz-terminus ( Fig. 1) and thus it is apparent that much of the acetylation is occurring on histories other than those synthesized during the pulse period.
The turnover of the acetate groups is quite rapid, as about 75% of the radiolabel has been removed within 20 min. Kubsequently almost all of the label other than that on the NH2 termini of F,,,, Fsa2, and Fi has been removed within 4 hours after the pulse. Details of the turnover are shown graphically for F3 histone in Fig. 2 which indicates that at least two turnover rates are involved. Approximately 75yc of the label has been removed within the first 20 min of the chase, indicating a tllz 5 10 min. An additional 20% of the label turns over with t1j2 = 30 min.  After 85 min, very little radiolabel remained associated with the acetylated forms of the F3 molecule.
The above experiment indicated that a substantial fraction of the acetate associated with F, (and other histone fractions) turns over with a half-life 5 10 min. In order to more precisely define the nature of this turnover rate, we incubated HTC cells with [3H]acetate for 1 min and chased for appropriate time intervals. Typical data are shown in Fig. 3 again using F, as an example. Each of the three levels of acetylation of F3 behave in a similar manner. These graphs contain two components. Approximately 60 to 70% of the radiolabel has a turnover time of tl/z = 3 min and most of the remaining acetate groups have tl/* = 30 min, in agreement with the previous experiments. A small fraction of acetate groups (5 to 80/c) may have a lower rate of turnover. This graph provides evidence that the acetate chase is initiated quite promptly, and is clearly evidenced within 6 min of the chase period.
The combined data for all histones from studies analyzed in the manner of Figs. 2 and 3 are shown in Table I.' Only histone Fzaz does not provide clear evidence of a fraction which turns over more slowly than tilz = 2 to 3 min. All other histones (except R) contain both a fraction which turns over rapidly and also a somewhat less metabolically active component. The relative contents of these fractions vary somewhat. Fz,i contains the least amount of the more rapidly hydrolyzed fraction and the largest amount of the slower fraction.
It was not possible to assess whether there was a small fraction of the histone acetate which turned over with a half-life much in excess of 30 to 40 min because the values of the residual counts fell so rapidly towards base-line values following the exceedingly short pulses described above. Accordingly, we incubated HTC cells in the presence of [3H]acetate for a full cell generation time (17 hours), and followed the turnover of the various histone fractions during an extended chase period. The results of such an approach are shown in Fig. 4 in which we show the specific activity of the various histone subfractions, corrected for isotope dilution due to the increase in cell (and histone) numbers. The critical difference between this figure and Fig. 1 lies in the amount of ('HIacetate associated with the unmodified F,,,, FZaz, and F1 molecules as a result of the substantial accumulation of acetate into the NHz-terminal position of these mole- Incorporation and Turnover-Cycloheximide possesses the capacity to rapidly inhibit DNA and protein synthesis in HTC cells (21). This provides us with a means to assess the extent of acetylation of histones which had not been synthesized immediately prior to the addition of radiolabel. The extent of t3H]acetate incorporation in the presence or absence of cycloheximide is shown in Fig. 5. Only small changes are observed, for instance there is a decrease of about 20% in the extent of incorporation into FZal and an increase of about 25% in labeling of FI. All other fractions remain unchanged. Analysis of turnover indicated the presence of fast and slower turnover rates of comparable degree to those reported above and the data are therefore not presented. Thus, the inhibition of DNA and histone synthesis exerts very little change in the nature of acetylation of histone fractions, except that NHz-terminal acetylation (which is synthesis dependent) is abolished. Evidently, a considerable proportion of acetate becomes associated with "old" histone, as was also suggested by the data presented above (Fig. 1).
Phosphorylation of Various Histone Fractions and Turnover-Modification of histones by phosphorylation has been an area of considerable interest for some time. We have surveyed all histone fractions for phosphorylation using the approach described 0 hr. I 3 hr.
1 FIN. 4. Turnover of histone acetate after long term incorporation of radiolabel. The experimental design was the same as that in Fig. 3 except that the pulse was for a period of 17 hours. All counts are normalized to a constant amount of histone applied to the gel and corrected for isotope dilution due to increase in cell number during the chase period. AC, and Aca indicate modifications due to mono-and diacetylation. in this paper, as high levels of incorporation were possible and we could analyze for low level acquisition of phosphate groups. Furthermore, using a short pulse, we could analyze for turnover rates of such rapidity that they might not have been observed 100 50 FIG. 7. Turnover of [3*P]phosphate associated with histones. The total radiolabeled phosphate associated with the various modified species of each of Ft, Fea, F2az, and F1 is shown as a function of time (min). Data are from the results of Fig. 6.

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previously. The value of this approach is documented in Fig. 6 in which we see the results of a IO-min pulse of "P followed by a chase period extended for 20 hours. The incorporation of 32P after a lo-min pulse is in many ways quite different from what we and several other groups have observed using longer pulse periods. In this experiment the most extensively labeled fraction is F2s2. The second highest labeled fraction is Fi followed by F3 and finally at a very low level by Fzal. Histone Fsb is not phosphorylated. If Fzb is isolated by the method of Johns (22), a vast quantity of 32P is present in this fraction; however, electrophoresis removes the label in its entirety from the FPb histone which migrates free from radioactivity. In addition, the high resolution gels (Fig. 6) leave no doubt that Fta2 and not F2b is the recipient of the phosphate groups. The turnover of the various histone phosphate fractions can be compiled from the data of Fig. 6 and is shown in Fig. 7. The reason that F2a2 shows a greater accumulation of label than Fi in short pulse experiments but not in longer pulses now becomes clear, inasmuch as FZa2 contains a fraction with a much more rapid turnover than that seen for FL Approximately 60% of Fzaz phosphate turns over with a t1/2 = 40 min. The F, phosphorylated in the short pulse turns over with a ti 2 = 3 hours which is significantly different from the long term tl,2 which has a value of 5 hours in these cells (23). Both F3 and Ftai, like F,,,, have two turnover rates for their phosphate modifications; a faster rate of hydrolysis with t112 = 30 min and a slower with tl12 = 2 to 3 hours. The initiation of the chase period is much slower for [32P]-than for [3H]acetate, presumably reflecting a greater time needed for depletion of the phosphate pool.

Effect of Inhibition of DNA and Protein Synthesis on Histone
Phosphorylation-A comparison of the incorporation of 32P into the various histone fractions, in growing cells and cells in which DNA and protein synthesis was inhibited, is shown in Fig. 8. Histone Fi shows a 50% decrease in phosphate incorporation in agreement with previous observations (21), histone F2,2 shows a small (15%) decrease, whereas histone F3 is apparently unaffected by this block in macromolecular synthesis. Although the level of incorporation of 32P into F2*l is low in the control cells, it appears that it is reduced by cycloheximide treatment. The effect of cycloheximide treatment on the turnover of the various phosphorylated histone molecules was also studied in a manner analogous to the studies with acetylated histones. Unfortunately FIG. 8. Effect on phosphorylation of inhibition of DNA and histone synthesis. Histones were labeled for 10 min with [32P]orthophosphate either in the presence (+CH) or absence (-CH) of cycloheximide. The cells were collected and histones analyzed for associated 32P as described above. Prior to labeling with [32P]orthophosphate, the cycloheximide-treated cells were incubated 45 min in medium containing cycloheximide (10 pg/ml). Pt and P, indicate modifications of mono-and diphosphorylation. the termination of the 3*P pulse period is much slower if cycloheximide was present during the pulse period and these studies were not pursued further. u1scuss10s The capacity for studying acetylation and phosphorylation of histones after very short pulses of radiolabel has been developed. The termination of a pulse of [3H]acetatc occurs promptly and the chase period can be initiated with some precision. This is not the case for phosphate incorporation and at least 40 min must elapse from the completion of the pulse period before the true chase begins and effective turnover values can be estimated.! All histone fractions are acetylated. Fractions F,,l, F2a2, and Fi arc SHz-terminally acetylated (1) and this is a stable modification, which does not turnover significantly irl a full cell cycle. Histones F2al, F,,,, F2b, and F, are all acetylated internally in a form which is metabolically highly active. This is in agreement with the observations of McCarty and his colleagues in the duck erythrocyte system (11). After a short 1-min pulse, about 700/, of the associated acetate turns over with a f112 = 3 min. This is a much shorter half-life than any value previously reported. However. analysis of earlier data (7,8,11) indicates that a significant fraction of histone acetate was turning over with a tilz of <20 min. A smaller fraction of the acetate incorporated into HTC histone molecules turns over with a tllz = 30 to 40 min.
The incorporation of labeled acetate into the NHz-terminal position of F,,, provides us with a means for estimating the extent of acetylation of the entire population of F2al molecules during a short pulse period. Thus, if after a lo-mm pulse of [3H]acetate we find "p" counts per min associated with the KHZ-terminal acetylated form of a molecule and "y" and "2" counts per min associated with internally mono-and diacetylated forms of the molecule, then the fraction of the total Fzal molecules which are internally acetylated is [(yp) + (x(zp))]/p x x6 (there are 96 units of 10 min in the 16.hour cell cycle of HTC cells). Using the data of Fig. 1 we find that 25 to 30y0 of all the FZal molecules in the cell have been acetylated during the IO-mm period. Obviously old histories must be extensively acetylated, though of course this does not at all exclude the possibility that newly synthesized histone is also acetylated.
Previously, although Dixon had estimated that newly synthesized FZal was acetylated at the time of its deposition (15), no means had existed to obtain estimates of the extent of acetylation of both new arid old histone such as those deduced above.
Inhibition of DNA and histone synthesis leads to a small but significant decrease in acetylation of the more extensively modified forms of F,,l. This is consistent with the observations by Dixon that this histone fraction is extensively acetylated shortly after its synthesis (15). We have recently confirmed this observation in HTC cells.2 Thus it is reasonable to expect a decrease in over-all acetylation of F2%, upon inhibition of histone synthesis with cycloheximide.
However, the acetylation of the other fractions does not respond in a similar manner, and thus the acetylation of new F,,, may be serving a different function to that of the acetylation of other histone fractions.
After short pulses of 3213, histone Fza2 is labeled more extensively than Fi, an observation in contrast to all previous reports. However, analysis of histone phosphate turnover explains the apparent discrepancy (23-25). F,,, has a substantial 2 V. Jackson, N. Tanphaichitr, A. Shires, and It. Chalkley, manuscript in preparation. amount of a phosphate fraction which is turning over about 4 to 5 times faster than F1 phosphate. Thus, even though F, is phosphorylatcd somewhat more slowly than Fznz (and thus shows a lower degree of 32P labeling in short pulses), after an extended pulse period, F1 accumulates a greater amount of 32P than F,,, because of its much lower rate of phosphate removal.
The effect of inhibiting DNA and histone synthesis upon histone phosphorylation in general produces a moderate decrease in 321' incorporation.
FI is most affected and F2a2 and F3 the least. The decrease in phosphorylation of Fi is due to inhibition of the more rapid phosphorylation of newly synthesized F,, whereas old R continues to be phosphorylated at a slower rate (26). It is not known whether this is true for the other three histone fractions which are phosphorylated.
Phosphorylation of F3 appears to be associated with a mitotic event (24), and it is perhaps not surprising that it is not dependent upon histone synthesis and is therefore but little affected by cycloheximide addition.
Several hypotheses have been presented concerning the biologic functions of histone modifications.
These include (a) gene activation through acetylation (8,27) or phosphorylation (28) ; (b) histone deposition through the combined action of acetylation and phosphorylation (12) ; and (c) chromosome condensation due to phosphorylation (24,29). To some extent the observations reported in this paper are consistent with proposal b, but it is clear that the acetylation is much too extensive and occurs to such a degree on old histone as to render this only a part of their total function. Furthermore acetylation has been reported in nondividing cells (11). The extent of the acetylation also appears to exclude a specific gene activating event, even though it could be working indirectly to facilitate RNA biosynthesis. However, it seems that the entire chromosome is probably exposed to this type of modification, as both euchromatin and heterochromatin are acetylated to the same degree (30). We envisage a 2-fold function for acetylation. On the one hand it may play a role in the deposition of specific histone fractions (mostly Fzal) as suggested by Dixon (la), and on the other hand it may serve to temporarily break a critical interaction to permit an additional function to be performed. For instance, it is conceivable that a structural role might depend upon a key electrostatic interaction and yet RNA polymerase movement along the chromosome might require the electrostatic bond be temporarily and briefly broken. Why the cell should elect to acetylate so extensively rather than at a few specific regions to be transcribed might simply reflect the high energy expenditure required for high precision acetylation.
I'hosphorglation and acetylation were first invoked to explain the electrophoretic microheterogeneity of histones in 1968 (19). It is now clear that the levels of such modified forms of histone detected in the polyacrylamide gels are the result of a vigorous dynamic equilibrium between an ongoing modification and its subsequent hydrolysis.